proceedings of the netherlands entomological society ... · attempts or pouncing behaviour. these...

Proceedings of theNetherlands Entomological Society

Meeting

Volume 19

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Proceedings of the Netherlands Entomological Society Meeting

[abbreviated as: Proc. Neth. Entomol. Soc. Meet.]

Volume 19 (2008)

Editor: Jan Bruin (Section Population Biology, University of Amsterdam)

ISSN: 1874-9542

ISBN: 978 90 71912 30 6

© 2008 by the Nederlandse Entomologische Vereniging (NEV)

Plantage Middenlaan 64, 1018 DH Amsterdam, The Netherlands

[http://www.nev.nl]

Copies of the Proceedings (and back volumes) may be ordered from the NEV atthis address

Printed in The Netherlands, by:

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This volume contains the proceedings of the 19th annual meeting of

entomologists in The Netherlands, organized by the Section

Experimental and Applied Entomology (SETE) of the Netherlands

Entomological Society (NEV)

14 December 2007

Ede

Organizing committee:L.W. Beukeboom (Chair), Groningen UniversityP.W. de Jong (Secretary), Wageningen UniversityJ.A.J. Breeuwer, University of AmsterdamJ. Bruin, University of AmsterdamJ. Ellers, Vrije Universiteit AmsterdamR.E. Kooi, Leiden UniversityM.J. Sommeijer, Utrecht UniversityK. Zwakhals, p/o Utrecht University

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Contents

7 Preface

INTRODUCTORY LECTURE9 N.J. VEREECKEN

Pollinator-mediated selection, reproductive isolation and floral evolution in Ophrys orchids

SOCIAL INSECTS23 H.H.W. VELTHUIS, M. CORTOPASSI LAURINO & F. CHAGAS

The nest of the Brazilian stingless bee Melipona quinquefasciata

31 D. MICHEZ

Monographic revision of the melittid bees (Hymenoptera,Apoidea, Melittidae sensu lato)

41 W.J. BOOT, J.N.M. CALIS & M.H. ALLSOPP

Selection for reproductive workers in the Cape honeybee population,Apis mellifera capensis, leads to social parasitism in bee colonies from thesavanna

53 J. PAALHAAR, W.J. BOOT, J.J.M. VAN DER STEEN & J.N.M. CALIS

In-hive pollen transfer between bees enhances cross-pollination ofplants

59 E.-L. ALANEN

Habitats and food plants of bumblebee queens in an agriculturallandscape

FUNCTIONAL BIODIVERSITY67 L.A. LANGOYA & P.C.J. VAN RIJN

The significance of floral resources for natural control of aphids

75 J. NOORDIJK, K.V. SÝKORA & A.P. SCHAFFERS

The conservation value of sandy highway verges for arthropods –implications for management

EVOLUTIONARY ECOLOGY95 J.J. SLOGGETT

Habitat and dietary specificity in aphidophagous ladybirds(Coleoptera: Coccinellidae): Explaining specialization

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115 K. PARKER, P. ROESSINGH & S.B.J. MENKEN

Effects of multiple mating and adult nutrition on longevity andfecundity in two Yponomeuta species

MEDICAL ENTOMOLOGY121 S. ONDIAKA, T. BUKHARI, M. FARENHORST, W. TAKKEN & B.G.J.

KNOLS

Effects of fungal infection on the host-seeking behaviour andfecundity of the malaria mosquito Anopheles gambiae Giles

129 N.O. VERHULST, W. TAKKEN & R.C. SMALLEGANGE

Structural design affects entry response of mosquitoes in olfactometers

137 J. BEEUWKES, J. SPITZEN, C.W. SPOOR, J.L. VAN LEEUWEN & W.TAKKEN

3-D flight behaviour of the malaria mosquito Anopheles gambiae s.s.inside an odour plume

INSECT ECOLOGY147 L. HEMERIK & E.H. VAN NES

A new release of INSIM: A temperature-dependent model forinsect development

MONITORING157 B.J. PIETERS, D. BOSMAN-MEIJERMAN, E. STEENBERGEN, E.-J. VAN

DEN BRANDHOF, P. VAN BEELEN, E. VAN DER GRINTEN, W.VERWEIJ & M.H.S. KRAAK

Ecological quality assessment of Dutch surface waters using a newbioassay with the cladoceran Chydorus sphaericus

165 B. WESSELS-BERK & E.-J. SCHOLTE

One beetle too many: The emerald ash-borer, Agrilus planipennis(Coleoptera: Buprestidae), threatens Fraxinus trees in Europe

169 Author index171 Subject index

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This volume contains the proceedings of the 19th annual meeting of – mostlyexperimental and applied – entomologists in The Netherlands, held on Friday 14December 2007, in Ede, near Wageningen. As every year, the SectionExperimental and Applied Entomology (SETE) of the NetherlandsEntomological Society (NEV) organized the meeting. The plenary lecture waspresented by Dr. Nicolas Vereecken (Behavioural and Evolutionary Ecology,Free University of Brussels, Belgium), on the exciting interaction between(pseudo-)pollinating hymenopterans and Ophrys orchids.

Amateurs, students and professional scientists who present their work at themeeting are given the opportunity to publish in the proceedings. Apart from fewcorrections of apparent mistakes, all manuscripts are being published essential-ly as they were handed in. Only in this way it is possible to bring out the pro-ceedings within three months after the meeting - and well before the next meet-ing.

All in all, sixteen papers make up this volume, presenting a highly diverseoverview of present-day entomological research - enjoy!

Jan BruinSection Population Biology, University of AmsterdamFebruary 2008

Preface

PROC. NETH. ENTOMOL. SOC. MEET. - VOLUME 19 - 2008 7

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Nicolas J. VereeckenBehavioural and Evolutionary Ecology, Free University of Brussels CP 160/12, Av.F.D. Roosevelt 50, B-1050 Brussels, Belgium; Institute of Systematic Botany,University of Zürich, Zollikerstrasse 107, CH-8008 Zürich, Switzerland. E-mail: [email protected], [email protected]

Pollination by sexual deception is arguably one of the most intriguingpollination syndromes among orchids. In this system, patrolling maleinsects, mostly hymenopterans, bring about cross-pollination whileattempting copulation with the female decoys of orchid flowers, a phe-nomenon known as ‘pseudo-copulation’. In this study we investigatefemale sex pheromones of bees and wasps and floral odours of a rangeof European orchids in a comparative approach, along with bioassaysperformed with male bees in situ, in an attempt at unlocking the basisand specificity of orchid-pollinator interactions.

Keywords: pollination, floral odours, sexual deception, bees, orchids

Mate choice and sex pheromones in the model: Colletescunicularius

Colletes cunicularius L. (Hymenoptera, Colletidae) is a vernal solitary bee thatnests underground in dense, conspicuous aggregations (Michener 1974, Hefetz etal. 1979b, Westrich 1990, Borg-Karlson et al. 2003, Pouvreau 2004). As frequentlyobserved in wild bees and wasps, males hatch before females and devote muchof their time and energy to patrolling over nesting sites where conspecific matesare soon to emerge (Alcock et al. 1978). In C. cunicularius, females are presumablymonandrous and not limited in their reproductive success by access to males,since sexually active males typically outnumber virgin, emerging females dur-ing the reproductive period (Fig. 1). Males are therefore thought to be understrong selection pressure to be first at finding and inseminating freshly emergedfemale partners (Alcock et al. 1978) and they frequently engage in intense scram-bles to access pre-emerging females (Borg-Karlson et al. 2003, Vereecken et al.2006).

Early studies on the chemical communication in C. cunicularius have shownthat olfactory cues are primarily responsible for releasing the males’ overall exci-

Pollinator-mediated selection, reproductiveisolation and floral evolution in Ophrys orchids


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tation and in directing them towards virgin females (Bergström & Tengö 1978).These works have mostly focused on linalool, the dominant odour compound inmandibular secretions of attractive females (Bergström & Tengö 1978, Hefetz etal. 1979a, Cane & Tengö 1981). Cane & Tengö (1981) have specifically reported onthe mate-attractant function of linalool, yet their bioassays carried out with C.cunicularius males using linalool-scented dummies failed to release copulationattempts or pouncing behaviour. These authors showed that head extracts of C.cunicularius females elicited more searching flights and more copulation attemptsin patrolling males than linalool alone. More recently, Borg-Karlson et al. (2003)showed that the (S)-(+)-linalool enantiomer was the key compound for mateattraction in mandibular secretions of C. cunicularius females. These authors alsopointed out the occurrence of a dramatic decrease of the amount of this com-pound in virgin vs. mated females, which they suggested to be responsible forthe loss of attractiveness of mated females to patrolling males.

During our research project on the chemical communication in C. cunicular-ius, we have performed a combination of chemical analyses by gas chromatogra-phy and electro-antennography (GC and GC-EAD) and we have been able to

INTRODUCTORY LECTURE

Figure 1. Two males of C. cunicularius attempting copulation with a conspecific, emerg-ing female (Photo NJ Vereecken).

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perform bioassays in situ with patrolling males of C. cunicularius. A first step inour analyses has been to investigate the level of attractiveness of head versuscuticle extracts of virgin C. cunicularius females to patrolling males and to testwhether any of these blends could release a stereotyped mating behaviour. Ourpreliminary results showed that, contrary to head extracts, cuticle extracts con-tained a mixture of fatty acids (cuticular hydrocarbons) and their derivatives. Byassaying the relative attractiveness of head vs. cuticle extracts, we have been ableto demonstrate that, contrary to the head extracts, the cuticle extracts triggereda significant number of pounces and copulation attempts of patrolling males ofC. cunicularius with the scented dummies (Figs 2 & 3). These results confirmedearlier studies by Cane & Tengö (1981) and Borg-Karlson et al. (2003) on theattractiveness of linalool, which indicated the long-range mate attractant func-tion of linalool without however stating which cues are involved in short-rangestimulation of male mating behaviour. Considering that (i) linalool is a highlyvolatile compound secreted by mandibular glands in females and that (ii) longchain hydrocarbons are less volatile compounds secreted on the female bodycuticle, the sex pheromone in C. cunicularius might be considered as multi-com-ponent, with linalool acting as long-range mate attractant and epicuticular com-pounds eliciting male mating behaviour at short range (Fig. 4) (Vereecken 2004,Mant et al. 2005a).

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Figure 2. Relative attractiveness of head versus cuticle extracts of virgin C. cuniculariusfemales to patrolling males. The cuticle extracts triggered significantly more pounces(i.e. short contacts) and copulation attempts with the scented source than headextracts. Modified after Brändli (2004) and Mant et al. (2005a).

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We have then performed another series of bioassays that have helped identi-fying a set of three compounds in the cuticle extracts as the key compounds formate attraction. No significant difference was found when testing these threecompounds vs. the full blend of cuticular hydrocarbons and their derivativeswith patrolling C. cunicularius males. The three key compounds of the female sexpheromone are mono-unsaturated fatty acids, namely (Z)-7-heneicosene (=C21ene), (Z)-7-tricosene (= C23ene) and (Z)-7-pentacosene (= C25ene) (Mant etal. 2005a).

We then sat out to investigate whether females from distant populations ofC. cunicularius used the same chemical communication channel or if there wasevidence for population differences in sex pheromone blends. This new projecthas led us to sample virgin females of C. cunicularius from 15 populations acrossWestern Europe, in different countries such as Austria, Belgium, England,France, Germany, Italy Switzerland and the United Kingdom. Our first resultswere published this year (Vereecken et al. 2007) and they show that distant pop-ulations are characterised by sex pheromone ‘dialects’, i.e. the females of differ-ent sites use population-specific ratios of the key compounds for the attraction


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Figure 3. Patrolling male of C. cunicularius landing on a dummy (plastic bead mountedon a pin) scented with a cuticle extract of a conspecific, virgin female (Photo NJVereecken).

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of their mates. We have also been able to provide evidence for a positive fitbetween the chemical differences among populations and the geographic dis-tance among the study sites, i.e. the more closely-located the populations are, themore likely they are to share a very similar sex pheromone ‘dialect’ (Vereeckenet al. 2007). These studies are very encouraging and they have allowed pushinginvestigations on sex pheromones in wild bees to a level never attained before,notably (i) by pinpointing a minimum set of compounds as the key odour signaland (ii) by evaluating the variation in this signal among populations.

Since females from different populations used different ‘dialects’ of sex phe-romones, we have set out to investigate whether males of a give population weremore attracted by their ‘local’ females or if ‘exotic’ females (i.e., from allopatricpopulations) were preferred by patrolling males. All these behavioural bioassaysand the results are described by Vereecken et al. (2007) (see references list). Therecordings of behavioural responses performed with C. cunicularius males usingsynthetic copies of sex pheromones indicate that male bees are able to discrimi-nate among the three synthetic copies of sex pheromones derived geographical-ly-distant populations. Our results showed differences in the intensity of thesexual stimulation released by the synthetic blends and indicate that synthetic

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Figure 4. Schematic sketch of mate attraction by multi-component female sexpheromone in Colletes cunicularius. Linalool is secreted by mandibular glands in C.cunicularius females and acts as longrange mate attractant (large circle). Contact aphro-disiacs (i.e. the contact sex pheromone) consist of cuticular hydrocarbons located onthe female body surface and stimulate the sexual arousal of patrolling C. cuniculariusmales at close range (small circle), leading to copulatory behaviour (Vereecken 2004).

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blends of the ‘local’ sex pheromones elicited significantly less approaching flightand less contacts with the odour source than blends of ‘exotic’ sex pheromones.As suggested in our original study, these preferences are likely to be the directoutcome of C. cunicularius populations experiencing significant levels of inbree-ding. This assumption rests upon reports of males patrolling restricted regionsof their emergence/reproductive site (Peakall & Schiestl 2004), and observationsof females initiating the construction of their nest in the near vicinity of theiremergence points early in their reproductive period. Populations of C. cunicula-rius are therefore expected to experience high degrees of philopatry, and inbree-ding as an immediate consequence.

A forthcoming step in our studies on the bees’ side will be to sample morepopulations of C. cunicularius in Western Europe to test the hypothesis that thecorrelation found between ‘chemistry’ and ‘geography’ can also be highlighted atthe genetic level, e.g. by using microsatellite markers. More populations aretherefore desirable, especially from countries that have not been sampled so far(Denmark, Germany, The Netherlands, Sweden, etc.) where large colonies arepresent to make sure that the females could be sampled without putting the per-sistence of these bees in jeopardy locally.

Pollinator attraction and floral odour in the mimic: Ophrysexaltata

Quite the most remarkable pollination mechanism to be found in any floweringplants is that of the ‘insect orchids’ of the genus Ophrys. The flowers of theseorchids are well-known for their striking resemblance to various insects. Whatfunction this resemblance serves has remained a mystery for a long time(Proctor et al. 1996). The remarkable similarity between some orchids andinsects has long been discussed and many explanations were given for thisresemblance, including the belief that the likeness to insects had nothing to dowith pollination, but served to frighten away browsing cows (Van der Pijl &Dodson 1966). Even though Darwin’s (1862) treatise provides a remarkable con-tribution in the field of orchid pollination, many of the mechanisms and phe-nomena that are now part of the scientific background have been discovered andunderstood only over the past two decades (Dafni 1983, 1984, Ackerman 1986,Peakall 1990, Nilsson 1992, Schiestl et al. 1999).

It was not until the early decades of the twentieth century that it was discov-ered that pollination in most of what we now know as sexually deceptive orchidspecies is brought about by insects attempting copulation or a pre-copulatoryroutine on the orchid labellum (Correvon & Pouyanne 1916, Pouyanne 1917,Kullenberg 1961, Proctor et al. 1996, Peakall & Beattie 1996, Schiestl et al. 1999).This procedure, often called pseudo-copulation (when the insect attempts to cop-ulate with the flower) (Fig. 5) or sexual stimulation (when the insect goes throughpart only of its mating behaviour during the pollination process) was first dis-covered by Pouyanne (Correvon & Pouyanne 1916, 1923, Pouyanne 1917) after


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some 20 years of observations carried out on the field in Algeria. His works haverevealed that the flowers of the ‘mirror orchid’, Ophrys ciliata (= Ophrys specu-lum) (Fig. 6), attract a male wasp (namely Dasyscolia ciliata – Hymenoptera:Scoliidae) by imitating its female partner (Fig. 7). In the course of a century,many controversies have arisen around sexual deception. Pouyanne’s observa-tions, perhaps because he was not a member of the scientific community, weremet with disbelief by laboratory biologists who denounced his findings as the‘nonsensical ideas of an amateur ecologist’ (Van der Pijl & Dodson 1966, Van derCingel 1995, Proctor et al. 1996). Later, Godfery (1922, 1929) and Wolf (1950) cor-roborated Pouyanne’s botanical field observations and deductions, whileKullenberg (1948, 1952a, 1952b, 1956, 1961, 1973) analysed the phenomenon fromthe zoological, physiological, and chemical perspective.

The attractiveness of a flower to its pollinating agents can be attributed tothree major categories of cues, namely (i) the olfactory stimulus; (ii) the opticalappearance; and (iii) the tactile stimulus (hairiness or surface villi) (Proctor etal. 1996). These cues act on the senses of the pollinator (Van der Pijl & Dodson1966), which associates them with an expected reward (Nilsson 1983, 1992, Dafni1984, Vogel 1993, Roy & Widmer 1999). Even though the three categories of flo-ral cues are commonly interdependent (i.e. appearance combines with odour toattract the pollinator), some of these stimuli may be prevailing in attracting

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Figure 5. A male of Colletes cunicularius attempting copulation (pseudo-copulation) on aflower of Ophrys arachnitiformis (Photo NJ Vereecken).

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Ophrys pollinators (Van der Pijl & Dodson 1966). For many decades sincePouyanne’s first reports (1917), the key issue in Ophrys pollination has undoub-tedly concerned the nature of the exact floral cues that have the ability to sexual-ly stimulate male Hymenopterans in search for a mate (Barth 1991, Nilsson1992).

Of the three main types of stimuli and their respective role in attracting maleHymenopterans, it seems that the visual cue is the least important (Borg-Karlson 1990). Being the least important does not mean that visual attractiondoes not take place. Correvon & Pouyanne (1916, 1923) reported striking decrea-se male visitation when Ophrys ciliata labella are placed upside down and on backto front: male insects seemed to spend more time finding the labella because thebrown hairs and the central blue patch (‘speculum’) of the orchid labellum(which was suggested to mimic the female’s crossed wings in the sunshine) wereno longer visible. It is not really surprising that olfactory cues are the mostimportant means of insect attraction in sexually deceptive orchids (in Ophrys asin other non-European orchid genera) since, as mentioned above, olfactory cuesare key stimuli in the sexual behaviour of numerous insect species includingAculeate Hymenopterans (Kullenberg & Bergström 1973, 1976, Alcock et al. 1978,Bergström 1978, Eickwort & Ginsberg 1980, Ayasse et al. 2001). Most investiga-tions on the chemistry of Ophrys pollination have stressed the similarity bet-


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Figure 6. Detail of an individual flower of the mirror orchid, Ophrys ciliata (= O. specu-lum) (Photo NJ Vereecken).

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ween the floral odour of these orchids and pheromonal cues of their pollinators’females (Borg-Karlson & Tengö 1986, Borg-Karlson 1990, Schiestl et al. 1999,2000, Ayasse et al. 2000, 2003, Schiestl & Ayasse 2000).

Chemical mimicry in Ophrys involves cuticular hydrocarbons similar tothose found in the attracting female Hymenopterans. These compounds mainlyconsist of long-chain fatty acids and their derivates similar to those found in therespective females’ cuticle extract, and trigger a mating behaviour in the targe-ted males (Borg-Karlson 1990, Schiestl et al. 2000, Schiestl & Ayasse 2002). TheOphrys floral odour bouquet is sometimes composed of up to 100 volatile com-pounds of which only a small proportion takes part actively in insect attraction(Schiestl et al. 1999, Ayasse et al. 2000, Schiestl & Marion-Poll 2002). Qualitativeand quantitative differences in floral odour and the numerous combinations inthese volatile compounds represent a considerable source of chemodiversitywhich enhance possibilities to attract pollinators on a species-specific basis.

Recent investigations have demonstrated that O. exaltata attracts patrollingmales of C. cunicularius by releasing the same set of key odour compounds as C.cunicularius females (Mant et al. 2005a), thereby providing further evidence forchemical mimicry in Ophrys pollination. Further studies on this species in thesouth of Italy have shown that in the orchid too, the pollinator-attracting odoursignals vary across populations (Mant et al. 2005b). In short, orchids belonging

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Figure 7. A female of the scoliid wasp Dasyscolia ciliata showing the red hairs coveringits abdomen (Photo NJ Vereecken).

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to the same species but occupying different populations are characterised by dif-ferent proportions of their key odour compounds, a situation identical to whatwas observed on the bees’ side.

Predictions for the evolution of the Ophrys-pollinatormimicry systemAfter we had discovered that the signals mediating the attraction of C. cunicular-ius males varies among populations both in the model (female bees) and in themimic (the orchid) of this plant-pollinator interaction, we have started wonder-ing how accurate the mimicry was in terms of the relative proportions of the keyodour compounds. Do the orchids imitate the local ‘dialect’ of their modelspecies? Or are there differences in odour between the orchids and the bees whensampled in sympatry? From the bioassays we have perfomed with C. cunicular-ius males and the available data on floral odour variation in O. exaltata, we havepredicted that selection should favour orchids that emit odour blends that wouldslightly differ from the local females’ mating signals, which would (i) allow theattraction of C. cunicularius and (ii) make the orchids more attractive than theirlocal models to patrolling C. cunicularius males (Vereecken et al. 2007). We arecurrently in the process of analysing our data from multiple populations wherethe bees and the orchids were sampled in sympatry, and our results indicate thatthe predictions made prove to be accurate. More details on the results and thebioassays that followed will be given during the keynote presentation.

Acknowledgements I am grateful to my supervisors, Pr Florian P. Schiestl(University of Zürich, Switzerland) and Pr Jean-Christophe de Biseau (Free Universityof Brussels, Belgium) for their help and guidance throughout my research. I am alsograteful to my colleagues for their help and collaboration in these research projects, aswell as to all my friends who have helped me collecting samples of bees & orchids acrossWestern Europe. Thanks are also due to the Belgian ‘Fonds pour la formation à laRecherche dans l’Industrie et l’Agriculture’ (F.R.I.A.) for having financially supportedmy research during my PhD.

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Nilsson LA, 1992. Orchid pollination biology. Trends in Ecology & Evolution 8: 255-259.Peakall R, 1990. Responses of male Zapilothynus trilobatus turner wasps to females and the

sexually deceptive orchid it pollinates. Functional Ecology 4(2): 159-167.Peakall R & Beattie AJ, 1996. Ecological and genetic consequences of pollination by sex-

ual deception in the orchid Caladenia tentactulata. Evolution 50(6): 2207-2220.Peakall R & Schiestl FP, 2004. A mark-recapture study of male Colletes cunicularius bees:

implications for pollination by sexual deception. Behavioural Ecology and Sociobiology56: 579-584.

Pouvreau A, 2004. Les Insectes Pollinisateurs. Delachaux et Niestlé, Paris.Pouyanne M, 1917. La fécondation des Ophrys par les Insectes. Bulletin de la Société

d’Histoire Naturelle d’Afrique du Nord 43: 53-62.Proctor M, Yeo P & Lack A, 1996. The natural history of pollination. Harper Collins

Publishers.Roy BA & Widmer A, 1999. Floral mimicry: a fascinating and yet poorly understood

phenomenon. TRENDS in Plant Science & Perspectives 4(8): 325-330.Schiestl FP & Ayasse M, 2000. Post-mating odor in females of the solitary bee, Andrena

nigroaenea (Apoidea, Andrenidae) inhibits male mating behavior. Behavioural Ecologyand Sociobiology 48: 303-307.

Schiestl FP, Ayasse M, Paulus HF, Löfstedt C, Hansson BS, Ibarra F & Francke W,1999. Orchid pollination by sexual swindle. Nature 399: 421-422.

Schiestl FP & Marion-Poll F, 2002. Detection of physiologically active flower volatilesusing gas chromatography coupled with electroantennography. Molecular Methods ofPlant Analysis, Volume 21: Analysis of Taste and Aroma (eds. J.F. Jackson, H.F.Linskens & R. Inman), pp 173-198. Springer, Berlin.

Van der Cingel NA, 1995. An atlas of orchid pollination – European Orchids. A.A.Balkema/Rotterdam/Brookfield.

Van der Pijl L & Dodson CH, 1966. Orchid flowers, their pollination and evolution. FairchildTropical Garden and the University of Miami Press, Coral Gables, Florida.

Vereecken NJ, 2004. Mate choice and odour preferences in the solitary bee Colletes cunicu-larius (L.) (Hymenoptera, Apoidea, Colletidae), the exclusive pollinator of the (sexuallydeceptive) orchid Ophrys exaltata (Orchidaceae). Diploma Thesis Dissertation,Gembloux University of Agricultural Sciences 87pp, 4 Tables and 27 Figures.


20

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Vereecken NJ, Mant J & Schiestl FP, 2007. Population differentiation in female sexpheromone and male preferences in a solitary bee. Behavioral Ecology and Sociobiology61(5): 811-821.

Vereecken N, Toffin E, Gosselin M & Michez D, 2006. Observations relatives à la biolo-gie et à la nidification d’abeilles psammophiles d’intérêt en Wallonie. 1.Observations printanières. Parcs et Réserves 61(1): 8-13.

Vogel S, 1993. Betrug bei Planzen: Die Täuschblumen. 1. Akademie der Wissenschaftenund der Literatur, Mainz, Franz Steiner, Stuttgart.

Westrich P, 1990. Die Wildbienen Baden-Württembergs. Ulmer Verlag, Stuttgart.Wolf T, 1950. Pollination and fertilization of fly-orchis Ophrys insectifera L. in

Allindellille fredskov, Denmark. Oikos 2: 20-59.

N.J. VEREECKEN

21

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Vereecken-2008.qxd 1/29/2008 10:04 AM


Hayo H.W. Velthuis, Marilda Cortopassi Laurino1 & Francisco Chagas2

Klemit 1, 5325 KG Wellseind, The Netherlands, E-mail: [email protected]; 1Laboratóriode Abelhas, Depto de Ecologia, Biociências, Universidade de São Paulo, Brazil; 2Melipo-nário São Saruê, BR-101 Norte Km 39, Zona Rural, CEP 53640-000 Igarassu, PE, Brazil

The neotropical genus Melipona, found from Mexico to Argentina, con-tains about 40 species. Most species live in woodland, some in savan-nahs. Nesting sites are found inside cavities within the trunk andbranches of trees. M. quinquefasciata is exceptional in that it lives under-ground, where it occupies abandoned nest cavities dug by other ani-mals. We describe details of the nest and discuss the impact of thesecluded nesting site for the physical conditions inside the nest.

Keywords: Melipona quinquefasciata, nest, regulation of physical condi-tions

The stingless bee genus Melipona is neotropical and comprises about 40 species(Michener 2000), 36 of which are found in Brazil (Silveira et al. 2002). Thesespecies typically nest in cavities in the trunk or branches of living trees. Withinthe nest, a brood area and a storage area can be distinguished. Brood cells,arranged in horizontal combs, are within an involucrum which, in most species,is multilayered. Outside this involucrum are aggregated food pots of extraordi-nary size. Some contain honey, while others harbour pollen. From within thecavity a long and narrow tube leads to the single one-bee-wide nest entrance onthe outside of the tree.

We assume that within a cavity of a tall living tree the temperature is fairlyconstant, due to the water flow through the xylem, from the roots towards theleaves. The cavity, therefore, is a kind of incubator, where apart from a stabilisedtemperature the relative humidity will be constant and high. When such a cav-ity is selected as a nesting site, the bees cover its inner side-wall with a resin-likelayer and separate the nest space from additional space in the cavity by heavybatumen plates. Such constructions suggest that the physical conditions insidethe nest are well isolated from thermal and humidity fluctuations in the outsideworld. This interpretation finds additional support in the long and narrow inter-nal entrance tube, which precludes air streams, and only allows a slow gasexchange to occur by way of diffusion.

The nest of the Brazilian stingless beeMelipona quinquefasciata

Velthuis-2008.qxd 1/29/2008 10:05 AM

24

Within the genus, Melipona quinquefasciata Lepeletier 1836 is exceptional inthat it nests in the soil, utilising nest cavities made and abandoned later by ants.The species is found on the plateaus of the Brazilian interior characterised by asavannah type of vegetation. The lack of trees of sufficient diameter in itsbiotope may have contributed to the peculiar evolutionary shift towards a sub-terranean nesting site. The considerable temperature differences between dayand night, and between the hot dry season and the cooler wet season, could alsohave played a part.

The species has been reported from the Southern and Central states of Brazil,ranging from Rio Grande do Sul to Mato Grosso, including Paraná, São Paulo,Minas Gerais and Goiás. Due to the expansion of agriculture, however, at manyplaces the species has disappeared or has become endangered. Recently, its occur-rence was reported in the NE Brazilian states of Pernambuco and Ceará (Lima-Verde & Freitas 2002). Interestingly, the inhabitants of this region probably doknow this bee already for centuries, long before its presence was established sci-entifically. They excavate nests to harvest approximately 1 litre of honey pernest, the pollen and the wax, a practise that kills the colonies discovered. Giventhe expansion of the human population and the reforms in agricultural activities,these excavations imply that this bee species is threatened in this area as well.Attempts are made to develop a more sustainable exploitation by means of amethod for keeping and cultivating it in hive boxes. We report here our obser-vations on the nests we encountered and excavated during a recent field trip.Characteristics of the nest site, nest construction, nest temperature and the tem-perature gradient in the soil are described.

MATERIALS AND METHODSThe field trip was made to the Chapada do Araripe. This plateau constitutes thephysical boundary between NW-Pernambuco and S-Ceará and reaches an alti-tude of 900 m (Lima-Verde & Freitas 2002). We visited the area during the firstweek of December 2006. In this period of the year the transition from the dry tothe wet season takes place. After the first rains excessive blooming occurs, andsoon thereafter the nests contain the largest quantity of honey. This is the peri-od of the year local people excavate the nests they had located earlier. Weplanned our trip just prior to such excavations.

Three nests were excavated in the vicinity of Moreilandia (PE), a locality notfar from Santana de Cariri (CE). Rural residents, which had been met andinstructed already during a previous visit, showed us the localities of the nestsand performed the digging. For each nest dug out, the temperature of the soilwas measured at several depths, using a digital thermometer, until the nest cav-ity was disclosed. From a number of sealed honey pots the moisture content ofthe honey was determined with a refractometer.

SOCIAL INSECTS

Velthuis-2008.qxd 1/29/2008 10:05 AM

RESULTS

The architecture of the nestOn the horizontal soil surface the presence of a nest was indicated by a small tur-ret made of soil particles, about 2 cm high. In the circular central opening, 8 mmin diameter, a guard bee could be present (Fig. 1a). About 5 cm below theentrance the tunnel widens to a diameter of 1.9 cm, thus allowing the departingand returning bees to pass each other. This part is not necessarily circular; wenoted ellipsoid cross sections. Over its entire length the wall of the entrance tun-nel was coated with a cerumen rich in resin, which may prevent sand grains toloosen and drop. The tunnels had small curvatures along its course and in oneinstance even ran horizontally over small distances. Upon reaching the cavity inthe soil where the nest is located it continued as a more fortified tunnel, extend-ing into the cavity. It ended at the involucrum of the nest, at 1/2-2/3 from thetop of the round-shaped nest.

H.H.W. VELTHUIS, M. CORTOPASSI LAURINO & F. CHAGAS

25

a b

c d

Figure 1. The nest of Melipona quinquefasciata (Photos by M. Cortopassi-Laurino). a) theturret and the guard bee; b) the narrow perforations in the sealed turret of the entrancetube of a colony that died; c) the brood combs of a colony, without the surroundinginvolucrum; and d) one of the two groups of food pots taken from the outside of theinvolucrum.

Velthuis-2008.qxd 1/29/2008 10:06 AM

The nest cavities of the three nests we excavated were located at 30 cm (nest2), 70 cm (nest 1) and 100 cm (nest 3) deep, respectively. They were of differentshapes. One nest cavity was spheroid, the two other cavities ovoid with theirlongest axis horizontal. In one instance at least one empty cavity of undeter-mined size, neighboring the nest cavity, was present. The nests proper did notfully occupy the cavities, there was an air space of up to 10 cm above and at thesides of the nest. The nests, composed of the involucrum and two groupings offood pots at the sides, had a diameter of about 30 cm. The involucrum is madeof several layers, and is rather brittle at the exterior but the innermost layer issoft and flexible. At the top of the nest the involucrum is much thinner com-pared to at its side and bottom parts.

The nest contentsThe nest encountered at 70 cm depth did not contain bees any more, and only asingle, empty brood cell was present. The turret was closed by a roof with anumber of rather small openings, apparently constructed by the bees (Fig. 1b).Within the nest two ants were seen, suggesting that the colony had died becauseof ants predating on the adults and brood. Interestingly, outside the involucrumsealed food pots were present that contained honey as well as pollen.

The other two colonies contained 7 and 8 brood combs, respectively (Fig. 1c),and young bees emerged from three combs in each colony. The combs wereirregular in shape, with a longest axis of 11-13 cm. At two opposite sides theinvolucrum had groups of food pots; in one colony at least 50 pots were present(Fig. 1d). From 30 of them, with honey, the volume and the water content of thehoney was measured. The volumes ranged 3-7 ml and the water content variedfrom 26.2-31.6%. The other 20 pots contained pollen.

Temperature aspectsThe temperature of the air, measured at 1 m above the ground during the exca-vations, was 26.3-30.0°C at 9.30 h, depending on the wind (nest 1); 28.8°C at 11 h(nest 2) and 34.5°C at 12.40 h (nest 3). In the morning hours the relative humid-ity of the air was around 40%; it dropped to 23% at 15.00 h. We were told nighttemperatures could be as low as 13°C.

In Figure 2 the temperature gradients in the soil are given. At the location ofthe empty nest 1 the soil temperatures were 2.5-3.5 degrees centigrade lower thanat the other two nests. At the other two localities the soil temperature at 10 cmfrom the surface was above 35°C, but at 30 cm deep it was found to be 30°C, andat 1 m depth it was only a further 0.7°C lower. The temperature inside the nest,in between the brood combs, was found to be 32.7°C (nest 2) and 31.6°C (nest 3),respectively; i.e. 2.1 and 2.2°C above the temperature of the surrounding soil. Thepots of nest 2 had a temperature of 31.1°C, in between the values for the broodand the soil surrounding the nest.

SOCIAL INSECTS

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Velthuis-2008.qxd 1/29/2008 10:06 AM

DISCUSSIONLike most other species of its genus, Melipona quinquefasciata builds its nest in awell-insulated place. Unlike the honeybees, which either nest in the open (theApis dorsata and A. florea groups of species), or occupy well-accessible cavitiesthat have a wide entrance (e.g. A. mellifera and A. cerana), this bee can only reachits nest through a long and narrow tunnel. A single tunnel of these dimensionsexcludes a regulatory system based on air currents, because for air currents tooccur separate in- and outgoing flows are needed. There is, however, just onesuch tunnel leading to the nest. It means that the air humidity and the carbondioxide and oxygen concentrations within the nest can change only by diffusion.As a consequence of the metabolic activities of the bees the concentrations ofthese gases change, and along with it the temperature is raised. Any differencewith the surrounding soil will be equalised during a slow process of dissipation.

We found the nests at 30-100 cm below the surface. These are depths nearerto the surface than is reported in the literature (1.5-3 m, Lima-Verde & Freitas2002, Nogueira-Neto 1997; and up to 5 m, B.M. Freitas, pers. comm.). Our exca-vators reported that most nests are at about 1.5 m depth, in rare cases at 4 m.Before starting the excavation, they blow air into the entrance and then listenwhether they can hear the bees’ buzzing. If not, the nest is deep, and perhaps notworth the efforts of digging.

In as far as the regulation of temperature within the nest is concerned, weinfer that the Melipona bees could easily increase the temperature of the broodnest by contractions of their flight muscles, in the same way as honeybees do.Reducing the temperature, however, is quite a problem. Honeybees combinewing movements with the exposure of diluted nectar or water inside the nest to


27

Soil depth

0 20 40 60 80 100 cm

Tem

pera

ture

36

34

32

30

28

26

°C

nest 1

nest 2

nest 3

BB

Figure 2. Temperature characteristics of the soil and the brood nests in relation to soildepth, of the three nests excavated. B = the upper part of the brood area, measuredwhen still surrounded by the involucrum.

Velthuis-2008.qxd 1/29/2008 10:06 AM

reduce the nest temperature, as the wide nest opening permits an efficient gasexchange with the exterior of the nest. In the Melipona nest, however, wing flut-tering will only cause the air to circulate within the nest; any evaporated waterwill necessarily condense nearby, and therefore, such activity will only raise thetemperature inside the nest instead of reduce it. Active reduction of temperaturewithin the nest appears impossible, so the only remedy to increasing tempera-ture seems to be reducing the activity level. We may suppose that this is a lim-iting factor for the size a colony can reach ultimately.

In this context it is of interest that the temperature within the nest of M.quinquefasciata was found to be only two degrees centigrade above the tempera-ture of the sand surrounding the nest. Dissipation of heat towards the lateralsides is obstructed by the presence of several layers of food pots; perhaps suchlateral dissipation is promoted by the concentration of these pots at only abouthalf of the perimeter of the nest, leaving parts of the side wall available forexchange. However, the best dissipation may occur at the top of the nest, wherethe involucrum is rather thin, above which there is an air space. The remainingair space in the nest cavity might be of importance. The greater this space, thelarger the inner wall of the cavity that conducts excess heat.

Stability in the physical aspects of the nest is favourable for the developmentof the brood. The optimal temperature for Melipona is unknown, but probablydoes not differ much from that of Apis mellifera and A. cerana, where the brood-nest is maintained at approximately 35°C (Kraus et al. 1998). At slightly lowertemperatures the development time for the brood increases, while at slightlyhigher temperatures there is no reduction in the time needed to complete devel-opment. Just a few centigrades above the optimum the temperature becomesharmful and lethal (Velthuis & Kraus 2002). Because the relative humiditychanges with temperature, a stabile temperature makes it easier to maintain apreferred level of humidity in the nest.

Not much is known about the temperatures of the brood nests of Meliponaspecies. In M. scutellaris nests, the combs had an average temperature of 30.7°C,varying from 26.6 to 34.6°C (n=58, Cortopassi, unpubl.). Occasional measure-ments in nests of the Amazonian species M. seminigra and M. rufiventris provid-ed values of 31.7°C and 31.3°C, respectively, while in a nest of M. quadrifasciata insoutheastern Brazil 29.3°C was measured (Cortopassi & Nogueira-Neto,unpubl.). These temperatures are similar or just below our values.

However, not all species of Melipona nest in a well-protected cavity with ahigh degree of temperature stability. For instance, M. subnitida and M. asilvaioften have their nest in the relatively thin branches or stems of shrubs. Asspecies of savannah-type vegetations with occasionally high day-time tempera-tures and contrastingly low temperatures at night, considerable fluctuations innest temperature should be expected. This was indeed found in some nests of M.subnitida and, interestingly, relative humidity turned out to be more constant

SOCIAL INSECTS

28

Velthuis-2008.qxd 1/29/2008 10:06 AM

(Cortopassi-Laurino, unpublished). There is, however, no detailed study on suchfluctuations in temperature and humidity within the natural nest, and neitherare there data on the impact of temperature differences on the development ofimmature stingless bees.

Acknowledgements We enjoyed the company and the stimulating help of Mrs.Selma Carvalho, dr. Renato Barbosa and dr. Tertuliano Aires Neto. Berend-Jan Velthuiscollaborated in the preparation of this manuscript.

REFERENCESKraus, B., Velthuis, H.H.W. & Tingek, S. 1998. Temperature profiles of the brood nests

of Apis cerana and Apis mellifera colonies and their relation to varoosis. J. ApiculturalResearch 37:175-181.

Lima-Verde, L.W. & Freitas, B.M. 2002. Occurrence and biogeographic aspects ofMelipona quinquefasciata in NE Brazil. Braz. J. Zool. 62:479-486.

Michener, C.D. 2000. The Bees of the World. Baltimore and London, Johns HopkinsUniversity Press.

Nogueira-Neto, P. 1997. Vida e Criação de Abelhas Indígenas sem Ferrão. São Paulo,Nogueirapis.

Silveira, F.A., Melo, G.A.R. & Almeida, E.A.B. 2002. Abelhas Brasileiras, Sistemática eIdentificação. Belo Horizonte, F.A.Silveira.

Velthuis, H.H.W. & Kraus B. 2002. The impact of temperature gradients in the broodnest of honey bees on the reproduction of Varroa jacobsoni Oud.: laboratory observa-tions. pp 224-234 in Erickson, E.J., Page, R.E., Hanna, A. A. (eds.), Proceedings ofthe 2nd international conference on Africanized honey bees and bee mites. MedinaOhio, A.I.Root Company.


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Velthuis-2008.qxd 1/29/2008 10:06 AM


Denis MichezLaboratory of Zoology, University of Mons-Hainaut, Avenue Maistriau 19, Mons,Belgium, E-mail: [email protected]

The popularity of bees is mainly due to a single species, the domestichoneybee although there are in fact thousands of different wild bees.Bees constitute a monophyletic group including 16,000 species in sevenfamilies. In this presentation, we propose an overview of one key groupin the bee phylogeny: the Melittidae s.l. We show information abouttheir diversity, biogeography and biology. Moreover, we investigatethe origin of Melittidae s.l. and the characteristics of their early diver-sification.

Keywords: diversification, monophyletic group, honeybee, biogeogra-phy, phylogeny

Bees are among the most common and familiar animals. This popularity ismainly due to a single species, the domestic honeybee (Apis mellifera) althoughthere are in fact thousands of other species of wild bees in the world. All beespecies constitute together a monophyletic group including more than 16,000described species and seven families currently acknowledged (Michener 2007).The extensive studies carried out on the honeybee contrast markedly with theglobal level of knowledge of most wild bees, which have received comparative-ly little attention so far. The ancestral states, the early diversification and thephylogeny of bees need particularly new advancements to propose a stronghypothesis on their evolution.

The phylogenetic relationships among bee families have been recently deeplyreconsidered. Traditional hypothesis presented the colletid bees as basal in theclade of bees (Michener 1944, Engel 2001). This hypothesis was mainly based ona few morphological similarities with the ancestral sphecid wasps. New robustphylogenies including morphological and molecular dataset have provided strongsupport to define the paraphyletic group of Melittidae s.l. as the real sister groupof all other contemporary bees (Danforth et al. 2006). This group includes threefamilies: Dasypodaidae, Melittidae s.str. and Meganomiidae (Fig. 1). This ‘melit-tid basal topology’ hypothesis calls for further research on the systematics, the

Monographic revision of the melittid bees(Hymenoptera, Apoidea, Melittidae sensu lato)

Michez-2008.qxd 1/29/2008 10:06 AM

32

biogeography, the biology and the host-plant associations of Melittidae s.l. tounderstand the ancestral states and the early diversification of all bees.

Systematic studies of Melittidae s.l. are limited to a few general revisions.Moreover, the information about all 16 melittid genera is generally scattered. Wefilled these gaps by undertaking a thorough systematic revision of the followingmelittid bee genera: Capicola, Dasypoda, Eremaphanta, Macropis, Melitta andPromelitta (Table 1; Michez 2007). In the same time, we compiled informationabout the general biology and the host-plants of Melittidae s.l. (Michez et al.2008). Using phylogenies and host-plants records of several genera, we exam-ined the inheritance of the host-plant choices throughout the evolution of melit-tid. Finally, we investigated the origin of Melittidae s.l. and the characteristicsof their early diversification. We carried out notably to a detailed examinationof the fossil specimens available and we included a new fossil record that we des-cribed and confronted to the current state of knowledge of bee systematics.

We present hereafter a review of the available information about melittidbees throughout our own works and a synthesis of the literature on this topic.

SYSTEMATICS AND BIOGEOGRAPHY OF THE MELITTIDAE S.L.

Melittidae s.l. includes 202 species: 198 contemporary species and 4 fossil species(Table 1). Dasypodaidae is the most diverse (101 species) while Meganomiidaecomprises only 12 species. Melittid bees occur in temperate and xeric ecosystems

SOCIAL INSECTS

Figure 1. Phylogeny of Melittidae s.l. from Michener (1981) adapted with taxonomicalhypothesis of Engel (2005) and Danforth et al. (2006). 1 = Dasypodaini Michener 1981, 2= Sambini Michener 1981, 3 = Promelittini Michener 1981, 4 = Afrodasypodaini Engel2005, 5 = Redivivini Engel 2001, 6 = Macropidini Robertson 1904, 7 = Melittini Schenk1860.

Michez-2008.qxd 1/29/2008 10:07 AM

D. MICHEZ

33

Tab

le 1.

Tax

onom

y, s

peci

es r

ichn

ess

and

dist

ribu

tion

of

the

Mel

itti

dae

s.l.

acco

rdin

g to

Mic

hene

r (2

007)

[*

foss

il ta

xa; (

*) fo

ssil

and

cont

empo

rary

tax

a; N

1= s

ubge

nera

div

ersi

ty; N

2= s

peci

es d

iver

sity

; Sou

th.=

Sou

ther

n; M

ad.=

Mad

agas

car,

Ken

.= K

enya

; M.=

Mal

i;O

W=

Old

Wor

ld; N

ear.

= N

earc

tic]

.

Fami

lies

Tribe

sGe

nera

Dive

rsity

Distr

ibutio

nMa

x. of

diver

sity

Main

refer

ence

s(N

1-N2

)Da

sypo

daida

eDa

sypo

daini

Dasy

poda

4 –

33Pa

laear

ctic

Med..

bas

inW

arnc

ke (1

973)

, Mich

ez e

t al.(

2004

a, b,)

Dasy

poda

iniEr

emap

hant

a2

– 9

Centr

al As

iaTu

rkesta

nPo

pov (

1957

), Mi

chez

& P

atiny

(200

6)Da

sypo

daini

Capic

ola1

– 13

South

. Afric

aCa

pe p

rovin

ceMi

chen

er (2

007)

, Mich

ez e

t al.(

2007

a)Da

sypo

daini

Hesp

erap

is7

– 38

Near

ctic

Califo

rnia

Stag

e (1

966)

, Mich

ener

(198

1, 20

07)

Samb

iniHa

plom

elitta

5 –

5So

uth A

frica

South

Afric

aMi

chen

er (1

981,

2007

)Sa

mbini

Sam

ba1

– 1

Keny

aKe

nya

Mich

ener

(198

1, 20

07)

Prom

elittin

iPr

omeli

tta1

– 1

North

Afric

aNo

rth A

frica

Mich

ez e

t al.(

2007

b)Pr

omeli

ttini

Afro

dasy

poda

1 –

1So

uth A

frica

South

Afric

aEn

gel (

2005

)Me

gano

miida

eMe

gano

miini

Cera

tom

onia

1 –

1Na

mibia

Nami

biaMi

chen

er (1

981)

Mega

nomi

iniM

egan

omia

1 –

5Et

hiopia

nSo

uth. A

frica

Mich

ener

(198

1)Me

gano

miini

Pseu

doph

il.2

– 4

Mad.

and

Ken.

Keny

aMi

chen

er (1

981)

, Mich

ener

et a

l.(19

90)

Mega

nomi

iniUr

omon

ia2

– 2

Mad.,

Ken

., M.

Mad.,

Ken

yaMi

chen

er &

Bro

oks (

1987

)Me

littida

eMe

littini

Meli

tta( *)

2 –

44OW

and

Nea

r.Eu

rope

Mich

ez &

Ear

dley (

2007

)Me

littini

Rediv

iva1

– 24

South

. Afric

aSo

uth A

frica

Whit

ehea

d &

Stein

er (2

001)

, Whit

ehea

d et

al.(

in pr

ess)

Melitt

iniRe

divivo

ides

1 –

3So

uth A

frica

South

Afric

aMi

chen

er (1

981,

2007

)Eo

macro

pidini

Eom

acro

pis*

1 –

1Ba

ltic a

mber

Baltic

amb

erEn

gel (

2001

)Ma

cropid

iniM

acro

pis( *)

3 –

16Ho

larcti

cEa

stern

Asia

Mich

ener

(198

1), M

ichez

& P

atiny

(200

5)Ma

cropid

iniPa

leom

acro

pis*

1 –

1Oi

se a

mber

Oise

amb

erMi

chez

et a

l.(20

07c)

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of the Nearctic and the Old World. Ethiopian region is the only region wherethe distributions of all families overlap. Ethiopian region shows the maximumof generic diversity but the maximum of species diversity is reached in thePalaearctic region. The African continent (Ethiopian region + North Africa)lumps clearly the maximum of both generic and specific diversity.

Dasypodaidae can be distinguished by an original combination of several fea-tures: short tongue with all segments of the labial palpus similar to one another,paraglossa reduced, submentum V-shaped and two submarginal cells with thefirst submarginal crossvein at right angles to longitudinal vein (Michener 1981).They include four tribes and eight genera (Table 1). The phylogenetic relationsamong genera and tribes are still dubious.

Dasypodaidae occur in both the Old World and the Neartic region. This fam-ily is absent in South America, Australia and tropical areas. The specific diver-sity is maximal in xeric areas: the southwestern deserts of North America(Hesperapis), the Mediterranean basin (Dasypoda and Promelitta), the Kyzyl kumin Central Asia (Eremaphanta) and the Southern Africa (Afrodasypoda, Capicolaand Haplomelitta). Dasypoda is the only widespread genus that occurs in the tem-perate to the xeric areas of the Palaearctic (Fig. 2). Dasypoda determines thenorthern limit of Dasypodaidae to the 62nd northern parallel. The otherDasypodaidae genera, Afrodasypoda, Capicola, Eremaphanta, Hesperapis andPromelitta are each one endemic in different Old World deserts.

Meganomiidae is the smallest family of Melittidae s.l. (Table 1). In ligth ofrecent molecular analyses, Meganomiidae is probably the sister group of theMelittidae s.str. (Danforth et al. 2006). Meganomiide species are robust bees with

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Figure 2. Global distribution of Dasypodaini including the genera Capicola, Dasypoda,Eremaphanta and Hesperapis.

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three sub-marginal cells, extending yellow marking on the whole body andmany unique modifications of legs and hidden sterna of male (Michener 1981).Meganomiidae is restricted to the Sub-Saharan Africa except one undescribedMeganomia species recorded in Yemen. Michener (1981), Michener & Brooks(1987) and Michener et al. (1990) reviewed the four included genera: CeratomoniaMichener 1981, Meganomia Cockerell 1931, Pseudophilanthus Alfken 1939 andUromonia Michener 1981.

Like Meganomiidae, most Melittidae s.str. have three submarginal cells(except Macropis), which set apart from the Dasypodaidae. Melittidae s.str. isalways smaller than Meganomiidae. The largest Melittidae s.str. is 15 mm longwhile the smallest Meganomiidae is 17 mm. The body of Melittidae s.str. ismainly black but some males of Macropis display yellow markings on the head.Designations of tribe are still unfixed in the Melittidae s.str. Michener (1981) didnot distinguish any tribe and included all genera in the Melittini. Engel (2005)considered two different subfamilies: Macropidinae and Melittinae. TheMacropidinae have been split into two tribes: Eomacropidini (including the fos-sil Eomacropis Engel 2001) and Macropidini (including the contemporaryMacropis Panzer 1809 and the fossil Paleomacropis Michez & Nel 2007). Twotribes have been recognized in the Melittinae, on the one hand the Rediviviniwith genera Rediviva and Redivivoides, on the other hand the Melittini with thegenus Melitta. We follow the tribe designation of Engel (2005). Melittidae s.str.is diverse (86 species) in the Old World and the Neartic region (Table 1). Unlikethe other melittid bees, the Melittidae s.str. show notable climatic preferences.As written above, most Dasypodaidae (Afrodasypoda, Capicola, Dasypoda,Eremaphanta and Promelitta) and all Meganomiidae are restricted to the xericareas of the Old World. By contrast the ecological optimum for Melittidae s.str.seems to live in cooler temperate climate. At least Melitta and Macropis prefer thecool temperate ecosystems. Both genera, Rediviva and Redivivoides, are restrictedto the coastal area of South Africa.

BIOLOGY OF THE MELITTIDAE S.L.

As far as known, all Melittidae s.l. are solitary and univoltine. All females canproduce offspring and each species completes one cycle of development duringone year. The general cycle of development is therefore relatively unchanged(Fig. 3). Males emerge from the ground some days before females. After femaleemergences, males mate with virgin females generally on host-plants aroundemergence site (i.e. rendez-vous flowers, Alcock et al. 1978). After mating, gravidfemales begin to dig a nest. At the bottom of lateral tunnels, females build oneor a few chambers where they bring pollen (Fig. 3D). When the pollen ball isformed, they lay one egg on the top. The larva eats the pollen during about tendays and grows fastly (Fig. 3E). After consuming all the pollen and after defeca-tion, larva overwinters and becomes pupa the following year (Fig. 3F).

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The mechanisms of the emergence are unexplored in Melittidae s.l..However, like most other specialist bees, the melittid bees probably need a min-imal overlap between their flight period and the host-plant(s) blooming (Thorp1979, 2000, Danforth 1999, Minckley et al. 2000). Flight collecting period must belong enough to produce the brood cells. In xeric areas like the southwesternAmerican desert, synchronisation between Hesperapis emergence and theirrespective host-plant blooming is probably possible thanks to the abilities ofHesperapis to feel the variation of the soil humidity after raining (Hurd 1957). Inmesic areas, Michez et al. (in press) showed that the emergence of Melitta nigri-cans females (Melittidae s.str.) overlaps the blooming peak of its host-plant,Lythrum salicaria L. However, they did not study the factors eliciting the emer-gence of M. nigricans.

Mating behaviour is only described for Dasypoda hirtipes (Dasypodaidae)(review of mating behaviour of bees see Ayasse et al. 2001). Males and femalesof D. hirtipes mate on their exclusive host-plants, yellow Asteraceae, (Bergmarket al. 1984). Bergmark et al. (1984) highlighted that mate recognition of male inD. hirtipes is driven by multiple factors as presence of scopae, scent of female andscent of host-plant. The other Melittidae s.l. could have the same kind of matingbehaviour on ‘rendez-vous flowers’.

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Figure 3. General cycle of development of Melittidae s.l.. A. Emergence of Dasypoda hir-tipes female (picture N.J. Vereecken). B. Copulation of a pair of bees (drawing M.Terzo). C. Female of D. hirtipes foraging on Hypochoeris radicata L. (picture N.J.Vereecken). D. Nest of Dasypoda braccata (from Radchenko 1987). E. Larva of D. hir-tipes (picture M. Gosselin). F. Pupa of Hesperapis trochanterata (from Rozen 1987).

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EVOLUTION OF THE MELITTIDAE S.L.

Inheritance of host-plants in the Melittidae s.l.

In most cases, closely related species visit similar host plants. These results con-firm previous studies on the evolution of flower relationships in non-melittidbees [Müller 1996 for Anthidiini (Megachilidae); Sipes & Tepedino 2005 for thegenus Diadasia (Apidae)]. However, floral choices have interestingly not alwaysbeen inherited among species in the course of the evolution of melittid bees. Weobserve independent shifts to different host plants (related or not) in the generaCapicola (Fig. 4), Dasypoda, Hesperapis, Macropis and Melitta (Michez et al. 2008).

Most Melittidae s.l. have a relatively narrow host range. Among the 108species with host-plant records, we record only 16 mesolectic or polylecticspecies making oligolecty a dominant condition within most groups. Our dataprovide strong evidence for the rarity of host breadth variations. Most cases ofhost-plant shifts involve shifts of host-plant use (shift from one specialisation toanother one).

Implication for the understanding of the early diversificationof beesMelittids constitute a group of specialist taxa, which are basal in the bee phy-logeny (see previous chapter). Likewise, we observe that a lot of other basal

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Figure 4. Phylogeny and host-plants of Capicola (from Michez & Kuhlmann 2007;Michez & Timmermann unpublished data). 1 = shift from Campanulaceae toAizoaceae; 2 = shift from Aizoaceae to Fabaceae; 3 = shift from Aizoaceae toCampanulaceae; 4 = shift from Aizoaceae to Asteraceae.

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groups are also oligolectic (i.e. Lithurginae, Panurginae and Rophitinae)(Danforth et al. 2006). The fact that the most primitive taxa within several beefamilies are oligolectic could be a hint that, in general, polylecty is the derivedforaging strategy that has evolved in bees. This hypothesis is supported by therecent discovery of the bee fossil, Paleomacropis eocenicus from the early Eocene(~53 myBP) (Michez et al. 2007c). This Melittidae s.str. presents oil-collectingstructures on its legs similar to those observed in contemporary oil-collectingbees. In light of these records, and since most contemporary oil-collecting beesare oligolectic, it can be reasonably assumed that this fossil bee was a specialisttaxon, which increases the likelihood for oligolecty to constitute an ancestralcondition in bees.

REFERENCESAlcock J, Barrows EM, Gordh G, Hubbard LJ, Kirkendall C, Pyle DW, Ponder TL &

Zalom FG, 1978. The ecology and evolution of male reproductive behaviour in beesand wasps. Zoological Journal of the Linnean Society 64: 293-326.

Ayasse M, Paxton RJ & Tengö J, 2001. Mating behavior and chemical communication inthe order Hymenoptera. Annual Review of Entomology 46: 31-78.

Bergmark L, Borg-Karlson A-K & Tengö J, 1984. Female characteristics and odour cuesin mate recognition in Dasypoda altercator (Hym., Melittidae). Nova Acta RegiaeSocietatis Scientiarum Upsaliensis 5: 137-143.

Danforth BN, Sipes SD, Fang J & Brady SG, 2006. The history of early bee diversifica-tion based on five genes plus morphology. Proceedings of the National Academy ofSciences of the United States of America 103: 15118-15123.

Engel MS 2001. A monograph of the Baltic Amber bees and evolution of the Apoidea(Hymenoptera). Bulletin of the American Museum of Natural History 259: 1-192.

Engel MS, 2005. Family-group names for bees (Hymenoptera: Apoidea). AmericanMuseum Novitates 3476: 1-33.

Michener CD, 1944. Comparative external morphology, phylogeny, and classification ofthe bees (Hymenoptera). Bulletin of the American Museum of Natural History 82: 1-326.

Michener CD, 1981. Classification of the bee family Melittidae with a review of speciesof Meganomiinae. Contribution of the American Entomological Institute 18: 1-135.

Michener CD, 2007. The bees of the world, second edition. Baltimore, 913 p.Michener CD & Brooks RW, 1987. The family Melittidae in Madagascar. Annales de la

Société entomologique de France 23: 99-103.Michener CD, Brooks RW & Pauly A, 1990. Little-known meganomiine bees with a key

to the genera (Hymenoptera: Melittidae). Journal of African Zoology 104: 135-140.Michez D, 2007. Monographic revision of the Melittidae s.l. (Hymenoptera: Apoidea:

Dasypodaidae, Meganomiidae, Melittidae). Ph-D, Université de Mons-Hainaut, Mons,50 p.

Michez D & Eardley CD, 2007. Monographic revision of the bee genus Melitta Kirby1802 (Hymenoptera: Apoidea: Melittidae). Annales de la Société entomologique deFrance (n. s.) 43: 379-440.

Michez D & Patiny S, 2005. World revision of the oil-collecting bee genus MacropisPanzer 1809 (Hymenoptera, Apoidea, Melittidae) with a description of a new

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species from Laos. Annales de la Société entomologique de France (n. s.) 41: 15-28.Michez D & Patiny S, 2006. Review of the bee genus Eremaphanta Popov 1940

(Hymenoptera: Melittidae), with the description of a new species. Zootaxa 1148: 47-68.

Michez D, Terzo M & Rasmont P, 2004a. Révision des espèces ouest-paléarctiques dugenre Dasypoda Latreille 1802 (Hymenoptera, Apoidea, Melittidae). Linzer biologischeBeiträge 36: 847-900.

Michez D, Terzo M & Rasmont P, 2004b. Phylogénie, biogéographie et choix floraux desabeilles oligolectiques du genre Dasypoda Latreille 1802 (Hymenoptera, Apoidea,Melittidae). Annales de la Société entomologique de France (n. s.) 40: 421-435.

Michez D, Eardley CD, Kuhlmann M & Patiny S, 2007a. Revision of the bee genusCapicola (Hymenoptera: Apoidea: Melittidae) distributed in the Southwest ofAfrica. European Journal of Entomology 104: 311-340.

Michez D, Else GR & Roberts SPM, 2007b. Biogeography, floral choices and redescrip-tion of Promelitta alboclypeata (Friese 1900) (Hymenoptera, Apoidea, Melittidae).African Entomology 15: 197-203.

Michez D, Nel A, Menier JJ & Rasmont P, 2007c. The oldest fossil of a melittid bee(Hymenoptera: Apiformes) from the early Eocene of Oise (France). ZoologicalJournal of the Linnean Society 150: 701-709.

Michez D, Patiny S, Rasmont P, Timmermann K & Vereecken N, 2008. Phylogeny andhost-plant of Melittidae s.l. (Hymenoptera, Apoidea). Apidologie 39: in press.

Müller A, 1996. Host-plant specialization in Western Palearctic anthidiine bees(Hymenoptera: Apoidea: Megachilidae). Ecological Monographs 66: 235-257.

Popov VV, 1957. New species and the geographical distribution of the genusEremaphanta. Zoologicheskii Zhurnal 36: 1706-1716.

Radchenko VG, 1987. Nesting of Dasypoda braccata Eversmann (Hymenoptera,Melittidae) in the southwestern Ukraine. Entomological review 67: 57-60.

Rozen JG, 1987. Nesting biology and immature stages of a new species in the bee genusHesperapis (Hymenoptera: Apoidea: Melittidae: Dasypodinae). American MuseumNovitates 2887: 1-13.

Sipes SD & Tepedino V, 2005. Pollen-host specificity and evolutionary patterns of hostswitching in a clade of specialist bees (Apoidea: Diadasia). Biological Journal of theLinnean Society 86: 487-505.

Stage GI, 1966. Biology and systematics of the American species of the genus HesperapisCockerell. Ph-D, University of California, Berkley, 464 p.

Warncke K, 1973. Die westpaläarktische Arten der Bienen Familie Melittidae(Hymenoptera). Polskie Pismo Entomologiczne 43: 97-126.

Whitehead VB & Steiner KE, 2001. Oil-collecting bees of the winter rainfall area ofSouth Africa (Melittidae, Rediviva). Annals of the South African Museum 108: 143-277.

Whitehead VB, Steiner KE & Eardley CD. Oil collecting bees mostly of the summerrainfall area of southern Africa (Melittidae, Rediviva). Journal of the KansasEntomological Society: in press.

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Willem J. Boot, Johan N.M. Calis & Mike H. Allsopp*Laboratory of Entomology, Wageningen University, PO Box 8031, 6700 EHWageningen, The Netherlands, E-mail: [email protected]; *ARC-Plant ProtectionResearch Institute, Private Bag X5017, Stellenbosch, 7599, South Africa

Colonies of Apis mellifera scutellata in the northern half of South Africasuffer from social parasitism by an A. m. capensis pseudo-clone. Insidescutellata colonies, these capensis workers increase their numbers byreproducing thelytokously. Infected colonies dwindle because normalworker tasks are neglected and eventually they die. We show that lar-vae of the pseudo-clone get more food in scutellata colonies than scutel-lata larvae do. Normal capensis larvae also get more food in scutellatacolonies but not as much as larvae of the pseudo-clone. Earlier wefound that more food results in more queen-like bees. Moreover, pseu-do-clones are on average more queen-like than normal capensis beeswhen both are raised in a scutellata colony. This confirms the linkbetween food and queen-like bees, because pseudo-clone larvae getmost food of all. We present a new theory, postulating that capensisworkers are selected to contribute to the next generation of queens bylaying in queen cups prior to swarming. Such reproductive bees proba-bly manage to get more food as larvae. At the same time nurse bees areselected to adjust the amount of food given, to maintain clear caste dif-ferences between workers and queens for optimizing colony perform-ance. Therefore, selection for reproductive workers inside the capensispopulation may have changed the communication between nurse beesand larvae on allocation of food. This changed communication resultsin capensis pseudo-clones being overfed in scutellata colonies.

Keywords: social parasitism, Apis mellifera capensis, Apis mellifera scutel-lata, selection, worker reproduction

Caste in honeybees is determined by the differential feeding of female larvae.The majority of the larvae develop into workers, whereas larvae destined tobecome queens receive more food which also has a different composition(Beetsma 1979). Hence, differential feeding leads to very distinct worker andqueen castes with unique characters. Queens typically show a short develop-

Selection for reproductive workers in the Capehoneybee population, Apis mellifera capensis,leads to social parasitism in bee colonies fromthe savanna


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ment time, a large spermatheca and a large number of ovarioles, compared toworkers where spermathecae are often absent and only a few ovarioles can befound. Pollen combs and pollen baskets are only found on the hind legs of work-ers (Snodgrass 1956, Wirtz & Beetsma 1972, Hepburn & Radloff 2002).

As worker and queen tasks differ completely, distinct worker and queencastes are expected. Fitness of both is increased by enhancing performance of thewhole colony, which in many respects is the unit natural selection is acting upon(Moritz & Southwick 1992). Usually, the queen is the sole reproductive in thecolony, whereas the workers do all other tasks like nursing the brood, buildingcombs and foraging. If workers do reproduce, they are expected to neglect theirworker tasks and hence decrease inclusive fitness of all the bees in the colony(Hillesheim et al. 1989, Moritz 1989, Dampney et al. 2004). In the Cape honeybee,Apis mellifera capensis, worker bees are relatively queen-like compared to work-ers of other A. mellifera races (Neumann & Hepburn 2002, Wossler 2002, Allsoppet al. 2003), suggesting that they are more often reproducing compared to work-ers of other subspecies. If so, direct fitness gains for the individual workers thatreproduce apparently outweigh their decreased inclusive fitness through selec-tion at colony level.

The most extreme situation where capensis workers are reproductive and havehigh fitness gains is when they interact with the bee from the savanna, A. m.scutellata, where they behave as social parasites. In 1989, colonies of capensishoney bee were moved out of their native range by beekeepers and introducedinto the Limpopo Province of South Africa (Allsopp 1993, 2004). They werehoused in apiaries with scutellata colonies, and some of the capensis workersinvaded the scutellata colonies. The capensis workers activated their ovaries andstarted laying eggs (Allsopp 1993, Martin et al. 2002a, Neumann & Hepburn2002). Unlike other honeybee subspecies where workers produce males byarrhenotokous parthenogenesis, capensis workers produce female offspringthrough thelytoky (Onions 1912, Anderson 1963). As a result, the numbers ofcapensis laying workers in the colonies increase. Eventually, this results in thedeath of the scutellata queen. Infected colonies dwindle, because normal workertasks are neglected, until the colony dies or absconds (Martin et al. 2002a,Neumann & Hepburn 2002). In the meantime the capensis bees in the dwindlingcolony may have infected new colonies, or absconded colonies may have mergedwith new scutellata colonies (Neumann & Hepburn 2002). The so-called‘Capensis problem’ has caused the loss tens of thousands of commercial scutella-ta honeybee colonies (Allsopp 1993, Martin et al. 2002a).

Capensis bees parasitizing scutellata colonies are probably more queen-likethan normal capensis workers for phenotypical reasons (Beekman et al. 2000,Calis et al. 2002, Allsopp et al. 2003). When capensis larvae are reared in coloniesof European honey bees or scutellata bees, they receive more food when com-pared to the larvae of the host colony, and also more than they would get in their

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own colony. Compared to capensis workers raised in capensis colonies, these beeshave enlarged spermathecae and higher numbers of ovarioles. They are alsoheavier than normal workers, develop faster and have reduced pollen combs(Beekman et al. 2000, Calis et al. 2002, Allsopp et al. 2003). Apparently, commu-nication between larvae and nurse bees over the amount of food provided is dif-ferent between capensis and other honeybee subspecies. This leads to queen-likebees, which can be expected to be more reproductive than normal workers.

It has been shown that the social parasites from central and northern SouthAfrica are now genetically almost identical (Kryger 2001, Baudry et al. 2004). Wefound that these parasitic capensis bees are on average even more queen-like com-pared to other capensis bees reared in scutellata colonies (Allsopp et al. 2003), butto date there has been no assessment of the level of feeding larvae of parasiticbees receive compared to other capensis workers. In this study, we investigate ifthe parasites being relatively more queen-like is related to the amount of foodreceived as a larva. In addition, we investigate how rapidly capensis and scutella-ta workers start laying upon queenlessness, and in which cell types they preferto lay their eggs. Based on the results of this study and earlier data (Allsopp etal. 2003), we present a new theory explaining why capensis workers are relative-ly queen-like compared to workers from other subspecies and why contactbetween capensis and scutellata has led to social parasitism.

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Pretoria

Kenhardt

Nieuwoudtville

Stellenbosch

Cape Point

capensis zone

hybrid zone

scutellata zone

Figure 1. Map of South Africa showing the places from which the bees were collected.Zones with A. m. capensis, with A. m. scutellata, and with their hybrids are indicated,following Hepburn and Radloff (2002).

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MATERIAL AND METHODS

Honey bee coloniesA capensis honeybee colony was obtained from a population at Cape Point in theTable Mountain National Park. Honey bees found here belong to a natural pop-ulation which is considered to be the least hybridized with scutellata bees(Allsopp & Hepburn 1997). Two scutellata honeybee colonies were obtained fromKenhardt in the Northern Cape (see Fig. 1). Colonies were kept in Stellenboschin one-storey Langstroth-hives and occupied about ten frames. The scutellatacolonies were kept in a separate apiary to prevent infestation with capensis work-ers. Combs with brood of parasitic capensis bees were collected from a scutellatahoneybee colony infested with parasitic bees in Pretoria (Fig. 1). The brood wastransported overnight from Pretoria to Stellenbosch and placed into an incuba-tor (35°C) to collect emerging bees. These emerging bees were introduced into asmall scutellata colony that had just been made queenless and was kept isolatedfrom all other colonies. We assumed that eggs laid after some days originatedfrom the parasitic capensis bees. They were harvested and used in the experi-ment.

Differential nursing measured by the amount of food inbrood cells just before cappingTwo pieces of comb (about 50 cm2) containing eggs from the ‘Cape Point’ capen-sis colony and two pieces of comb (about 50 cm2) with eggs from the parasiticcapensis bees were cut from their original combs, and inserted into combs con-taining eggs from the two scutellata colonies. The manipulated combs werereturned to the scutellata colonies. After five days food was sampled from cellscontaining larvae that were about to be capped by the bees. These cells were rec-ognized by the size of the larvae and by wax deposited on the cell rims. The larvawas gently removed from each cell to avoid larval fluid from flowing into thelarval food. Then the larval food was scooped out of the cell with a fine spatulaand put into a pre-weighed Eppendorf tube. Twenty five samples were collectedfrom each scutellata colony for both of the source brood types. The tubes wereweighed directly after sampling in order to determine the weight of the foodsampled. As a control 25 samples of scutellata larval food were taken from bothcolonies.

Egg-production and cell-type preference of workers afterbecoming queenlessIt has often been noticed that after becoming queenless capensis workers start egglaying within a few days and prefer to oviposit in queen and worker cells.Scutellata workers start laying eggs later and seem to prefer to lay there eggs indrone cells (Onions 1914, Johannsmeier 1983, Neumann et al. 2000, Martin et al.2002a, personal observations). We documented these phenomena more closely,

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to help interpretation of our results in relation to egg-laying by workers. Twosmall queenless colonies were made, one with capensis bees and another withscutellata bees. The hives contained one comb with pollen and honey, and oneempty comb with areas of both drone and worker cells. In addition, a top-barwith 20 queen cups made of wax was placed between the two combs. Once everyday, all cells were inspected for the presence of eggs.

RESULTSSignificantly larger amounts of food were collected both from cells with ‘CapePoint’ capensis larvae and from cells with parasitic capensis larvae, when com-pared to the amounts found in cells with scutellata larvae (Fig. 2; t-test, p<0.05).When ‘Cape Point’ larvae were raised by scutellata workers the amount of foodsampled per cell was almost 1.5 times higher (6.7 and 7.4 mg on average, com-pared to 4.9 and 5.1 mg on average in cells with scutellata larvae). When parasiticcapensis larvae were raised by scutellata workers the amount of food collected wasalmost twice as high as samples from scutellata brood cells (9.9 and 8.6 mg onaverage; Fig. 2). The amount of food present in cells with parasitic capensis lar-vae was also significantly higher than in the cells with ‘Cape Point’ capensis lar-vae (Fig. 2; t-test, p<0.05).


45

Colony 1 Colony 2

Larv

al fo

od (

mg)

parasitic capensis

Cape Point capensis

scutellata

0

2

4

6

8

10

12

14

16

a b c

parasitic capensis

Cape Point capensis

scutellata

0

2

4

6

8

10

12

14

16

a b c

Figure 2. Amount of larval food found in 25 worker brood cells shortly before capping.The cells contained parasitic capensis larvae, ‘Cape Point’ capensis larvae and scutellatalarvae in two scutellata colonies. The actual data distribution is shown as a box com-prising the midrange, i.e. the 2nd and 3rd quartile ranges, divided by a horizontal linerepresenting the median. Values within 1.5x of the midrange are shown as ‘whiskers’.Values at 1.5-3x the midrange, measured from the edge of the box, are shown as a cir-cle. Different letters below the box-plots indicate if average amounts of larval food aredifferent using the t-test (P<0.05).

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In the queenless colonies of capensis and scutellata bees, worker laid eggs werefound after two and four days, respectively (Table 1). Initially, capensis beesclearly preferred to lay in queen cells, although they did not use the cups offeredby us. New queen cups were made on the surface of the comb. After four days,all types of cells were used to lay eggs in and large numbers of eggs were found.In contrast to the capensis workers, scutellata workers preferred to lay eggs indrone cells. The scutellata bees also made new queen cups on the surface of thecomb, a few of which were used for egg-laying.

DISCUSSIONThis study shows that the larvae of the parasitic capensis honey bees in the northof South Africa, the agents of the ‘Capensis Problem’ (Allsopp 1993), receivemore food from the scutellata workers than the scutellata larvae receive. Becauseof this excessive feeding, they develop into worker-queen intermediates(Allsopp et al. 2003). Therefore, they are expected to be more reproductivelyactive than capensis bees reared in their own colonies, and to be treated in a morequeen-like fashion by other workers (Calis et al. 2002). Additionally and impor-tantly, the parasitic capensis larvae receive even more food from scutellata nursebees than do normal capensis larvae (Fig. 2). In an earlier study, we found thatparasitic capensis bees are on average more queen-like than normal capensis beeswhen reared in scutellata colonies, although the characters of the parasitic beesare within the range that was found for the ‘Cape Point’ capensis bees (Allsoppet al. 2003). Hence, the differences in the amount of food in the cells confirm thelink between the amount of food given by nurse bees and the more queen-likedevelopment of the resulting bees.

It has been shown that the parasitic capensis honey bees from central andnorthern South Africa are now genetically almost identical, and these bees havebeen termed a pseudo-clone (Kryger 2001, Baudry et al. 2004). When capensisworker bees have a serious chance to reproduce, like the parasitic capensis bees in

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Table 1. Egg-production and cell-type preference in queenless capensis and scutellatacolonies. At day 0 the colonies were made queenless. Numbers of eggs observed in thedifferent cell-types are presented.

Eggs in queenless capensis colony Eggs in queenless scutellata colonyDays Queen cells Drone cells Worker cells Queen cells Drone cells Worker cells1 0 0 0 0 0 02 1 0 1 0 0 03 18 0 1 0 0 04 >50 50 25 1 1 05 >50 >50 >50 2 1 06 >50 >50 >50 1 >50 0

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scutellata colonies, bees will be selected that contribute more than average to thenext generation of social parasites. Left for a few further generations, it is easyto imagine that all current parasitic bees now descend from a single worker,especially since only a limited number of workers will have started parasitizingscutellata colonies in the first place. This has resulted in the social parasites cur-rently found, which as larvae receive even larger amounts of food from the nursebees than other capensis larvae and subsequently develop to be more queen-like(Calis et al. 2002, Allsopp et al. 2003). Within-colony selection of capensis work-ers was also described by Moritz et al. (1996), who found that the number ofpatrilines reproducing in queenless capensis colonies quickly decreased frommore than twenty to only a few.

In comparison with other A. mellifera subspecies, normal capensis workersreadily synthesize and secrete queen-like pheromones under queenless condi-tions, have higher numbers of ovarioles, and activate their ovaries extremelyrapidly (Table 1; Martin et al. 2002b, Wossler 2002). Apparently, selection forreproductive workers is not only acting in the social parasites, but also in thecapensis bee population itself. Moreover, Moritz et al. (1998) found that workerreproduction actually has a large impact in the hybrid zone between the capensisand the scutellata populations. They showed that the nuclear hybrid zone begins200 km south of the mitochondrial hybrid zone. This can only be explained ifcapensis workers contribute significantly to the gene pool, because mitochondri-al DNA-markers are only inherited by the female line.

As capensis workers lay diploid eggs by thelytoky and do not typically pro-duce drone offspring, they can only contribute to the gene pool if queens are pro-duced from their brood. In queenless colonies new queens are usually raisedfrom the old queen’s brood. If this fails, new queens may eventually be producedfrom worker-laid eggs. This implies high direct fitness gains for the workersfrom whose offspring these queens are produced. Hence, more reproductiveworkers may be selected naturally via this route. In addition, we hypothesizeanother route for queen production from worker-laid eggs that may be muchmore important: some of the new queens raised in queenright colonies prior toswarming may be produced from worker-laid eggs. This would imply selectionfor more reproductive workers during each generation.

Several arguments suggest that capensis workers may be able to producequeens in queenright colonies. In European A. mellifera colonies, a few workerswith fully activated ovaries have been found (Ratnieks 1993, Visscher 1996). Incapensis colonies, numbers of workers with activated ovaries may be higher:Anderson (1963) and Hepburn et al. (1991) report about 2% of workers with par-tially activated ovaries. We observed queenless capensis colonies to produce anegg within a day, suggesting that workers with activated ovaries were alreadypresent before the queen was removed. So, production of a few worker eggs maybe normal in queenright colonies. In addition, increased numbers of workers


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with activated ovaries have been reported to coincide with swarm preparation(Perepelova 1929). After capensis workers have activated their ovaries, theypreferably lay in queen cups (Table 1; Onions 1914, Johannsmeier 1983, Martin etal. 2002a). Worker-laid eggs in queen cells have a high chance to be reared intonew queens, because evasion of egg-policing is the rule in capensis bees (Moritzet al. 1999, Martin et al. 2002b, Calis et al. 2003, Allsopp et al. 2008). During theswarming season queen cups containing many eggs were observed in capensiscolonies, which is indicative for laying worker activity, and a few days later thesame cells contained a larva being raised into a queen (M.A., personal observa-tion). Note that there are no relatedness benefits at stake for the queen, becausethe queen is just as related to a new queen produced by a worker as to her ownprogeny. The definite test for our hypothesis is to compose colonies in which thequeen is unrelated to the workers, and study the progeny in queen cells geneti-cally. In the meantime, this has been done: 23 out of 39 new queens in 7 colonieswere of worker origin (Jordan et al. 2007)

When queens are produced from worker-laid eggs, they must be recessivehomozygous for the gene needed for thelytoky (Ruttner 1988, Lattorff et al.2005). This is meaningful, because it may explain why multiple introductions ofarrhenotokous bees inside the capensis area (Ruttner 1977); personal observa-tions) seem to dissolve without a trace. In the hybrid colonies of the next gener-ations after an introduction, a few thelytokous workers may produce queens,thus restoring thelytoky. Otherwise, the dominant allele resulting in arrheno-toky of the workers would remain in the gene pool, which is one of the reasonswhy the thelytokous capensis bees were considered endangered before 1990(Ruttner 1977). The same may play a role in the natural hybrid zone (Fig. 1) andexplain the strong introgression of capensis mitochondrial DNA into the scutel-lata population (Moritz et al. 1998).

In our view, the selection for reproductive workers originates from inside thenatural capensis population. Since capensis workers reproduce thelytokously,individual workers can have high fitness returns when they contribute to thenext generation of queens. These fitness gains will be much higher than that ofarrhenotokous reproductive workers in other honeybee races because the chancethat a worker-produced drone will mate with a queen is extremely small (Page& Erickson 1988). Moreover, as queens are mated with many drones their con-tribution is even lower. Selection for reproductive workers will, however,decrease colony performance (Hillesheim et al. 1989, Moritz 1989). Therefore, weexpect a balanced selection. On one side workers are selected to be more repro-ductive which is related to the amount of food they get from the nurse bees. Onthe other side nurse bees are selected to adjust the amount of food given, tomaintain clear caste differences between workers and queens for optimizingcolony performance. Earlier we found that when larvae are raised in their owncolonies, be it capensis larvae, scutellata larvae or hybrids between them, they

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always receive about the same amount of food from the nurse bees (Allsopp etal. 2003). However, capensis larvae receive more food from scutellata nurse beesand scutellata larvae receive less food from capensis nurse bees (Allsopp et al.2003). In other words, capensis larvae cry louder for food, but capensis workers arehard of hearing. Apparently, selection for reproductive workers inside the capen-sis population has changed the communication between nurse bees and larvae onthe allocation of food. This changed communication results in capensis parasitesbeing overfed in scutellata colonies.

Hence, social parasitism by capensis in scutellata colonies may be an acciden-tal result of selection inside the capensis population for workers that contributeto the next generation of queens. Conversely, a number of studies have suggest-ed that social parasitic tactics may have evolved as such (Neumann & Moritz2002, Dietemann et al. 2006, Härtel et al. 2006), and that the present social para-sites of northern South Africa are threatening capensis colonies as well. Sinceworkers produce new queens prior to swarming, the system indeed seems vul-nerable to parasites. In fact, Härtel et al. (2006) showed that non-nestmate work-ers reproduce in queenless colonies. Moreover, Jordan et al. (2007) not onlyshowed that queens were produced from worker-laid eggs prior to swarming, butalso that many of these eggs had been laid by non-nestmates. Possibly, this rep-resents highly reproductive capensis workers parasitizing neighbouring colonies.It may also represent the natural drift of workers between neighbouringcolonies, however, whereas the most reproductively capable of all the workers inthe group of colonies reproduce. Workers compete to become (and stay) repro-ductive, and some will have a higher chance than others (Moritz et al. 1996). Byitself this cannot be regarded as social parasitism, unless tactics to invadecolonies followed by laying eggs from which eventually a queen is produced canbe shown. In our view there is no evidence that the social parasites in the pres-ent ‘Capensis Problem’ differ from normal capensis workers, except that the mostreproductive pseudo-clone of the founder population has been selected, and thatthey are more queen-like due to overfeeding as larvae (Allsopp 2004). Thiswould imply they are not threatening to the capensis population itself. Problemssimilar in nature if not in scale to the present ‘Capensis Problem’ have beenreported previously in South Africa (on two occasions), Zimbabwe and Brazil(Onions 1914, Lundie 1954, Kerr 1967, Johannsmeier 1983) and may have beendeveloping in Arizona (DeGrandi-Hoffman & Schneider 2001, DeGrandi-Hoffman et al. 2004). We therefore caution that normal capensis bees are capableof the type of devastation witnessed in the ‘Capensis Problem’ (Allsopp 2004).

Acknowledgements W.B. and J.C. were supported by the Netherlands Foundationfor the Advancement of Tropical Research (WOTRO; W 84-503). Dawid Swart isthanked for providing parasitic capensis pseudo-clones from Pretoria. Pam van Stratumis thanked for editing the graphs.


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REFERENCESAllsopp, M.H. 1993. Summarized overview of the Capensis Problem. S. Afr. Bee J.

65:127-136.Allsopp, M.H. 2004. Cause or Consequence: The pseudo-clone and the Capensis

Problem. S. Afr. Bee J. 76:2-5.Allsopp, M.H., Boot, W.J. & Calis, J.N.M. 2008. Eggs of Apis mellifera capensis workers

survive the ‘worker police’. Submitted.Allsopp, M.H., Calis, J.N.M. & Boot, W.J. 2003. Differential feeding of worker larvae

affects caste characters in the Cape honeybee, Apis mellifera capensis. Behav. Ecol.Sociobiol. 54:555-561.

Allsopp, M.H. & Hepburn, H.R. 1997. Swarming, supersedure and the mating system ofa natural population of honey bees (Apis mellifera capensis). J. Apic. Res. 36:41-48.

Anderson, R.H. 1963. The laying worker in the Cape honeybee, Apis mellifera capensis. J.Apic. Res. 2:85-92.

Baudry, E., Kryger, P., Allsopp, M., Koeniger, N., Vautrin, D., Mougel, F., Cornuet,J.M. & Solignac, M. 2004. Whole-genome scan in thelytokous-laying workers of thecape honeybee (Apis mellifera capensis): Central fusion, reduced recombination ratesand centromere mapping using half-tetrad analysis. Genetics 167:243-252.

Beekman, M., Calis, J.N.M. & Boot, W.J. 2000. Parasitic honeybees get royal treatment.Nature 404:723-723.

Beetsma, J. 1979. The process of queen-worker differentiation in the honeybee. Bee Wld.60:24-39.

Calis, J.N.M., Boot, W.J. & Allsopp, M.H. 2003. Capensis honeybees: crucial steps lead-ing to social parasitism. Proc. Exper. Appl. Entomol. NEV Amsterdam 14:39-43.

Calis, J.N.M., Boot, W.J., Allsopp, M.H. & Beekman, M. 2002. Getting more than a fairshare: nutrition of worker larvae related to social parasitism in the Cape honey beeApis mellifera capensis. Apidologie 33:193-202.

Dampney, J.R., Barron, A.B. & Oldroyd, B.P. 2004. Measuring the cost of worker repro-duction in honeybees: work tempo in an ‘anarchic’ line. Apidologie 35:83-88.

DeGrandi-Hoffman, G., Chambers, M., Hooper, J.E. & Schneider, S.S. 2004.Description of an intermorph between a worker and queen in African honey beesApis mellifera scutellata (Hymenoptera: Apidae). Ann. Entomol. Soc. Am. 97:1299-1305.

DeGrandi-Hoffman, G. & Schneider, S.S. 2001. Worker behaviours in queenless african-ized honey bee colonies. Proc. 2nd Int. Conf. on Africanized honey bees and beemites, Medina, Ohio, The A.I. Root Company:104-108

Dietemann, V., Pflugfelder, J., Hartel, S., Neumann, P. & Crewe, R.M. 2006. Social par-asitism by honeybee workers (Apis mellifera capensis Esch.): evidence for pheromon-al resistance to host queen’s signals. Behav. Ecol. Sociobiol. 60:785-793.

Härtel, S., Neumann, P., Raassen, F.S., Moritz, R.F.A. & Hepburn, H.R. 2006. Socialparasitism by Cape honeybee workers in colonies of their own subspecies (Apis mel-lifera capensis Esch.). Ins. Soc. 53:183-193.

Hepburn, H.R., Magnuson, P., Herbert, L. & Whiffler, L.A. 1991. The development oflaying workers in field colonies of the Cape honey-bee. J. Apic. Res. 30:107-112.

Hepburn, R. & Radloff, S.E. 2002. Apis mellifera capensis: an essay on the subspecific clas-sification of honeybees. Apidologie 33:105-127.

Hillesheim, E., Koeniger, N. & Moritz, R.F.A.1989. Colony performance in honeybees(Apis mellifera capensis Esch) depends on the proportion of subordinate and domi-

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nant Workers. Behav. Ecol. Sociobiol. 24:291-296.Johannsmeier, M.F. 1983. Experiences with the Capensis bee in the Transvaal. S. Afr. Bee

J. 55:130-138.Jordan, L.A., Allsopp, M.H., Oldroyd, B.P., Wossler, T.C. & Beekman, M. 2007.

Cheating honeybee workers produce royal offspring. Proc. R. Soc. B doi:10.1098/rspb.2007.1422.

Kerr, W.E. 1967. The history of the introduction of African bees to Brazil. S. Afr. Bee J.39: 2-5.

Kryger, P. 2001. An obligate social parasite in honey bees: the pseudo-clone of Apis mel-lifera capensis. Proc. 13th Entomol.Congr. S. Afr.:38

Lattorff, H.M.G., Moritz, R.F.A. & Fuchs, S. 2005. A single locus determines thelytok-ous parthenogenesis of laying honeybee workers (Apis mellifera capensis). Heredity94:533-537.

Lundie, A.E. 1954. Laying worker bees produce worker bees. S. Afr. Bee J. 29:10-11.Martin, S., Wossler, T. & Kryger, P. 2002a. Usurpation of African Apis mellifera scutel-

lata colonies by parasitic Apis mellifera capensis workers. Apidologie 33:215-231.Martin, S.J., Beekman, M., Wossler, T.C. & Ratnieks F.L.W. 2002b. Parasitic Cape hon-

eybee workers, Apis mellifera capensis, evade policing. Nature 415:163-165.Moritz, R.F.A. 1989. Colony Level and within Colony Level Selection in Honeybees – a

2 Allele Population-Model for Apis mellifera capensis. Behav. Ecol. Sociobiol. 25:437-444.

Moritz, R.F.A., Beye, M. & Hepburn, H.R. 1998. Estimating the contribution of layingworkers to population fitness in African honeybees (Apis mellifera) with molecularmarkers. Ins. Soc. 45:277-287.

Moritz, R.F.A., Kryger, P. & Allsopp, M.H. 1996. Competition for royalty in bees.Nature 384:31-31.

Moritz, R.F.A., Kryger, P. & Allsopp, M.H. 1999. Lack of worker policing in the Capehoneybee (Apis mellifera capensis). Behav. 136:1079-1092.

Moritz, R.F.A. & Southwick, E.E. 1992. Bees as superorganisms. Springer-Verlag, Berlin.Neumann, P., Hepburn, H.R. & Radloff, S.E. 2000. Modes of worker reproduction,

reproductive dominance and brood cell construction in queenless honeybee (Apismellifera L.) colonies. Apidologie 31:479-486.

Neumann, P. & Hepburn, R. 2002. Behavioural basis for social parasitism of Cape hon-eybees (Apis mellifera capensis). Apidologie 33:165-192.

Neumann, P. & Moritz, R.F.A. 2002. The Cape honeybee phenomenon: the sympatricevolution of a social parasite in real time? Behav. Ecol. Sociobiol. 52:271-281.

Onions, G.W. 1912. South African fertile worker bees. Agric. J. Union S. Afr. 1:720-728.Onions, G.W. 1914. South African ‘fertile’ worker bees. Union S. Afr. Agric. J. 7:44-46.Page, R.E. & Erickson, E.H. 1988. Reproduction by worker honey bees (Apis mellifera L).

Behav. Ecol. Sociobiol. 23:117-126.Perepelova, L.I. 1929. Laying workers, the oviposition of the queen, and swarming. Bee

Wld. 10:69-71.Ratnieks, F.L.W. 1993. Egg-laying, egg-removal, and ovary development by workers in

queenright honey-bee colonies. Behav. Ecol. Sociobiol. 32:191-198.Ruttner, F. 1977. Problem of cape Bee (Apis mellifera capensis Escholtz) – Parthenogenesis

– Size of Population – Evolution. Apidologie 8:281-294.Ruttner, F. 1988. Biogeography and taxonomy of honeybees. Springer, Berlin.


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Snodgrass, R.E. 1956. Anatomy of the honey bee. Cornell University Press, New York.Visscher, P.K. 1996. Reproductive conflict in honey bees: A stalemate of worker egg-lay-

ing and policing. Behav. Ecol. Sociobiol. 39:237-244.Wirtz, P. & Beetsma, J. 1972. Induction of caste differentiation in the honey bee (Apis

mellifera) by juvenile hormone. Entomol. Exper. Appl. 15:517-520.Wossler, T.C. 2002. Pheromone mimicry by Apis mellifera capensis social parasites leads

to reproductive anarchy in host Apis mellifera scutellata colonies. Apidologie 33:139-163.

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J. Paalhaar, W.J. Boot, J.J.M. van der Steen* & J.N.M. CalisLaboratory of Entomology, Wageningen University, PO Box 8031, 6700 EHWageningen, The Netherlands, E-mail: [email protected]; *Applied Plant Research(PPO), Bee Unit, PO Box 69, 6700 AB Wageningen, The Netherlands

In-hive transfer of pollen was studied by analysing the pollen on thebodies of young in-hive bees and foraging bees of the same honeybeecolony. The colony was placed and opened in a bee-tight greenhousewith flowering carrot, celery and endive, and samples of bees weretaken in the course of one day. The density of pollen on the bodies ofyoung bees inside the hive rapidly built up to about 0.5 % of that foundon the foragers on each of the three plant species. A similar amountwas apparently transferred to the foragers. Therefore we conclude thatin-hive transfer of pollen between honey bees enhances cross-pollina-tion of plants.

Keywords: Apis mellifera, cross-pollination, pollen, in-hive transfer

Plant species have different strategies to attract insects for pollination. Self-incompatible plant species need pollinators, like bees, for seed production. Self-compatible plants may use pollinators to achieve cross-pollination. When beesvisit flowers, pollen adheres to their hairs and may stay there for a long time,depending on the characteristics of the specific pollen grains (Free & Williams1972). While bees fly from flower to flower, transfer of pollen leads to cross–pol-lination (Roubik 1999).

In social bees, like honey bees Apis mellifera L., the individuals usually focuson specific patches of flowers. Pollen may be transferred from a forager to otherbees in the hive by brushing against each other, or because all bees reside in thesame pollen-contaminated hive (Free & Williams 1972). Bees of one hive forageon different patches of flowers that can be more than nine kilometres from thehive in every direction (Beekman & Ratnieks 2000). Different bees from onehive may simultaneously visit patches of plants of one species that are flower-ing many kilometres from each other. In theory, transfer of pollen inside thehive from one forager to another forager may therefore lead to cross-pollinationover distances unequalled to pollination by other insects.

In-hive pollen transfer between bees enhancescross-pollination of plants


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Several studies on fruit-set in self-incompatible plants suggest that in-hivetransfer of pollen enhances cross-pollination, but leave unanswered to whatextent (Dag et al. 2001, DeGrandi-Hoffman et al. 1992). In other studies, bees in-and outside the hive were analysed instead of the pollinated plants. These stud-ies showed that pollen was transferred between bees, but it was unclear wherethe pollen came from and if the pollen were still viable and could lead to polli-nation (Free & Williams 1972, Hatjina et al. 1998).

The purpose of this study is to determine to what extent in-hive transfer ofpollen may contribute to cross-pollination. We studied the distribution of previ-ously inexperienced pollen over foraging bees and young in-hive bees of onehoneybee colony in the course of one day. Pollen on bees was quantified to gaininsight into in-hive transfer of pollen from foragers to other bees in the colony.

MATERIALS AND METHODSA bee-tight greenhouse with three flowering crops, carrot, celery and endive,was used as an experimental set-up. At 8:00 a.m. a bee colony (ca. 12,000 individ-uals) was placed and opened inside the greenhouse. The first samples of beeswere taken directly and they showed that the pollen of the three plant speciesindeed were absent on the bees at the beginning of the experiment. More sam-ples of bees were taken in the course of one day, every two hours, from youngbees inside the hive (n=20), water foragers and foragers from the flowers of thethree crops (n=10). To examine whether pollen stayed overnight on the bees,another sampling of young bees (n=20) was done at the beginning of the nextday prior to bee flight. All bees were sampled individually and were put inEppendorf tubes and stored in a freezer until analysis of the pollen.

Division of labour inside a colony depends on age of the bees (Seeley 1995).Young bees start performing tasks inside the hive, whereas older bees explorethe environment. To obtain marked young in-hive bees, emerging bees were col-lected and marked from various intervals prior to the experiment. As a result,four different cohorts of bees with the age of 1-2 days, 8-9 days, 15-16 days and 22-23 days, had different colours on their thoraxes at the day of the experiment.Marked bees were returned to the original hive, except for the young bees of twodays old. These newly emerged bees had had no contact with their other nest-mates before the experiment started, at which moment they were released intotheir original hive. Together with the marked bees with an age of 8-9 days, thesebees formed the group of young in-hive bees, because they were not observedflying outside the hive. Older marked bees were observed to be foraging. Beeslanding during five minutes on the landing board of the hive were counted everytwo hours to get an impression of the foraging population.

To determine the pollen density on the bees’ body hairs, the stored bees werebrought on room temperature and the pollen was collected as follows. Firstly,the hind legs of foraging bees were cut off to prevent that pollen in the corbicu-

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lae would be analysed. The bees were individually sonified for 10 seconds in 1.0ml of a mixture of ethanol, water and detergent (1% vol./vol. Triton-X100 in a70% ethanol solution) with a Sonifier B-12, micro-tip level 3. After removing thebees the tubes with the ethanol solution were centrifuged for 3.5 minutes on level14 (*1000 min-1). The supernatants were discharged, and 50 μl distilled water wasadded to the pellet of pollen in each tube. Tubes were shaken for 1 minute in thehighest rotation (vortex) to ensure a homogeneous distribution of pollen in thesolution. Then 18 μl from each tube was dropped with a pipette on a haemocy-tometer slide, ‘Neubauer improved’, and analysed with a Euromex-microscope.When the density of pollen in the solution was low, for example in the samplesof young bees, the number of pollen grains on the total slide grid (3.6% of the 50μl) was counted, and the type of pollen was determined. When samples with ahigh density of pollen were analysed, for example samples of foragers, only 1.0%or 1.8% of the solution was analysed. The pollen of endive, carrot and celerycould easily be distinguished from each other. The total number of grains in theoriginal pollen precipitate of each tube was derived under the assumption thatthe analysed solution was representative for the whole sample. Note that part ofthe pollen remained on the bees’ body. After repeating the procedure three timeswith 10 test samples we deduced that on average 67% of the pollen on the beeswere collected after one sonification. Other loss of pollen was limited because nopollen could be found in the supernatants after centrifuging. Additionally, hard-ly any pollen (2%) was found when tubes were shaken again to resuspend pal-lets that had possibly stayed behind.

RESULTSThe bees foraged between 8:00 a.m. till 9:00 p.m. The period with the highestactivity was around noon (Fig. 1). Integrating the counts on the landing boardwe calculated that approximately 25,000 forage flights were made during the day.

J. PAALHAAR, W.J. BOOT, J.J.M. VAN DER STEEN & J.N.M. CALIS

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Two hours after foraging started, celery and endive pollen was found onyoung bees that stayed inside the hive, 1-2 days and 8-9 days old. Carrot pollenwas found in the next sample, two hours later. Hence, pollen was transferredfrom foragers to bees inside the hive. Pollen numbers varied during the day, butboth on foragers and young bees there was no clear build up of pollen over time(Fig. 2). Foragers that were taken from a flower carried on average about 3500pollen grains from that plant (Table 1). The pollen numbers on young bees willdepend on how many bees are foraging on the flowers. In our setting, 9 to 18

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Figure 2. Number of pollen grains from carrot, celery and endive in a greenhouse, onsamples of bees taken in the course of a day from young in-hive bees (n=20), water for-agers and foragers from the three crops (n=10). The colony was introduced from out-doors and the bees were released at 8:00 a.m.

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pollen grains were found on average per plant species, which is about 0.5% of thenumbers found on the foragers (Table 1). Note that the young bees acquiredpollen from all three plant species.

Celery pollen was most abundant on the bodies of foraging bees. Numbers ofendive pollen were relatively low. Foragers also had pollen from the plants theywere not collected from. In the case of foragers from carrot with male-sterileflowers and endive, these numbers were similar to the numbers found on youngbees (Fig. 2). This suggests that these foragers specialized on one type of flower.Foragers from carrot with male flowers and celery apparently switched betweenflowers, because high numbers of both types of pollen grains were found. Thenext morning, pollen of both carrot and celery were still found on young bees.Endive pollen were absent on these bees (Fig. 2).

Pollen grains other than from the three crops were also found on the bodiesof foraging bees and to a lesser extent on young bees. Even the newly emergedbees from the incubator carried some pollen grains. Especially the small-sizedpollen of sweet chestnut was abundantly found. These pollen grains must havebeen acquired before the start of the experiment and could still be found onyoung bees the next morning.

DISCUSSIONThis study shows that pollen carried by foragers to a honeybee colony is trans-ferred rapidly among the honey bees inside a hive. Within two hours of forag-ing activity on previously inexperienced plant species, young bees inside thehive had acquired significant numbers of pollen. Related to the numbers ofpollen on a forager collected from one of the three plant species in the green-house, about 0.5% could be found on the young bees. Foragers may have acquiredall three types of pollen by flying directly to the respective flowers. The num-bers of pollen found indeed suggest that bees switch between celery and carrot.However, bees foraging on male-sterile carrot and endive seem specialized,because numbers of strange pollen types are similar to the numbers on the youngbees, suggesting transfer inside the hive.

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Table 1. Average numbers of pollen grains on foragers on a specific crop and on youngbees (in brackets the numbers on young bees as percentage of the numbers on for-agers).

Crop species Average number of pollen grains onForagers (n=60) Young bees (n=140)

Carrot 4297 12 (0.3%)Celery 5173 18 (0.4%)Endive 1363 9 (0.7%)

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Pollen may adhere for a long period on a bee’s body. The size of the grainsseems an important factor defining how long pollen grains can be found. Thebigger endive pollen was absent on young bees on the next morning, whereas thesmaller celery and carrot grains were still present. Moreover, many chestnutgrains were found. These grains are relatively small compared to the othergrains, and they must have been acquired in the days before the experimentstarted. How long pollen grains remain viable on the bee’s body is unknown andprobably varies for different pollen species. This viability will be important forcross-pollination through in-hive transfer of pollen. However, even if pollenremains viable for only a few hours, in-hive transfer may lead to cross-pollina-tion, because transfer between bees seems to be fast.

Different bees from one hive may simultaneously visit patches of plants ofone species that are flowering many kilometres from each other. As in-hivetransfer of pollen takes place at a significant level, transfer of pollen from oneforager to another forager can lead to genetic contacts between plants over dis-tances unequalled to pollination by other insects. Additionally, in hybrid-seedproduction plots, where individual bees may specialize on different strains ofone plant species, in-hive transfer of pollen enhances effectiveness of honeybees.

Acknowledgements Rijk Zwaan Breeding B.V. is greatly acknowledged for facili-tating the experiment by allowing the use of one of their greenhouses used for breeding.

REFERENCESBeekman, M. & Ratnieks, F.L.W. 2000. Long-range foraging by the honey-bee, Apis mel-

lifera L. Functional Ecology. 14(4): 490-496.Dag, A., Degani, C. & Gazit, S. 2001. In-hive pollen transfer in Mango. Acta Horticulture.

561: 61-64.DeGrandi-Hoffman, G., Thorp, R., Loper, G. & Eisikowitch, D. 1992. Identification and

distribution of cross-pollinating honey-bees on almonds. Journal of Applied Ecology.29: 238-246.

Free, G.B. & Williams, I.H. 1972. The transport of pollen on the body hairs of honey-bees (Apis mellifera L.) and bumblebees (Bombus spp.). Journal of Apicultural Research.9: 609-615.

Hatjina, F., Free, J.B. & Paxton, R.J. 1998. Hive-entrance pollen transfer devices toincrease the cross-pollination potential of honey bees. I. Examination of six materi-als. Journal of Apicultural Research. 37: 231-237.

Roubik, D.W. 1999. The foraging and potential outcrossing pollination ranges of Africanhoney bees (Apiformes: Apidae; Apini) in Congo Forest. Journal of the KansasEntomological Society. 72(4): 394-401.

Seeley, T.D. 1995. The wisdom of the hive. The social physiology of honey bee colonies.Harvard University Press, London.

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Eeva-Liisa AlanenDepartment of Applied Biology, PO Box 27, FI-00014 University of Helsinki, Finland,E-mail: [email protected]

The objective of this study was to estimate the suitability of a largearea of agricultural land for the starting phase of bumblebee colonies.The densities and food plants of bumblebee queens in eight differenthabitat types were recorded. In early May the highest densities of for-aging queens were encountered in stream sides and in late May on leys.In these habitat types the availability of the most important food plants(Salix and Taraxacum) was highest.

Keywords: Bombus, queens, habitats, Salix, Taraxacum

In temperate species of bumblebees (Bombus: Hymenoptera, Apidae) the annualcolony is started by an overwintered queen in the spring. In Finland the timingof emergence from hibernation varies between species in the southern and cen-tral parts of the country e.g. B. lapidarius emerges in late April and B. distinguen-dus in late May (Pekkarinen & Teräs 1977).

Bumblebees are important pollinators in agricultural ecosystems (Free 1993,Kwak et al. 1996). Due to their large size and thermoregulatory ability, bumble-bee queens fly even in cold temperatures in early spring (Heinrich 1979).However, bumblebee populations, especially those of long-tongued species, havesuffered declines in several countries in the last few decades. This is now wide-ly regarded as a consequence of intensified agriculture (Williams 1986, Goulson2003).

The resources needed by the overwintered bumblebee queens are suitableflowers providing nectar and pollen (Fussell & Corbet 1992a, Mänd 2000) and asuitable site for starting a colony (Fussell & Corbet 1992b, Svensson 2002).Bumblebee nests are often found in old rodent holes, which are commonly situ-ated in forest boundaries (Svensson 2000). After finding a nesting site, the queenlays her first batch of eggs on a clump of pollen she has collected and then incu-bates it (Heinrich 1979). This is an energy consuming process and a large enoughsupply of forage plants is therefore essential for a successful start of the colony.

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MATERIALS AND METHODS The data were collected in the spring of 2000 between 25 April and 2 June. Thestudy area [situated in Lammi, southern Finland (61°05’ N, 24°00’ E)] consistedof ten large (from 92.9 to 240.3 ha) areas of farmland, which in turn consisted ofvarious landscape elements, such as cultivated fields, forest islands and fieldmargins. These individual study areas were separated from each other by mainroads, lakes and forest belts, and had different landscape structures (Bäckman &Tiainen 2002).

Weather conditionsThe counts were made between 9.00 h and 18.00 h in good weather conditions.In general, it was fairly warm and the precipitation was low during the studyspring (Fig. 1). At the end of April the daytime temperatures were higher thanusual in southern Finland and at Lammi 21.0°C was reached on 20 April. In Maythere were two periods of warm weather and at Lammi the maximum was 23.1°Con 10 May. The mean temperature of May was normal, but due to some unusu-ally warm days in April and May, the growing season advanced rapidly. By thebeginning of June the season was ten days earlier compared to the long-termaverages. During the study period daily precipitation exceeded 2 mm on threedays: 25 May (3.1 mm), 26 May (11.6 mm) and 2 June (2.7 mm). In June a colderand rainier period began.

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Figure 1. Weather conditions (daily maximum and minimum temperatures in °C, rain-fall in mm) during the spring of 2000. Normal max. and normal min. are long-termaverage temperatures over ten years.

°°CC

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Bumblebee countsThe bumblebee counts were carried out three times in six, and four times in fourstudy areas using the line-transect method (Banaszak 1980, Teräs 1983). Thecaste (queen or worker) based on the individual’s size and the behaviour (search-ing for food or a nesting site) were recorded. The nomenclature followsSöderman & Leinonen (2003).

Each transect was placed into one of the following habitat types: forestboundary between a forest and a field, forest boundary between a forest and aroad, ley (cultivated with grass and/or clovers), yard (garden), field marginbetween two fields, field margin between a field and a road, stream side (ripari-an corridor) and ruderal patch (uncultivated land, such as a meadow). The widthof the transects was five meters, except on leys, where the width was twometers.

The food plants of food-collecting bumblebees were identified according toHämet-Ahti et al. (1998). During the study period flowering shrubs and treeswere drawn on a large scale map so that the area on the map equalled the cover-age (m2) of that of shrub/tree individual in the field. The coverage of flowers(%) of Taraxacum spp. and Corydalis solida were also estimated for each line-tran-sect.

Data analysisThe Shannon’s diversity index (H’) and evenness index (J’) (Hutcheson 1970)both for the plants and for the bumblebees were calculated. Evenness indiceswere only calculated for species with more than 10 observations (Teräs 1985). Theformulae used were: H’ = –Σ pi ln pi and J = H’/ln S. For the plants the terms inthe indices were: pi = the proportion of visits paid by the ith bumblebee species andS = the total number of bumblebee species visiting the plant species. For the bum-blebees the terms were: pi = the proportion of visits paid to the ith plant speciesand S = the total number of plant species visited by the bumblebee species.

Bumblebee densities (individuals per hectare) ([number of bumblebees/tran-sect length* width]/10000) were calculated for each line-transect and count. Thecalculations were made separately for the food-collecting and nest-seekingqueens. Species were also divided into ecological species groups (open landscapespecies/forest species/generalists) according to literature (see Bäckman &Tiainen 2002) and the corresponding densities were then calculated.

Before performing the statistical analyses, the data was log-transformed.Bumblebee densities in different habitats were compared by ANOVA and t-testwas performed for ruderal patches (two types: open and bushy). Densities werealso compared according to some other characteristics of the transects e.g. ditchtype (three types: main ditch, other ditch and no ditch between the field mar-gins). Stepwise linear regression analysis was performed for the bumblebee den-sities and the availability of food plants.

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Landscape structureLandscape indices were calculated using the ArcViewTM PatchAnalystTM exten-sion in order to describe the landscape structure of each study area. The exten-sion uses the FragstatsTM program (McGarigal & Marks 1994) for the calculationof landscape variables. The indices were calculated using large scale digital mapsbased on aerial orthophotographs (scale 1:5000) (Bäckman & Tiainen 2002).There were 15 classes of landscape elements.

The calculated variables were: total landscape area (TLA, ha), class area (CA,ha), number of patches (NumP), mean patch size (MPS, ha), total edge (TE, m),edge density (ED, m/ha), landscape diversity (Shannon’s diversity index, SDI)and landscape evenness (Shannon’s evenness index, SEI). TLA, SDI and SEIwere calculated for the whole study areas and the other indices also for each ele-ment class separately. Correlations (Kendall rK) were then calculated in order toreveal any interdependence of the landscape indices and the bumblebee densitiesin each study area during each count.

RESULTS

BumblebeesEleven species of bumblebees and two species of cuckoo bumblebees wererecorded. The total number of individuals was 3711, of which 3526 were queensand 185 workers. 189 of the queens were nest-seeking individuals. Out of all indi-viduals, 3371 bumblebees were identified at species level, 181 as cuckoo bumble-bees and 159 remained unidentified. Both food-collecting and nest-seekingqueens were encountered from the first count onwards and workers were pres-ent during the last two counts. The percentage of B. lucorum, the most abundantbumblebee of agricultural landscapes in southern Finland, diminished towardsthe end of the study period (Alanen 2008b).

Food plantsThe plant species that were visited by the bumblebees belonged to 28 species orgenera. Salix spp., Taraxacum spp., Acer platanoides, Ribes rubrum and Caraganaarborescens were the most visited ones. Figure 2 shows B. lucorum on Salix and fig-ure 3 shows B. pascuorum on Taraxacum. The number of visited plant species var-ied between five during the first and 17 during the fourth count. There were dif-ferences in the diversity and evenness of flower visits, as regards to both theplants and the bumblebees (Alanen 2008b).

Bumblebee densitiesDuring the first two counts the highest densities of food-collecting queens werecounted in stream sides and during the last two on leys. This seemed to be main-ly due to the availability of flowering food plants (Salix and Taraxacum). Theseresults were supported by the regression analyses, although the R-values were

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Figure 2. Bombus lucorum foraging onSalix.

Figure 3. Bombus pascuorum foraging on Taraxacum.

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generally small. The preferred habitat types by nest-seeking queens were some-what different (Alanen 2008a).

Pair-wise comparisons (Games-Howell test) showed some significant resultse.g. different types of field margins supported the highest densities of foragingbumblebees during different counts. The effect of food plants was also visible onruderal patches There were also differences in the densities of the ecologicalspecies groups among the habitat types. Open landscape species seemed to beespecially abundant in yards (Alanen 2008a).

The correlations of the landscape indices and bumblebee densities were weakin general. However, there was a significant correlation between the edge densi-ty of yards and the density of B. hypnorum during the first count. This species issaid to be associated with human habitation and is often the most commonspecies in city areas. During the fourth count, the edge density of forests and thedensity of forest species correlated significantly.

DISCUSSIONMy study reveals a clear pattern in the mass flowering of Salix and Taraxacumduring the starting phase of the bumblebee colonies. Stream sides are the mostvaluable habitats for the queens during the first weeks of their activity, due tothe flowering of willows. Later the foraging bumblebees switch to leys, as theflowering period of dandelions starts. Notably, gardens supported rather highdensities during the whole study period.

Willows Salix offer large amounts of both nectar and pollen (Pekkarinen &Teräs 1977, Teräs 1985, Edwards 1996, Svensson 2002). Bumblebee queens drinkup their nectar in order to gain energy and eat their pollen, which is essential forthe development of their ovaries. In early May Norway maples Acer platanoidesflowering in forest boundaries and yards also attracted large numbers of bumble-bees. Dandelions Taraxacum are the most important food plants after the flow-ering of willows has ended (Teräs 1985).

Later in May the queens visited Ribes rubrum and the ornamental Caraganaarborescens in relatively high numbers. The latter is evidently a good food plantfor queens initiating nest building (Teräs 1985). In late May and early Junevetches Vicia are important food plants both for the queens and the first work-ers (Teräs 1985). The intensification of agriculture has negatively affected espe-cially vetches and other bumblebee plants with deep corollas (Fussell & Corbet1992a). These are typically plants of permanent patches of open vegetation out-side cultivation, such as field margins, meadows and pastures.

REFERENCESAlanen, E.L. 2008a. A landscape ecological approach for comparing densities of bumble-

bee queens in agricultural habitats. manuscript, in preparation.Alanen, E.L. 2008b. Phenology and food plants of bumblebee queens in an agricultural landscape.

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submitted to Journal of Apicultural Research.Banaszak, J. 1980. Studies on methods censusing the numbers of bees (Hymenoptera,

Apoidea). Polish Ecological Studies 6: 355-366.Bäckman, J.P.C. & Tiainen, J. 2002. Habitat quality of field margins in a Finnish farm-

land area for bumblebees (Hymenoptera: Bombus and Psithyrus). Agriculture,Ecosystems and Environment 89: 53-68.

Edwards, M. 1996. Optimizing habitats for bees in the United Kingdom – a review ofrecent conservation action. In: Matheson, A., Buchmann, S.L., O’Toole, C.,Westrich, P. & Williams, I.H. (eds.) The conservation of bees, pp. 35-45. AcademicPress, London.

Free, B. 1993. Insect pollination of crops. Academic Press, London.Fussell, M. & Corbet, S.A. 1992a. Flower usage by bumble-bees: a basis for forage plant

management. Journal of Applied Ecology 29: 451-465.Fussell, M. & Corbet, S.A. 1992b. The nesting places of some British bumble bees. Journal

of Apicultural Research 31 (1): 32-41.Goulson, D. 2003. Bumblebees behaviour and ecology. Oxford University Press, Oxford.Heinrich, B. 1979. Bumblebee economics. Harvard University press, Cambridge.Hämet-Ahti, L., Suominen, J., Ulvinen, T. & Uotila, P. 1998. Retkeilykasvio. Luonnontie-

teellinen keskusmuseo, Kasvimuseo, Helsinki.Kwak, M.M., Velterop, O. & Boerrigter, E.J.M. 1996. Insect diversity and the pollination

of rare plant species. In: Matheson, A., Buchmann, S.L., O’Toole, C., Westrich, P.& Williams, I.H. (eds.) The conservation of bees, pp. 115-124. Academic Press, London.

McGarigal, K. & Marks, B.J. 1994. Fragstats: spatial pattern analysis program for quantifyinglandscape structure. Reference manual. Forest Science Department, Oregon stateUniversity, Corvallis, Oregon.

Mänd, M. 2000. Bumblebees in agricultural landscapes of Estonia. PhD Thesis, University ofTarto.

Pekkarinen, A. & Teräs, I. 1977. Suomen kimalaisista ja loiskimalaisista. Luonnon tutkija81: 1-24.

Svensson, B., Lagerlöf, J. & Svensson, B.G. 2000. Habitat preferences of nest-seekingbumble bees (Hymenoptera: Apidae) in an agricultural landscape. Agriculture,Ecosystems and Environment 77: 247-255.

Svensson, B. 2002. Foraging and nesting ecology of bumblebees (Bombus spp.) in agricul-tural landscapes in Sweden. Acta Universitatis Agriculturae Sueciae, Agaria 318.

Söderman, G. & Leinonen, R. 2003. Suomen mesipistiäiset ja niiden uhanalaisuus. TremexPress Ltd, Helsinki.

Teräs, I. 1983. Estimation of bumblebee densities (Bombus: Hymenoptera, Apidae). ActaEntomologica Fennica 42.

Teräs, I. 1985. Food plants and flower visits of bumblebees (Bombus: Hymenoptera,Apidae) in southern Finland. Acta Zoologica Fennica 179: 1-120.

Williams, P.H. 1986. Environmental change and the distribution of British bumble bees(Bombus Latr.). Bee World 67: 50-61.

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L.A. Langoya & P.C.J. van RijnCentre for Terrestrial Ecology, Netherlands Institute for Ecology, NIOO-KNAW, POBox 40, 6666 ZG Heteren, The Netherlands, E-mail: [email protected],[email protected]

The main objective of the Functional Agro-Biodiversity project in the‘Hoeksche Waard’ is the creation of favourable conditions both at thefarm and landscape level for promoting bio-diversity and the role ofnatural enemies of pests.Aphids constitute one of the major crop pests in Dutch arable and veg-etable farming. The common syrphid, Episyrphus balteatus, is one of theimportant predators of this polyphagous pest complex. Since only itspredatory larval stage subsists on aphids, while the adult live primari-ly on nectar and pollen, agro-biodiversity enhancement in the form offield margin plants can be particularly essential for syrphid survivaland reproduction and for sustaining a viable natural enemy population.Field and laboratory investigations show that selected plant speciesstrongly differ in their suitability as food sources for the syrphid E.balteatus, even within specific families. Aphid honeydew is an impor-tant supplementary food source. E. balteatus can, on the basis of ouranalyses, be regarded as an effective predator of aphids.

Keywords: conservation biological control, aphids, hoverflies, Episyr-phus balteatus, nectar, pollen and honeydew.

Functional Agro-Biodiversity (FAB) is a concept that aims at substantiallyreducing pesticide use in arable and field vegetable farming through exploitationof the potentials of natural control. One of the goals of FAB is the enhancementof natural control by: 1) improving the quality of semi-natural landscape ele-ments such as hedgerows and dykes, 2) creating field margins for food and shel-ter for entomophagous insects, and 3) minimal and selective use of pesticidesbased on monitoring pests and natural enemies.

Syrphids (hoverflies) feature conspicuously among the natural enemy popu-lations in field crops. Detailed studies of their contribution to pest managementare limited (Schmidt et al. 2004). Compared to other natural enemies, e.g. ladybeetles or lacewings, hoverflies seem to be better equipped in locating aphid

The significance of floral resources for naturalcontrol of aphids


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colonies, because of their swift flight patterns and ability to hover to quicklyassess preys. Worldwide there is a fairly large number of species that are aphi-dophagous (Bugg 1992). Among these, Episyrphus balteatus is one of the mostabundant species in arable fields (van Rijn & Smit 2007). Aphidophagous hover-flies alternately feed on plant-derived foods (nectar and pollen) and prey onaphids during different stages of their life cycle. The exploitation of these foodsources is of practical significance for realising sustainable pest control strate-gies. These attributes were relevant for the functional choice of field marginplants in this research.

We examined three key questions: (1) the suitability of flowering plantspecies for field margins, (2) the importance of nectar, pollen and honeydew forsyrphid survival, and (3) the impact of syrphids on aphid populations.


Field experimentsField experiments were conducted with both annual and perennial field marginplants. Annual plants produce comparatively large quantities of flowers, whileperennials essentially function as sanctuaries for various insects. Careful pheno-logical analysis of these prospective plants provided us with a select group ofspecies capable of guaranteeing a steady supply of nectar and pollen for naturalenemies from week 22 to week 40, during which aphids can be present in thefield crops. The margins were laid adjacent to the crop fields.

Aphid and syrphid population densities per leaf were monitored on potatocrops at 3.5, 15, 40 and 90 metres distances from the perennial margins and annu-al plant strips.

Laboratory experimentsComplementary to field observations, laboratory experiments were conductedwith the aim of understanding the extent to which flowering plants in farmingenvironments can contribute to the survival and reproduction of natural enemies.

The role of floral resources of the different plant species in the life history ofE. balteatus was investigated. Cage experiments in climate chambers were car-ried out under controlled conditions (22°C and 80% RH). Each cage (130 dm2)was provided with a flowering plant, two freshly emerged males and females ofE. balteatus and a bottle with wet cotton wool for water supply. E. balteatus pupaewere provided by Koppert B.V. (Berkel and Rodenrijs, The Netherlands). Thesurvival of the syrphids was checked every other day.

For studying the impact of honeydew and for reproductive studies a sixweeks-old Brussels sprout plant (bearing 5-7 leaves), initially infested with 10cabbage aphids (Brevicoryne brassicae), was added to each cage. The honeydewproduced by aphids is known to be essential for stimulating syrphid oviposition(Scholz & Poehling 2000).

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RESULTS

Field ExperimentsThe results of field observations of 2007 in potato fields can be summarised bythe following:

1) Aphis nasturtii was the most observed aphid species, followed byMacrosiphum euphorbiae.

2) In the potato crop the eggs and larvae of hoverflies were the most numer-ous of all natural enemies, followed by lacewings (esp. Chrysoperla spp.). Ladybeetles (Coccinella spp.), predatory bugs (Orius spp.) and parasitoids (Aphidiusspp.) were of minor importance.

3) Among the adult hoverflies visiting the flowering strips Melanostomamellinum and E. balteatus were the most numerous.

4) The mean total aphid density was always below 1 per (compound) leaf;well below economic threshold levels. No pesticide applications were needed.

5) In June and July the aphid densities were never higher than 0.3 per leaf,clearly lower than in the previous two years on these farms. The predator-to-prey ratio was clearly higher compared to previous years, indicating an increas-ing effectiveness of local natural enemy populations since field margins andintegrated pest management schemes were incorporated.

6) Only at the field with the highest aphid numbers spatial relations withfield margins were observed that are similar to the results from the previous year(LTO 2007).

Laboratory experiments

Syrphid survival on different plant speciesSurvival studies show that for four out of 16 plant species tested (Table 1),longevity of E. balteatus males and/or females is not significantly higher thanwith water only (1.9 days at 22°C). For the other 12 selected species, longevity ishigher than with water only, with the averages ranging from 8 to 14 days. Theperformance of this group of plants did not differ significantly (P>0.05) com-pared to the 1M sucrose control, which resulted in an average of 11.9 days.

Syrphids forage especially on plants with easily accessible nectar. The flower-ing plants tested as food plants for E. balteatus are hence broadly represented in anumber of plant families, particularly Asteraceae, Apiaceae and Fabaceae. Evenwithin these selected families, plant species strongly differ in their suitability asfood source (Table 1). Within the family of Apiaceae, Carum carvi clearly under-performed, whereas within the family of Asteraceae, Calendula officinalis wasunsuitable as food source for this syrphid species. These differences are associat-ed with the compatibility of the floral morphology with the size and structure ofthe insect mouthparts. Within Fabaceae, only Vicia sativa is suitable, but this isexplained not by differences in floral attributes but by the presence of extrafloralnectaries on the stipulae, which are readily accessible for hoverflies.

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The importance of pollen, nectar and honeydew for syrphid longevityA preliminary assessment of adult E. balteatus survival when reared exclusivelyon pollen shows only half the life span of one reared on sucrose.

The relative importance of sucrose (a substitute for nectar for this experi-ment) and honeydew as food sources are shown in Figure 1. A strong positiveimpact of honeydew on adult longevity was observed when compared to a treat-


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Table 1. The effect of flowering plants on the longevity of E. balteatus compared to acontrol with water only (P<0.05). Plant species in order of decreasing syrphid longevi-ty.

Food plant species Plant family n Impact on longevityPastinaca sativa Apiaceae 16 +Borago officinalis Boraginaceae 44 +Daucus carota Apiaceae 10 +Matricaria recutita Asteraceae 15 +Fagopyrum esculentum Polygonaceae 49 +Achillea millefolium Asteraceae 15 +Vicia sativa Fabaceae 33 +Malva sylvestris Malvacaeae 12 +Centaurea cyanus Asteraceae 39 +Coriandrum sativum Apiaceae 46 +Foeniculum vulgare Apiaceae 21 +Chrysanthemum segetum Asteraceae 22 +/0Medicago sativa Fabaceae 18 0Vicia cracca Fabaceae 13 0Calendula officinalis Asteraceae 20 0Carum carvi Apiaceae 12 0

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Figure 1. Effect of honeydew in different combinations with other food sources on adultlongevity (22°C and 80% RH) ANOVA indicates significant effects of food-source(P<10-6) and prey (aphid/honeydew) (P=0.003) and their interaction (P=0.045).

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ment with water only (P<10-6). A parallel result was seen when honeydew wasadded to a treatment with flowering buckwheat (Fagopyrum esculentum) (P=0.01).This indicates that honeydew is used by the adult syrphids and directly impactson their longevity. In the presence of sucrose, however, no effect of honeydewis observed. The lack of synergy in the latter combination can be explained byboth substances functioning mainly as energy sources.

Syrphid reproduction and aphid controlIn a small scale experiment egg production is observed for four out of eight sur-viving females in cages with cabbage aphids on Brussels sprout plants and pollenand nectar providing plants (of four different plant species – buckwheat, corn-flower, coriander and borage). The results (Fig. 2A) show that oviposition com-mences one week after emergence of females from pupae and continues until theend of the third week.

The larvae of viable eggs normally emerge within 2 to 3 days after oviposi-tion. The predator-prey population dynamics on cabbage plants show that aphidinfestation is effectively compromised and diminishes substantially in the pres-ence of syrphid larvae (Fig. 2B). In their absence, the pest population increasesunabated to an extent that the cabbage plants no longer can sustain them.Wilting eventually ensues, marking the subsequent decline in aphid numbers(Fig. 2B).


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The observed population dynamics indicate that the offspring of a singlefemale syrphid can check the growth of a colony of about 500 aphids during 3weeks. Coupled with controlling aphid infestation we witness a correspondingdecline in larval predator numbers.

DISCUSSIONThe complexity of the trophic interactions under semi-natural conditions neces-sitated that we take a closer look at the mechanisms behind field observations.We came to the following conclusions: (1) both pollen and nectar are essentialfor syrphid survival and reproduction, (2) diverse field margins are important inproviding a stable supply of food for syrphids and other natural enemies, (3)selected plant species strongly differ in their suitability as food sources for thesyrphid E. balteatus, even within selected families, (4) aphid honeydew is animportant supplementary food source, and (5) E. balteatus is potentially an effec-tive predator of aphids. These conclusions are briefly discussed below.

The availability of plant provided food is inevitable for the completion of thelife cycle of natural enemies such as syrphids. The females in particular requirepollen during the adult stage. Pollen is primarily a source of proteins and aminoacids. In addition, it contains lipids, steroids and carbohydrates (Wäckers et al.2007).

Diverse field margins provide additional food for sustaining a viable aphi-dophagous natural enemy population. Plant species that flower at differenttimes of the season offer a continuous availability of floral resources in the vicin-ity of crop fields throughout the farming season, facilitating continued presenceof natural enemies as long as pest control is required in the field.

Plant species strongly differ in their suitability as food sources for E. baltea-tus. Suitability is partially determined by the morphological match between themouthparts and flowers. The short tongue of E. balteatus as compared to otherflower visiting insects, limits it to plant species with short or open corollas (vanRijn & Wäckers 2007). But even within the group of plants with accessible nec-tar, some species appeared unsuitable, indicating that other factors such as foodquality are important as well. When nectar is not available, syrphids may relyon the more easily accessible pollen. Preliminary experiments, however, indicatethat pollen is not equally significant in sustaining adult longevity.

Syrphid oviposition in or near aphid colonies is advantageous for the femalethat benefits from the available honeydew. This can otherwise be seen as a pru-dent strategy to avail emerging larvae easy access to aphids. Still, aphid honey-dew appears to have a strong impact on adult longevity. As a supplementaryfood source it is valuable not only for being rich in carbohydrates, but can pro-vide some amino-acids as well (Wäckers et al. 2008). Hogervorst et al. (2007)showed that honeydew specific sugars can be traced in field-collected E. baltea-tus. Even second and third instar larvae of syrphids appear to feed on honeydew


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and sucrose too (Bargen et al. 1998). More research in the reproductive physiolo-gy of E. balteatus could probably provide us with valuable information about theimportance of honeydew in the predator-prey interrelationship.

The practical implications of the above observations for biological control canbest be viewed against the background of three sets of realities: 1) A good naturalenemy does not exclusively rely on a single prey species under field conditions.Being a facultative omnivore that feeds on the nymphs of scale insects (Bargen etal. 1998) and on other homopterous taxa (personal field observations), E. balteatuscan survive on alternative preys; 2) Aphids are under natural conditions ephemer-al resources due to their susceptibility to a wide range of biotic and other influ-ences such as predation, parasitism, declining host qualities, changes in weatherand dispersal (Bugg 1992). Polyphagy apparently has suited them for their rela-tive success in agrarian environments. A successful aphid pest management musttherefore embrace the use of efficient predators. Whereas the relatively slowernatural enemies such as coccinelid beetles, chrysophids and anthrocorids may bepreferred in augmentative bio control (ABC) in covered crops, the highly mobileand effective E. balteatus appears to be suitable for conservation bio control(CBC) under open field conditions; 3) The presence of natural enemies in fieldmargins should be well synchronised with the earliest onset of crop infestationsin order to realise the practical benefits of timely biological pest control.

Acknowledgments The authors would like to thank Jurgen Kooijman and AnitaDulos for their valuable contributions to laboratory experiments and André Kamp forpractical assistance in field monitoring. We are particularly grateful to the departmentof Multitrophic Interactions (MTI) of the NIOO for providing the opportunities foraccomplishing this work.

REFERENCESBargen, H., Sandhof, K. & Poehling, H.-M. 1998. Prey finding by larvae and adult

females of Episyrphus Balteatus. Entomologia Experimentalis et Applicata 87 (3): 245-254.

Bugg, R.L. 1992. Habitat manipulation to enhance the effectiveness of aphidophagoushoverflies. Sustainable Agricultural Technical Reviews. University of California.

Hogervorst, P.A., Wäckers, F.L. & Römeis, J. 2007. Detecting nutritional state and foodsource use in field-collected insects that synthesize honeydew oligosaccharides.Functional Ecology 21 (5): 936-946.

LTO. 2007. Rapportage FAB 2006. Functionele AgroBiodiversiteit. www.lto.nl/fab.Rijn, P.C.J. van & Smit, J.T. 2007. Zweefvliegen (Diptera: Syrphidae) voor de natuur-

lijke bestrijding van bladluizen. Entomologische Berichten 67(6): 253-256.Rijn, P.C.J. van & Wäckers, F.L. 2007. Bloemrijke akkerranden voeden natuurlijke vijan-

den. Entomologische Berichten 67(6): 226-230.Schmidt M.N., Thewes U., Thies, C. & Tscharntke, T. 2004. Aphid suppression by

natural enemies in mulched cereals. Entomologia Experimentalis et Applicata 113:87-93.


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Scholz, D. & Poehling, H.-M. 2000. Oviposition site selection of Episyrphus balteatus.Entomologia Experimentalis et Applicata 94(2): 149-158.

Wäckers, F., Rijn, P. van & Heimpel, G. 2008. Honeydew as a food source for naturalenemies: Making the best of a bad meal? Biological Control (in press).

Wäckers, F.L., Römeis, J. & Rijn, P.C.J. van 2007. Nectar and pollen feeding by insectherbivores and implications for multitrophic interactions. Annual Review of Ento-mology 52: 301-23.


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Jinze Noordijk, Karlè V. Sýkora & André P. SchaffersWageningen University, Nature Conservation and Plant Ecology Group, PO Box 47,6700 AA Wageningen, The Netherlands, E-mail: [email protected]

Sandy highway verges in the Veluwe region can be of importance tolight demanding, pioneer grassland and heathland communities, char-acteristic of nutrient-poor sandy soils. The last decade however, theopen character of these road verges is negatively influenced byencroaching trees and shrubs. The responsible authorities are now con-sidering restoring the open and low vegetation. Although ample knowl-edge exists about the botanical aspects, information on the importanceof these road verges for arthropod species and the management neededis insufficient. We surveyed reference sites in these verges, especiallyfor ground beetles, ants and spiders, and found many specialized, rare,protected, and red list species. It is apparent that management changesare required, and the reference sites are to be taken as the target situa-tion for the rest of the verges. We formulated a set of management rec-ommendations focused on arthropods. We advise to restore open situ-ations in highway verges by removing trees and shrubs. The nutrient-poor zone with mosaics of low vegetation types should be maximized.If applied along the overall length, verges can potentially become suit-able habitat corridors connecting different nature reserves. When largescale removal of the top soil is performed, high quality vegetation refu-gia should be left intact as a source from which arthropods can spreadagain. Subsequent management should consist of fine scale and phasedmowing and removal of encroachment; yearly a fraction of the vergesshould be managed so that in approximately ten years time all siteshave been managed and the cycle can be repeated.

Keywords: Araneae, Carabidae, corridors, ecology of infrastructure,Formicidae

The Netherlands are covered by an extensive network of highways. Despitemany detrimental effects of roads themselves (Spellerberg 2002), the accompa-nying verges may contribute to the diversity of flora and fauna (Sýkora et al.1993, Noordijk et al. 2005). When compared to the surrounding agricultural orurban areas, many plant and animal species can be found to live in the unfertil-

The conservation value of sandy highwayverges for arthropods – implications formanagement


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ized semi-natural plant communities in verges. For traffic safety and for vegeta-tion management most highway verges are yearly mown. This way, encroach-ment by shrubs and trees and succession to productive tall plant communities isprevented. Plant communities with a low standing biomass, like hay meadows,often bear relatively high biodiversity (Parr & Way 1988, Schaffers 2002). Thesecommunities declined considerably in the Netherlands and have become quiterare. From a conservation point of view, nutrient-poor situations merit particu-lar attention, as they harbour relatively many threatened and/or rare species(Kleukers et al. 1997, Turin & Heijerman 1997, Mabelis 2004).

In the Veluwe region, four highways (the A1, A12, A28 and A50) were con-structed between 1950 en 1980. As these roads and accompanying verges were con-structed on the original nutrient-poor sandy soils it was assumed that little man-agement was needed. Consequently, in general a mosaic of nutrient-poor grass-lands, heathlands, grey hair-grass vegetation, trees and shrubs developed. Thesesites are usually bordered by a strip of nutrient-rich soil with grassy vegetationright next to the asphalt and by forest on the other side (Fig. 1). These vergesbecame a suitable habitat for organisms like reptiles and lichens (Lemmens 1984,Zuiderwijk 1989). As such, they provided welcome extensions of a threatenedopen landscape type. Heathlands and drift sands are of high conservation concern(European Community 1992), but they are declining severely in surface and arehighly fragmented in the Netherlands (Van Duuren et al. 2003). It has often beensuggested that these particular highway verges could function as movement orhabitat corridor (Bekker & De Vries 1992, Vermeulen 1994, Smeenge et al. 2005,Keizer et al. 2006). When un-interrupted suitable vegetation is provided betweenseparate nature reserves, the corridor function seems very plausible and recom-mendable (Beier & Noss 1998, Ries et al. 2001, Anderson & Jenkins 2006).

Recently, the increasing dominance of trees and shrubs has become a majorproblem for the open and nutrient-poor vegetation in the highway verges of theVeluwe (Noordijk et al. 2005; Fig. 1). The Road and Hydraulic EngineeringDivision (at present: Centre for Traffic and Navigation) from the Ministry ofTransport, Public Works and Water Management aims to restore the open andheathy conditions of the verges on a large scale. In this scope it is important toobtain more insight in:

1) the importance of remaining high quality verges for specialized arthropodspecies, which therefore act as target situations for the verges to be restored;

2) the presence of protected or red list species. As large scale management ispossibly needed, it is important to know about the presence and location ofspecies from the national Flora and Fauna legislation and red lists in order toavoid their disturbance;

3) the need for a management specifically aiming at arthropod conservation;4) the species most suitable for monitoring the success of the restoration

measurements.


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In this contribution we present the main results of our arthropod surveys inthese particular highway verges and propose some easily recognisable species tobe used for monitoring restoration or management success. We conclude with arestoration plan followed by recommendations for a consequent managementscheme.


Study areas – reference sitesWe selected six relatively high quality sites with nutrient-poor and low vegeta-tion patches alongside highways on the Veluwe to determine species composi-tion of ground beetles, ants and spiders (Noordijk 2005, Noordijk & Boer 2007,Noordijk et al. in press a). These sites were located close to nature reserves, andwere almost the only locations where open and low vegetation of good qualitycould still be found in the verges. These sites act as references for other verges,indicating the potential arthropod communities after restoration.

J. NOORDIJK, K.V. SÝKORA & A.P. SCHAFFERS

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Figure 1. Example of a sandy highway verge with open vegetation on the Veluwe. Sandlizards, the spider Eresus sandaliatus, the red list ants Anergates atratulus, Formicoxenusnitidulus, Formica pratensis and F. rufa/polyctena, and the highly characteristic carabidbeetles Amara equestris, Olisthopus rotundatus and Harpalus smaragdinus are abundanthere. Photo: J. Noordijk.

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Sampling methodsIn the high quality patches arthropods were sampled in different ways. Mostspiders, ants and ground beetles were collected using pitfall traps (ø 100 mm, halffilled with a 3% formalin solution, a sample is collected during five months peryear, Noordijk et al. in press a). Three sites were sampled for four consecutiveyears, while the three other sites were sampled only one year. In total, this con-stitutes of 99 pitfall trap samples being collected. In three of these sites we alsoplaced four window traps for one year (window 80-by-60 cm, positioned at 1 mabove the ground, gutter under window 18 cm broad and filled with salt solution,sampling for six months) to collect flying ground beetles and winged sexuals ofants. Some additional sight observations were done on ant species. For eachobserved species we noted whether we found it occasionally (only at one loca-tion or at two locations but in low numbers) or frequently (at two locations inquite high numbers, or at more than two locations).

Species of conservation concernIt is very difficult to make reliable and well funded statements on the conserva-tion status of arthropods, due to the many species, the relatively few data, theinfluences of observational biases and the lack of monitoring programs (New1999, Stewart & New 2007, Conrad et al. 2007). No national conservation policyexists for spiders and ground beetles, and insufficiently so for ants. Therefore –from the pool of collected species in the highway verges – we ourselves designat-ed arthropods of conservation concern in the Veluwe region to illustrate the conser-vation value of the studied sites.

For ants we used three criteria, each of which indicates a significant conser-vation concern: species from the international red list of threatened species ofthe IUCN (2006), species that are protected by national law (Flora and FaunaAct, Ministry of Agriculture, Nature and Food Quality 2002), or specializedspecies which are highly characteristic of nutrient-poor and open plant commu-nities (pers. comm. P. Boer). For the ground beetles we used two criteria: specieshighly characteristic of heath/drift sand areas or other low nutrient-poor vege-tations on sand (Turin 2000), or species from habitats with low vegetation show-ing a declining trend in the Netherlands (Desender & Turin 1989). For the spi-ders we used two criteria: species restricted to heath/drift sand areas or otherlow nutrient-poor vegetations (according to Bauchhenss 1990, Hängi et al. 1995,Roberts 1998, Bonte et al. 2003), or species from habitats with low vegetation,which are rare in the Netherlands (Roberts 1998).

Finally, for each of the three arthropod groups we propose some specieswhich can easily be used for monitoring the success of restoration or manage-ment. The selection was done according to guidelines presented by Noss (1990):the species to be monitored should be vulnerable and indicative (i.e. largelyrestricted to the habitat to be monitored and therefore vulnerable to changes inthis habitat), easily recognisable, and preferably an umbrella species (i.e. requires


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a relatively large area or is a poor disperser, and therefore is expected to indicatethe presence of other species).

RESULTS

Ants (Hymenoptera: Formicidae)A total of thirty-five ant species was observed in the highway verges (Noordijk& Boer 2007). Among these, eighteen species (or twenty if Formica lusatica andF. rufibarbis, and F. rufa and F. polyctena are considered separate species) of con-servation concern could be recognized (Appendix I), including Myrmica schenck-ioides; a new species found in a highway verge (Boer & Noordijk 2005). Togetherthese species form a very rich species pool, highly characteristic of heathlandand drift sand. We actually found most of the ant species known to occur else-where in the Veluwe region; only six ant species could not be found in any ofthe studied verges. Some sampled species are rarely observed in the Veluwe, likethe thermophilous species Tapinoma ambiguum and Strongylognathus testaceus(Van Loon 2004), but were quite frequently collected in the highway verges.

Formica pratensis, F. rufa/polyctena and F. truncorum are protected by thenational Flora and Fauna act. This means that it is not permitted to disturb boththe nests and the surrounding habitat without legal exemption. We found noless than five ant species mentioned on the international red list of threatenedspecies of the IUCN; i.e. Formicoxenus nitidulus, Myrmica hirsuta, Anergates atrat-ulus, Formica pratensis, and F. rufa/polyctena. Three of these, F. nitidulus, M. hirsu-ta and A. atratulus, are socially-parasitic species. They have small colonies situ-ated in the nests of other ant species.

To monitor the effects of restoration and management measures, the focusshould be on the protected and red list species. The protected species – theFormica’s or red wood ants – are easy to find, this is however not the case for thethreatened socially parasitic ants. These socially parasitic species live in thenests of other ants, and an appropriate practise to protect these species is to pro-mote high densities of the host species (Boer & Noordijk 2004, Mabelis 2007).The nests of Tetramorium caespitum (Fig. 2) can host small colonies of the threat-


Figure 2. Tetramorium caespitum, thehost species of two rare socially para-sitic ant species. Photo: Th.Heijerman.

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ened A. atratulus, but also of the rare Strongylognathus testaceus. Formicoxenusnitidulus makes small colonies in nest mounds of red wood ant species. Both thenest mounds of red wood ant species and the dug up sand of T. caespitum nests –often deposited in circular way around the entrance – are easy to find (Fig. 3).These two species offer suitable monitoring targets. Before management prac-tices are applied, the nests of red wood ant species and of T. caespitum should beinventoried and located. The persistence of red wood ant nest mounds indicatesthe caution taken during previous management activities, but says not much onthe xero-thermic habitats. The un-interrupted presence of T. caespitum nests


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Figure 3. Nest mounds of Formica-species (in this case F. rufa) and nest entrances ofTetramorium caespitum colonies are easy to find. Photos: J. Noordijk.

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alongside the highway indicates the existence of a – nutrient-poor and open –corridor for two rare socially parasitic ant species. Unfortunately, the nests ofthe host species of Myrmica hirsuta – M. sabuleti and M. lonae – are difficult tofind.

Ground beetles (Coleoptera: Carabidae)In total, seventy-nine ground beetle species were sampled in the studied verges.Many species, thirty-two, could be labelled as of conservation concern(Appendix II). These include habitat specialists, for example Amara equestris,Calathus ambiguus, Bradycellus ruficollis and Harpalus servus, and beetles that arein decline in the Netherlands, for example Harpalus neglectus, H. anxius (Fig. 4),H. solitaris and Laemostenus terricola (Turin 2000). The species rich carabid com-position of the verges clearly characterises the mosaics of heathland, nutrient-poor grasslands and forest edges (see also Vermeulen 1993). For many species,recently emerged adults (so-called tenerals) were collected, indicating the impor-tance of verges for their reproduction; verges appear not only to be temporaryrefugia but also reproduction sites.

Almost every location harbours ground beetles. Many species are very rapidcolonisers of newly created sites (Haeck 1971). Quick colonisation will almostcertainly take place at restoration sites in the highway verges as well. However,the carabid species that really need ecological corridors in fragmented landscapes


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Figure 4. The ground beetle Harpalus anxius shows a decreasing trend in theNetherlands. Photo: Th. Heijerman.

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are habitat specialists which are incapable of dispersal by flight (De Vries et al.1996, Den Boer 1990). During our inventories we found several of these species:Carabus arvensis, Poecilus lepidus, Notiophilus germinyi, Olisthopus rotundatus andMasoreus wetterhallii. These non-flying specialist species of heathy vegetationsare good indicators of site quality and accessibility for carabids. Their appear-ance will therefore reflect the success of restoration or management practises.Pitfall trapping during spring and summer is an appropriate method to establishthe presence of these species before and after the practises. Their identification,however, asks for some entomological expertise.

Spiders (Araneae)We found seventy-four spider species at the six highway verge sites (only non-webbuilding spiders belonging to the families Atypidae, Eresidae, Gnaphosidae,Liocranidae, Corinnidae, Clubionidae, Miturgidae Zoridae, Thomisidae,Philodromidae, Salticidae, Lycosidae, Pisauridae, Agelenidae, Mimetidae andTheridiidae were identified). Thirty-seven species can be regarded as of conser-vation concern (Appendix III, Fig. 5), since they are very characteristic of nutri-ent-poor situations or rare in the Netherlands. These include the spectacularEresus sandaliatus, which in the Netherlands is restricted to the southern part ofthe Veluwe (Van Helsdingen 2005). In addition, some very rare species werecollected like Micaria silesiaca, Kishidaia conspicua, Phaeocedus braccatus andOzyptila scabricula (see also Noordijk 2005).

Figure 5. The spider Aelurillus v-insignitus; a common but highly characteristic speciesin the studied verges. Photo J. Lissner.


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Spider species are relatively difficult to identify, due to the high number ofclosely resembling species (Van Helsdingen 1999). However, one species seemsto fulfil all requirements that would make it suitable for monitoring purposes:Atypus affinis. This mygalomorph spider is characteristic of largely undisturbed,mosaics of nutrient-poor vegetations. It is a large species (females up to 18 mm)and probably has a relatively low dispersal capacity (Pedersen & Loeschcke2001). Therefore, the presence of this spider most likely indicates the accessibil-ity of the site for spiders and that it is large enough to harbour many otherspecies as well. In addition, it is easily recognisable (Fig. 6) and widespread onthe Veluwe (Noordijk 2005, Tutelaers 2008). Inventory of this species – directlyin the field or using pitfall traps – should take place in late summer, when themales leave their underground tubular webs in search of females. Another suit-able spider to be monitored is Pardosa monticola. This species is restricted to xero-thermic nutrient poor vegetation, a relatively poor disperser (Bonte et al. 2001),and during the early summer easy to find because of its conspicuous huntingbehaviour. This spider knows, however, some closely resembling species and istherefore not as easily identifiable.


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Figure 6. The spider Atypus affinis is an appropriate species to monitor restoration andmanagement success. Photo Th. Heijerman.

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DISCUSSIONThe observed species and their conservation concern indicate that entomologicalvalues of the selected sites in highway verges are very high and should not beneglected. The habitat characteristics of the studied verges indicate that poten-tially all highway verges on the Veluwe can be restored into suitable habitatareas as well. To stimulate the presence of species with high conservation con-cern, open and nutrient-poor vegetation should be restored and the complete areaof highway verge should be managed.

Our results do not suggest that highway verges bear some intrinsic character-istics that make them unsuitable for a wide array of specialized species of heath-land and drift sand situations. However – as could be shown in a previous study(Noordijk et al. in press a) – arthropod species composition in selected grey hair-grass vegetation patches (Violo-Corynephoretum; pioneer vegetation on acidicsand) in highway verges differs from nearby nature reserves, and some carabidsand spiders occurring in the reserves are lacking in the verges. In particular,some characteristic species preferring sites with bare sand were missing. Also,characteristic carabid species with large body-size were less numerous in vergesthan in the nature reserves, probably indicating the patches in the verges to betoo small for some species. Also the vegetation in the verges, even when of thesame type, differed from the vegetation in the nature reserves. In the pioneervegetation of the verges the proportion of bare sand appeared to be lower, whilethe cover of herbs and trees was higher. Concluding from the prevailing speciesand the vegetation characteristics, the last remaining grey hair-grass patches inthe verges are already of lower quality than in the nature reserves (see alsoAngold 1997). Likely key factors capable of increasing the presence of specialistspecies in the verges are the increase of openness of the sward, the prevention ofencroachment by tall herbs, grass, trees and/or shrubs, and extension of the totalsurface of suitable open vegetation (see also Morris et al. 1994).

Many species will benefit quickly from restoration practises. Mostly thisconcerns the good disperses – e.g. small ballooning spiders and insects with goodflight capacities – and species which are not so much habitat specialists(Tscharntke et al. 2002). An example of a quickly colonising species is Oedipodacoerulescence (Fig. 7), a red list grasshopper which can fly over considerable dis-tances. This grasshopper was found in a verge one year after sod-cutting and inanother verge one week after the burning away of the vegetation. Most wingedsexuals of ant species probably also fly considerable distances to start newcolonies (Duelli et al. 1989). For example, we found Lasius psammophilus – aspecies that shows a decreasing trend in the Netherlands – in suitable habitat inthe middle of a highway junction, a site completely isolated from other habitatsby asphalt.

However, many other species might have more problems to reach (far-away)highway verge sites, for example flightless species or habitat specialists.


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Although most spiders can disperse quite well as spiderlings, there are indica-tions that specialist species tend to balloon to a lesser extent, since their risk toland in unsuitable habitat is higher than for generalist species (Bonte et al. 2003).Sensitive and poorly dispersing ants are likely to be found under the socially par-asitic species. These species often have few winged sexually active individualsper year, and need high densities of the host ant species to start new populations,while the chance of starting a new colony is likely to be lower than in otherspecies. Among the carabids, stenotopic species unable to fly are poor dispersers.Species like these would benefit greatly from connections consisting of appro-priate vegetation – and the appropriate host ant species for the socially parasiticants – to reach suitable sites.

Possibly, our dataset is biased towards higher species diversity. The sampledhighway verges were quite close to nature reserves; this possibly had a positiveeffect on species diversity (Vermeulen 1994, Koivula 2005). On the other hand,in the Veluwe region almost all verges are close to natural heathland/drift sandareas. A landscape design where many connections are created between naturereserves and close-by verges is feasible, and may therefore very well contributeto the realisation of local ecological networks (Keizer et al. 2006). Since high-ways are one of the main causes of fragmentation of the landscape, it seems logicto construct and manage the accompanying verges in such a way that they can


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Figure 7. The red-list grasshopper Oedipoda coerulescence is a fast immigrant of newlycreated sites with bare sand. Photo: J. Noordijk.

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counteract fragmentation. Such ecological compensation seems well possiblebecause verges form an extensive network, stretch over long distances and afterrestoration and under right management can harbour many characteristicspecies – as shown in the current study. However, two conditions should be metin order to function as a corridor. First, connections with nature reserves shouldfacilitate species to reach the verges. A second – important – condition is that theverges should be managed as habitat corridor. This means that the entire stretchof the road (up to the next nature reserve) should consist of high quality habitatfor xero- and thermophilic arthropod species. Sites with high vegetation or largeareas with closed swards are probably already barriers for some habitat special-ist (Vermeulen 1994).

Implications for managementIt has already been mentioned that nutrient-poor habitats deserve much atten-tion in Dutch nature conservation practices. The nutrient-poor vegetations inthe Veluwe region are threatened and deserve most attention (Houdijk et al.1993, Riksen et al. 2006). The heathy situations here studied are European targetvegetations (European Community 1992), and Veluwe verges potentially pro-vide extensive areas of open habitats. Our inventories make it very clear that theverges should be managed to maintain the open and nutrient-poor vegetationthat can still be found occasionally, and that it is highly advisable to restore thishabitat in all other densely vegetated verges. Although, a temporal and spatiallyphased management is more profitable for many arthropod species (Morris2005), the initial restoration takes some extensive measures. This way, a species-rich and highly characteristic arthropod fauna, including protected and threat-ened species, will be preserved and extended. Obviously, there are also interest-ing species to be found in the forest edge and forest. However, these habitats willalways be amply available; forest is abundant along the verges and the forestedge will merely be shifted further from the road. The total area of forests habi-tats are actually increasing and gaining in quality in the Netherlands (Reemer etal. 2003, Van Duuren et al. 2003).

To restore and maintain the highway verges as important habitat for xero-and thermophilic arthropods, we suggest (see also Bell et al. 2001, Van Turnhoutet al. 2001, Noordijk et al. in press b):

> An extensive restoration of the open, nutrient-poor and sandy situations inhighway verges in the Veluwe region. Encroaching trees and shrubs should beremoved over the full width of the verges. At selected sites the topsoil should beremoved.

> Leaving sites still containing nutrient-poor vegetation patches intact. Fromhere, arthropod species can colonize other parts of the verges.

> Creating connections between nature reserves and the adjacent verges. Inthis way, verges can be used as an extension of existing nutrient-poor habitatand even function as ecological corridors between nature reserves.


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> A periodical management scheme with relatively small scale measures tobe implemented subsequently. Each year, encroaching trees and shrubs shouldbe removed in a different part of the total area of highway verge in such a waythat all sites receive this treatment once every eight to ten years. The manage-ment cycle can be restarted after this period.

> The nutrient-richer grassy vegetation close to the asphalt to be mown reg-ularly – preferably every autumn – and the cuttings to be removed.

> During reconstructions of road verges (or construction of new ones) theoriginal soil or soil from local sources to be used. The application of a nutrientrich top-soil – used during some previous highway reconstructions – is highlyinadvisable.

> ‘Red wood ant’ nest mounds – which are legally protected – and the sur-rounding plants should be avoided during management. Before management theeasily recognizable nest mounds should be inventoried.

Acknowledgments Many persons helped in some way during the presented study:Maurits Gleichman, Louis de Nijs, Theodoor Heijerman, Roy Morssinkhof, Peter-JanKeizer, Rikjan Vermeulen and Frits Hollander. We owe special thanks to Peter Boer forthe identification of the ants and the useful discussions concerning these insects. Petervan Helsdingen and Theodoor Heijerman made useful comments on an earlier versionof this article.

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APPENDIX IAnts (Formicidae) of conservation concern in highwayverges on the veluweThe reason for the conservation concern status is given: red list (IUCN), pro-tected (FF) or characteristic (char) thermo- and xerophilic species. Indicated iswhether we encountered the species frequently (freq) or occasionally (occas).

Species Conservation concern Frequent or occasionalAnergates atratulus (Schenck) IUCN, char occasFormica cunicularia Latreille char freqFormica lusatica Seifert / rufibarbis Fabricius char freqFormica pratensis Retzius IUCN, FF freqFormica rufa Linnaeus / polyctena Förster IUCN, FF freqFormica truncorum Fabricius FF occasFormicoxenus nitidulus (Nylander) IUCN occasLasius psammophilus Seifert char freqMyrmica hirsuta Elmes IUCN, char occasMyrmica lonae Finzi char freqMyrmica rugulosa Nylander char occasMyrmica sabuleti Meinert char freqMyrmica schencki Viereck char freqMyrmica schenckioides Boer & Noordijk char occasMyrmica specioides Bondroit char freqPonera coarctata (Latreille) char occasStrongylognathus testaceus (Schenck) char freqTapinoma ambiguum Emery char freq


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APPENDIX IIGround beetles (Carabidae) of conservation concern inhighway verges on the VeluweThe reason for the conservation concern status is given: characteristic (char)thermo- and xerophilic species or species which show a decreasing trend (decr)in the Netherlands. Indicated is whether we encountered the species frequently(freq) or occasionally (occas).

Species Conservation concern Frequent or occasionalAmara consularis (Duftschmid) char occasAmara convexior Stephens decr occasAmara equestris (Duftschmid) char freqAmara lucida (Duftschmid) char, decr occasAmara ovata (Fabricius) decr occasBradycellus ruficollis (Stephens) char, decr freqCalathus ambiguus (Paykull) char, decr occasCalathus erratus (C.R. Sahlberg) decr freqCalathus micropterus (Duftschmid) char occasCarabus arvensis Herbst char occasCicindela campestris Linnaeus char, decr occasCicindela hybrida Linnaeus char, decr occasHarpalus anxius (Duftschmid) char, decr freqHarpalus griseus (Panzer) decr occasHarpalus latus (Linnaeus) char, decr freqHarpalus neglectus Serville char, decr freqHarpalus rufipalpis Sturm char, decr freqHarpalus servus (Duftschmid) char, decr freqHarpalus smaragdinus (Duftschmid) char, decr freqHarpalus solitaris Dejean char, decr freqHarpalus tardus (Panzer) decr freqLaemostenus terricola (Herbst) char, decr occasMasoreus wetterhallii (Gyllenhal) char freqNebria salina Fairmaire & Laboulbene char freqNotiophilus aesthuans Motschulsky char occasNotiophilus germinyi Fauvel char freqOlisthopus rotundatus (Paykull) char freqPoecilus cupreus (Linnaeus) decr freqPoecilus lepidus (Leske) char, decr occasPterostichus diligens (sturm) char freqPterostichus quadrifoveolatus Letzner char freqSyntomus foveatus (Geoffroy) decr freq


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APPENDIX III

Spiders (Araneae) of conservation concern in highwayverges on the veluweThe reason for the conservation concern status is given: characteristic (char)thermo- and xerophilic species or rare species (rare) in the Netherlands. Indicatedis whether we encountered the species frequently (freq) or occasionally (occas).

Species Conservation concern Frequent or occasionalAelurillus v-insignitus (Clerck) char freqAgroeca proxima (O.P.-Cambridge) char freqAlopecosa barbipes (Sundevall) char freqAlopecosa cuneata (Clerck) char freqAlopecosa fabrilis Clerck) char freqAtypus affinis Eichwald char freqCheiracanthium erraticum (Walckenaer) char occasCheiracanthium virescens (Sundevall) char occasClubiona diversa O.P.-Cambridge char occasDrassodes cupreus (Blackwall) char freqDrassodes pubescens (Thorell) char occasDrassyllus pusillus (C.L. Koch) char freqEresus sandaliatus (Martini & Goeze) char, rare occasEvarcha arcuata (Clerck) char, rare occasKishidaia conspicua (L. Koch) rare occasMicaria dives (Lucas) char freqMicaria fulgens (Walckenaer) char freqMicaria silesiaca L. Koch char, rare freqOzyptila scabricula (Westring) char, rare occasPardosa monticola (Clerck) char freqPellenes tripunctatus (Walckenaer) char, rare freqPhaeocedus braccatus (L. Koch) char, rare freqPhlegra fasciata (Hahn) char freqPhrurolithus festivus (C.L. Koch) char freqSteatoda albomaculata (De Geer) char occasSteatoda phalerata (Panzer) char freqTalavera petrensis (C.L. Koch) char, rare freqTegenaria agrestis (Walckenaer) char freqThanatus formicinus (Clerck) char, rare occasTibellus oblongus (Walckenaer) char occasTrachyzelotes pedestris (C.L. Koch) char, rare occasXysticus erraticus (Blackwall) char occasXysticus ferrugineus Menge char, rare occasXysticus kempelini Thorell char, rare occasZelotes electus (C.L. Koch) char freqZelotes longipes (L. Koch) char freqZelotes petrensis (C.L. Koch) char freq


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John J. SloggettTussen Beide Markten 45, 9712 CC Groningen, The Netherlands, E-mail:[email protected]

In this paper I review the factors responsible for the evolution ofdietary and habitat specialization in the aphidophagous Coccinellidae.Trade-offs related to foraging and capture efficiency, particularly bodysize, are probably more important in determining diet breadth thantrade offs related to aphid chemistry/nutritional suitability. Habitatspecialization occurs when a species is able to persist on aphids in a par-ticular habitat type for a long period of time: this is more importantthan aphid abundance. The trade-offs underlying habitat specializationremain a matter for speculation. The underlying factors responsible forecological specialization in ladybirds may differ from those in otheraphidophagous insects, due to adult feeding on aphids, which occurs toa much greater extent in ladybirds than in many other aphidophages.

Keywords: Coccinellidae, aphids, specialization, trade-offs

In one sense all aphidophagous ladybirds are specialized because their primaryfood consists of one particular group of homopteran insects. Aphids consideredas a group are ubiquitous and it is unsurprising that ladybirds and members ofother insect groups, such as lacewings and syrphids, have become specialized tofeed on them. Much work on ladybirds has addressed how aphidophagy affectsthe life histories, ecology and behaviour of ladybirds and the adaptations thatladybirds use to effectively exploit aphid prey (e.g. Dixon 2000).

However, even within this already specialized mode of life there is also a fur-ther diversity of specialization. The ladybird Myzia oblongoguttata feeds onaphids on pine trees and other conifers and is rarely found elsewhere (Majerus1994, Klausnitzer & Klausnitzer 1997). By contrast Adalia bipunctata feeds onaphids on a diversity of trees, shrubs and herbaceous plants and consumes a widediversity of aphid species (e.g. Banks 1955, Mills 1981, Majerus 1994). Coccinellamagnifica, on the other hand, feeds on a diversity of aphid species, but its habi-tat, in western Europe at least, is limited to the immediate vicinity of coloniesof wood ants (Formica rufa group) (Sloggett et al. 2002).

We may divide this specialization into two types, diet-related and habitat-

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related. Dietary breadth is the easier of the two to describe, and may be definedas the number of different aphid species that a ladybird species consumes and,more particularly, breeds on in the wild. It is not feasible to accurately quantifythis for most species, particularly generalists, which may feed on tens or evenhundreds of different prey species, and this simple definition glosses over someof the complexities of ladybird diet (see Hodek 1996, Michaud 2005 for reviews);however, within this framework it is generally possible on the basis of knowndata to establish how specialized the dietary breadths of individual species are inrelation to each other.

A clear definition of habitat specialization is yet more elusive. The problemarises because there are many different ways of describing the habitat (Honek1986, Honek & Hodek 1996): we might, for example, use microclimate, planttype or some other feature of the biotic or abiotic environment. Generally thehabitat is described as we perceive it ourselves, based on host plant or some otherimportant feature, for example the presence of ants for myrmecophiles (e.g.Majerus 1994), and this approach is used here. Even more than dietary breadth,it is not possible to provide a quantifiable measure of habitat specialization;however, again it is possible to say how specialized different species are in rela-tive terms.

It is the dispersive adult ladybird that determines the diet and habitat of itslarvae through oviposition preferences. Although the relationship betweendietary and habitat specialization is close, it is not simple. The assumption thatall aphids within the chosen habitat are suitable prey [i.e. (habitat) preferenceand (dietary) performance are linked] may be impossible to test rigorously: evenwithin habitats as perceived by the researcher, both aphid and ladybird microcli-matic and microhabitat preferences may be operating, thus aphids which appearto co-occur with certain ladybird species may not actually do so. However theevidence available does not support an invariable association between preferenceand performance. Although in England A. bipunctata feed and breed on the blackbean aphid, Aphis fabae (e.g. Banks 1955), the aphid is a poor quality prey for thisspecies (e.g. Blackman 1965, 1967a, El-Hariri 1966); this is also the case for someA. bipunctata prey on trees and shrubs (Hodek 1996). Conversely, many aphidswhich ladybird species do not encounter naturally seem to be suitable prey forbreeding and larval growth in the laboratory (e.g. Majerus & Kearns 1989,Kalushkov & Hodek 2001). Although in many cases, dietary specialization is cor-related with habitat specialization, such as in M. oblongoguttata and A. bipunctatamentioned above, there are a number of cases where species that consume a widevariety of prey exhibit restricted habitat preferences, such as C. magnifica andCoccinella quinquepunctata, which despite a catholic diet is limited to shingle habi-tats near water in Britain and nearby areas (Majerus & Fowles 1989, Sloggett &Majerus 2000a). It is noteworthy that the reverse is never the case: habitat gen-eralists do not appear to exhibit dietary specialization.

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In this paper I attempt to some extent to tease apart dietary and habitat spe-cialization and to provide an overview of what we do and do not yet understandabout what factors are responsible for ecological specialization (i.e. both types ofspecialization together) in aphidophagous ladybirds. The division I makebetween diet and habitat is to some extent artificial as an understanding of bothis necessary to fully understand either. In some cases the reader may feel thatsome things which I assign to one section could be equally assigned to the other(for example foraging behaviour which is not only related directly to the preybut also the host plant habitat). This is certainly true: however, I here adopt autilitarian approach, to hopefully provide a clear progression of the narrative.This is also not a comprehensive review of all aspects of ladybird specialization,merely its underlying causes. For further information the reader is referred toseveral older reviews and books (Majerus 1994, Hodek & Honek 1996, Sloggett& Majerus 2000a).

DIET BREADTHWork on dietary breadth in ladybirds, like other insects, is centred around trade-offs: that is, a specialized ladybird that performs very well on aphid A will, as aconsequence, perform badly on aphid B and vice versa. A generalist will performmoderately well on both aphids, but will not perform as well as a specialist onits preferred aphids. Trade-offs are a central tenet of evolutionary biology andunderlie many important biological processes (e.g. Stearns 1992); their role indietary specialization in ladybirds is unquestionable. However the nature ofthese trade-offs, i.e. what exactly is being traded off, has been a matter of debate.

Prey suitability trade-offsA large proportion of the interest in dietary specialization in ladybirds and otheraphidophagous insects has been focused on trade-offs related to diet suitabilityfor growth or reproduction, as a consequence of nutritional differences or aphidchemistry (e.g. Albuquerque et al. 1997, Sadeghi & Gilbert 1999, Rana et al. 2002).There are two underlying reasons pertinent to ladybirds for this. Most work ondietary trade-offs has been concentrated on phytophagous insects, where dietsuitability, especially the effects of plant chemistry, appears to be very impor-tant (Jaenike 1990, Schoonhoven et al. 1998): researchers on predatory insectshave thus tended to wish to draw parallels between their own studies and thoseon phytophages. Additionally in aphidophagous ladybirds diet suitability hasbeen extensively studied from the 1950s on, partly due to the ease of testing dif-ferent aphid diets in the laboratory, and this approach has and continues to formthe basis for the majority of discussion and debate about ladybird diet (Hodek1996, Dixon 2000, Michaud 2005).

Apart from indirect evidence from other aphidophagous groups(Albuquerque et al. 1997, Sadeghi & Gilbert 1999), evidence for suitability-relat-

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ed trade-offs operating in ladybirds has relied on a single study that claimed thattrade-offs in performance related to aphid chemistry were of primary impor-tance in determining dietary breadth (Rana et al. 2002). In this study, two groupsof Adalia bipunctata, a generalist, exhibited increased fitness on one of twoaphids, Acyrthosiphon pisum and Aphis fabae, after selection for enhanced per-formance on that aphid as might occur when dietary specialization evolves.They were also found to perform better on their own prey than ladybirds fromthe other group. However, this result only demonstrates that performance onthe two prey is not positively correlated; performance on the two prey speciesmight be uncorrelated (i.e. there is no trade-off), in which case it is hardly sur-prising that lines selected for enhanced performance on one prey type performbetter on it than ladybirds from the other group. Some evidence that this mayactually be the case can be seen when the final fitness parameters for the select-ed lines on the alternative prey are compared to those of the other selectiongroup from early in the selection process. Here differences in fitness are smallor non-existent rather than being lower in the selected lines, as would be expect-ed were the trade-off necessary for dietary specialization to be operating.

Even were Rana et al.’s method to be modified to include unselected lines,which would act as a better comparator for the selected ones, these are difficultto sustain in the laboratory. Ladybirds provided with both types of prey mightfeed more or less on one of them and inadvertent selection might occur, ulti-mately giving misleading results. A different and arguably more reliableapproach has been used by Ueno (2003) and Fukunaga & Akimoto (2007) bothusing the generalist Harmonia axyridis. In both cases they used a large number ofgenetic lines to examine how performances on two different prey types werecorrelated across these lines. Both Ueno, using Acyrthosiphon pisum and Aphiscraccivora, and Fukunaga & Akimoto, using Aulacorthum magnoliae from two dif-ferent host plants differing in their dietary suitability for H. axyridis, found pos-itive correlations between performance on their two aphids. However in bothcases, only larval characters (development time, growth rate, pupal size) wereexamined. Because different life history components may respond differently todietary treatments (e.g. Sadeghi & Gilbert 1999, Michaud 2005), additional meas-ures of prey suitability, such as adult fecundity, are desirable: only with thesemeasures can the overall fitness on the different prey be calculated.

Circumstantial data arguing against an important role for prey-suitabilityrelated trade-offs in determining diet breadth may also be found in more basicstudies of feeding and natural history in ladybirds. In contrast to phytophagousinsects, there are no ladybirds that are known to be specialized on particularlytoxic prey, as might be expected were prey chemistry to be a significant factorin determining specialization. Furthermore, as discussed above, ladybirds mayperform poorly on aphids that they feed on naturally and well on aphids thatthey do not. While other explanations such as evolutionary lag (Sloggett &


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Majerus 2000a) may be tenable to explain this pattern and some more support-ive feeding data can also be found in the literature (Kesten 1969, Majerus 1993),overall the evidence acquired thus far does not support a major role for prey suit-ability related trade offs playing in dietary specialization. However, because ofthe limitations of studies thus far conducted on prey suitability trade-offs inaphidophagous ladybirds (Rana et al. 2002, Ueno 2003, Fukunaga & Akimoto2007), additional work is required to verify that this is indeed the case.

Prey switchingIt has been suggested that switching between different prey might in itselfimpose a cost and that specialists trade-off a lowered fecundity consequent onremaining with resources for longer with a cost to generalists of regularly chang-ing prey species (Sloggett & Majerus 2000a). It is known from coccidophagousladybirds that such switching can lead to a temporary decrease in fecundity(Hattingh & Samways 1992), although no such experiments have been carriedout with aphidophagous species.

Specialist aphidophages are known to be found associated with lower densi-ties of aphids than generalists (Sloggett & Majerus 2000a, b). However, morerecent work on body size and on foraging adaptations in specialist ladybirds (seenext sections) suggests that there is no intrinsic cost to this association, but thatit arises due to increased efficiency; thus one side of this putative trade-off doesnot appear to be supported. Additionally, while it remains possible or even prob-able that generalists do suffer some cost of frequent prey switching, it is ques-tionable whether within their lives individuals of even generalist ladybirdsswitch prey sufficiently often that a major cost would be incurred: although gen-eralist species may feed on many types of aphids, not all individuals feed on allprey types during their lives. Thus, while prey switching is certainly of interestin the context of prey specialization, it seems unlikely that in itself it can explainthe evolution of specialized feeding in aphidophagous ladybirds.

Finding and catching prey: foraging adaptationsIn spite of the wealth of data in the literature describing how ladybirds locateand catch aphid prey (reviews in Hodek 1996, Dixon 2000), this aspect of lady-bird feeding ecology has received relatively little attention as a potential factordetermining specialization until relatively recently. This is perhaps surprising:almost thirty years ago, Mills (1979) argued that the suitability of an aphidspecies as ladybird prey was most influenced by capture efficiency. Althoughthis work is sometimes cited, the historical adherence to prey suitability and thegreater difficulty of designing and carrying out realistic experiments on preyforaging and capture compared to prey suitability have arguably meant thatMills’ conclusions have not been carried through to their logical endpoint.

Our knowledge of the foraging behaviour of dietary specialists has alsolagged a long way behind that of generalists. Most research on aphidophagous

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ladybirds, including on feeding, has in fact been carried out on a rather narrowrange of generalist ‘model’ species, primarily due to their perceived importancein aphid biological control (Sloggett 2005). However, in a recent paperBethiaume et al. (2007) compared Anatis mali, which feeds predominantly onconifer aphids, with the generalist Harmonia axyridis, which also sometimesfeeds on conifers, on the aphid Mindarus abietinus in Christmas tree plantations.Both adults and larvae of A. mali were more active in foraging for M. abietinus,which generally occurs at rather low densities, and adult A. mali exhibited acharacteristic behaviour of using its head to open up compact conifer needles andpenetrate bursting buds in order to feed on concealed aphids there. In conse-quence A. mali was able to feed earlier on developing aphid colonies than H.axyridis, exhibited greater reproductive synchrony with aphid populations andhad an adult-to-adult reproductive rate three times that of H. axyridis. Both thedistribution of A. mali on the trees and microhabitat ovipositional preferencesalso differed from those of H. axyridis.

Although Berthiaume et al. emphasize the specialist-generalist difference,they also conclude that these differences may be a consequence of a long periodof coevolution between the North American A. mali and M. abietinus comparedto that for H. axyridis, which has only been established in North America forabout 20 years. However, in its native range H. axyridis also predates aphids onconifers (e.g. Sasaji 1980), thus the difference seems more likely to be a case of adietary specialist evolving specific behaviour to more effectively forage withinits more limited prey spectrum. Neither, has a trade-off specifically been identi-fied: to do this it would be necessary to show that on non-conifer aphids, A.mali’s foraging behaviour was less efficient than that of H. axyridis. Nonethelessit is easy to envisage that A. mali’s needle loosening behaviour, for example,might result in a lower foraging efficiency on other types of plants, if aphidswere not concealed in buds.

In addition to Berthiaume et al.’s study, work on myrmecophilous ladybirdshas followed that on chrysopids (Eisner et al. 1978, Milbrath et al. 1993) in iden-tifying a number of behavioural and chemical adaptations to potentially aggres-sive aphid-tending ants that will consequently increase foraging efficiency onant-tended aphids (reviewed in Majerus et al. 2007), but again the underlyingtrade-offs have not been fully investigated. It is clear that unlike prey suitabili-ty and body size (discussed below) no one single trade-off is involved in suchcases as a diverse collection of differing adaptations in behaviour and physiolo-gy are involved; in consequence their evaluation must proceed on a specific case-by-case basis.


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Catching prey: body sizeThe role that body size plays in ladybird specialization has been overlooked fora long period of time, although recent work suggests it may be a key trade-off.For a long time it was considered that there was no relationship between thebody size of aphidophagous ladybird species and the size of the prey that theyconsumed (Dixon & Stewart 1991, Stewart et al. 1991, Dixon 2007). Predator sizewas considered to be exclusively related to prey density, with smaller speciesfeeding and reproducing on lower aphid densities (Dixon 2007). Recently, it hasbeen shown that the relationship between predator size, prey size and prey den-sity is more complex than was previously assumed (Sloggett 2008). Small lady-bird species can feed on low densities of small aphid species, which they can eas-ily catch. However, large ladybird species cannot because there is insufficientaphid biomass to sustain high levels of reproduction for a large ladybird that willconsume more prey; large ladybirds thus require high densities of small aphids.However, large ladybirds can feed on low densities of large aphid species becausethey can easily capture even the biggest aphids, which are much greater energysources than small ones, by virtue of their size. Small ladybirds cannot catch thelarger instars of big aphid species and require high densities of large aphidspecies, where there will be sufficient numbers of small instars that they can eas-ily capture.

This means that while small ladybirds can generally exploit small aphidspecies, they can only sometimes exploit large ones, and that the reverse is truefor large ladybirds. Some degree of size matching between predator and prey isexpected. Specialists that exploit a narrower prey spectrum should more closelymatch the size of their prey than generalists, which will adopt a ‘one-size-fits-all’ medium-sized strategy to exploit a large range of prey sizes. Because a spe-cialist’s body size will be optimized for exploiting its particular prey, specialistscan thus exploit lower densities of their prey than generalists, allowing them toremain associated with fewer prey for longer periods of time (Sloggett 2008).Since body size is known to be heritable in ladybirds (Ueno 1994), it would thuscomprise a clear trade-off influencing specialization.

Because specialists are expected to more closely match the size of their prey,they should exhibit a greater diversity of body sizes than medium sized general-ists. This has been tested for native British aphidophagous Coccinellini, whichare all ecologically well-characterized (e.g. Majerus 1994). After they weredichotomized into dietary specialists and generalists this prediction was con-firmed: the specialist group showed a greater size range with some specialistspecies being bigger and smaller than generalists (Sloggett 2008). Some special-ists do occur within the size range of generalists: however, as dietary breadthobserved today is also a consequence of evolutionary history and phylogeny thisis unsurprising and in no way invalidates the contention that body size trade-offsare significant in determining dietary breadth and specialization (Sloggett 2008).

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Indirect evidence, related to aphid density, also supports the assertion thatbody size and dietary breadth are related. It has been known that specializedladybirds tend to exploit lower densities of aphids than generalists for some time(Sloggett & Majerus 2000a, b). Gagné & Martin (1968) showed that the coniferaphid specialists Anatis mali and Mulsantina picta both tend to exploit aphids onolder pine trees where densities are lower, whereas more generalist Coccinellaspecies utilize younger trees with higher aphid densities. Although the data ismore scattered, the same is also true for European conifer aphid specialists(Sloggett & Majerus 2000b and incl. refs). Differences in foraging behaviour(Berthiaume et al. 2007; see above) certainly also contribute to this pattern; how-ever, it is notable that some conifer aphid specialists (Anatis, Myzia) areextremely large as are a number of common conifer aphids, notably the mem-bers of the genus Cinara. A more phylogenetically controlled example of therelationship between specialization and aphid density comes from Adalia bipunc-tata, which is a broad aphidophagous generalist and its sibling species Adaliadecempunctata, which is more specialized, feeding and breeding almost entirelyon tree and shrub aphids (e.g. Hemptinne & Naisse 1988, Majerus 1994). In thecourse of characterizing the habitat preferences and niche overlap of a numberof Czech ladybird species, Honek (1985), showed that the more specialized A.decempunctata is associated with lower aphid densities than A. bipunctata.

It is also noteworthy that body size and capture efficiency provide an auto-matic link between preference and performance (Sloggett 2008). Ladybirds thatare not satiated are likely to disperse elsewhere to find food. If body size is amajor determinant of capture efficiency then ladybirds of the ‘wrong’ size in apatch of aphids of a particular size and density will not catch a sufficient num-ber of aphids, will lay few or no eggs there and will disperse from the patch: thusa preference could be a passive satiation-mediated process. Additional means oflocating the ‘right’ aphid colonies would be likely to evolve over evolutionarytime to reduce patch searching time: for example Anatis ocellata use of pinevolatiles to locate conifer aphids (Kesten 1969). However such additional mech-anisms are not initially necessary in the evolution of a ladybird-aphid associa-tion to provide a means for a ladybird to find suitable (in terms of capture effi-ciency) prey. By contrast, it is unlikely that such a mechanism would work ifprey were dietarily unsuitable as satiation remains possible even if what hasbeen consumed is of poor quality or even toxic.

The broad scale evidence obtained thus far clearly supports body size being asignificant trade-off influencing diet. Future studies need to be directed at spe-cific systems to further test the role that body size plays in the evolution of spe-cialization. It is also necessary to look at other life history stages, which thus farhave received little attention: immature size appears to play a role in prey spe-cialization in chrysopids (Tauber et al. 1995, Albuquerque et al. 1997).


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The relative importance of different trade-offs indetermining diet breadthThe studies conducted thus far are more supportive of trade-offs related to for-aging and capture of prey being important in determining dietary breadth, ratherthan trade-offs related to prey suitability and (if any) prey switching. Body sizeand its relationship with capture efficiency seems to be a particularly importantunifying factor across a broad range of species, given the ability of such argu-ments to explain the size distributions of dietary specialists and generalists. Asdiscussed above, behavioural and other foraging adaptations comprise a diversecollection of trade-offs and generalities are difficult to make: while they are like-ly to be of considerable significance in certain cases, such as in cases of myrme-cophily (e.g. Sloggett et al. 1998, Sloggett & Majerus 2003) they may not be ofuniversal importance. However, it may require many differing studies to estab-lish whether this is indeed so.

Although further work is required to verify this, the evidence for dietary suit-ability playing a significant role in specialization is exceedingly weak and preysuitability does not appear to be primary importance, although it may be a sec-ondary factor. Reasons for this may be sought in the feeding ecology of lady-birds. Unlike many other aphidophagous insects, including syrphids (Gilbert1993) and to a lesser extent chrysopids (Principi & Canard 1984, Canard 2001),adult ladybirds, like the larvae, rely predominantly on aphids as food (Hodek1996). Ladybird fecundity and consequent fitness are strongly influenced by theamount of aphid prey that they consume (e.g. Dixon & Guo 1993) and thus thecapture efficiency of adults. By contrast in syrphids, for example, in which theadults do not feed on aphids, adult fecundity and fitness will be more stronglyinfluenced by larval fitness parameters and thus larval aphid prey suitability.The adult requirement for aphids means that throughout much of the year, lady-birds must continuously feed on aphids if they are available. As aphids are anephemeral and sometimes unpredictable food source, the majority ladybirdsmust on occasions also exploit atypical prey, notably late in the season whentheir own prey are scarce and they are not breeding (Sloggett & Majerus 2000a).Because of this necessity to utilize unusual food sources, the underlying diges-tive physiology of ladybirds might have evolved in a way that strong negativeprey suitability-related trade-offs do not operate. Ultimately ladybird depend-ence on aphid prey as adults probably explains why prey suitability relatedtrade-offs might play a significant role in some other aphidophagous groups (e.g.Sadeghi & Gilbert 1999) but do not appear to do so in ladybirds.

HABITAT UTILIZATION AND SPECIALIZATIONHabitat specialization and prey specialization are closely intertwined, thoughnot perfectly correlated, and it is difficult to delineate a fixed border between thetwo. In aphidophagous ladybirds, the primary factor responsible for habitat pref-

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erence appears to be resource-based. Although natural enemies or competitorsmay exert an effect on, for example, microhabitat ovipositional preferences orpatch choice (e.g. R°u�icka 1997, Schellhorn & Andow 1999), there is little evi-dence that they exert any strong role in broad species habitat preferences: thatis, enemy free space (Jeffries & Lawton 1984) is not a primary concern.

Two examples serve to support this view. A comparison of Adalia bipunctata,which occurs in trees, shrubs and herbaceous habitats, and Adalia decempunctata,which is largely restricted to trees and shrubs, showed that their natural enemyspectrum was largely the same and thus that natural enemies were unlikely tohave played a role in their habitat preferences (Sloggett & Majerus 2000a).Similarly although the myrmecophile Coccinella magnifica exhibits exceedinglylow parasitization rates by the coccinellid parasitoid Dinocampus coccinellae,unlike the generalist Coccinella septempunctata (Majerus 1989, 1997), this isbecause the ladybird is an unsuitable host for the parasitoid, rather than becauseof enemy free space provided by the ants with which it lives (Sloggett et al.2004). This is unsurprising: the majority of ladybirds are chemically defended(Daloze et al. 1995, King & Meinwald 1996) and current evidence suggests thatchemical defence evolves to provide the primary protection for ladybirds againstthe natural enemies occurring in the habitat in which they have evolved to live(Sloggett 2005).

It is therefore necessary to return to ladybird relationships with their aphidprey. Trade-offs determining dietary breadth can explain why specialized lady-birds do not eat other types of prey, but do not fully explain why specialists spe-cialize to eat the aphids they do and thus why they live where they do. To under-stand this it is necessary to know what features of certain types of aphids andtheir environments make them suitable for specialization. Two schools ofthought exist on this subject, one centred on aphid abundance and the other con-centrated on longevity of the aphid resource.

Aphid abundanceRana et al. (2002) have argued that aphid abundance is important in determiningspecialization. They point out, correctly, that a high abundance renders an aphidsource of high value for oviposition and larval development. Their argument isconsistent with their assertion that prey suitability trade-offs are responsible fordietary specialization; if aphids of a particular type are extremely abundant thenmany ladybirds will feed there and the fittest of these will be the least welladapted to feed other prey sources. However their arguments are less consistentwith body-size related dietary trade-offs. With an abundant food source, captureefficiency related selection is likely to be relaxed and the largest individuals arelikely to be invariably the fittest. This does not match the observed pattern ofbody size in dietary specialists, which may also be very small. Similarly the evi-dence that many specialists feed on low aphid densities is also inconsistent withtheir arguments.


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Furthermore, as conceded by them, an aphid source which is abundant butshort-lived will not alone lead to enhanced fitness if the ladybirds feeding on itcannot exploit an alternative resource before or afterwards. One possibility isthat the breeding seasons of specialists are shorter than generalists and synchro-nized with abundant prey. This is known in chrysopids, in which the myrme-cophilous Chrysopa slossonae is primarily univoltine whereas its generalist siblingspecies Chrysopa quadripunctata is often multivoltine (Tauber & Tauber 1987,Tauber et al. 1993); however, there is little evidence that this is true in ladybirds.Both Adalia bipunctata and Adalia decempunctata undergo a single generation inBritain and sometimes produce a partial second generation (Majerus 1994) andin Bayreuth (Bavaria, Germany) both species are bivoltine (J.J. Sloggett pers.obs.). Nor is there any evidence that the length of specialist ladybird breedingseasons is shorter. Both A. bipunctata and A. decempunctata reproduce in Britainbetween about mid-April and early July and in Bayreuth between about midApril and early August (J.J. Sloggett pers. obs.). The breeding season of themyrmecophile Coccinella magnifica is considerably longer than that of its gener-alist congener Coccinella septempunctata in Britain: while C. septempunctata gener-ally breeds from mid-April to late June, C. magnifica regularly breeds until lateJuly (J.J. Sloggett unpublished data).

Thus attempts to relate ladybird specialization to aphid abundance are incon-sistent with a number of other factors associated with specialization in lady-birds. It therefore does not seem that habitat specialization is closely linked toprey abundance.

Aphid resource longevityThe original arguments in favour of habitat preferences being linked to aphidresource longevity sprang from the observation that certain types of habitat spe-cialization were associated with particularly long-lived aphid resources: exam-ples include ant-tended aphids, which tend to persist in substantial numberslater in the year than untended ones, and aphids in wetland areas, which alsoappear to persist for longer than ones in drier biotopes (Sloggett & Majerus2000a, b). A long-lived resource permits continued reproduction in the samehabitat over a long period of time; such a resource may not consist of one typeof aphid but a number of aphid species occurring together in the same habitat.This can explain why some dietary generalists, such as the myrmecophileCoccinella magnifica exhibit a habitat specialization: C. magnifica may be foundbreeding around the same ant colonies, although on different aphid species,throughout their entire breeding season (J.J. Sloggett pers. obs.). The mecha-nism for the evolution of such habitat preferences is probably very simple.Ancestral ladybirds exploit a long-lived resource when aphids in their more typ-ical habitats are scarce and ultimately specialize to use this resource all the time:this appears to be the case for C. magnifica’s myrmecophily (Sloggett & Majerus2000b).

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Additional support for this argument comes from a handful of Europeanspecies that appear to change their habitat preferences across their range. In allcases these ladybirds exhibit greater habitat specificity in north western Europeand decreased habitat specificity towards the south and east (Sloggett & Majerus2000a). Two particularly well researched cases are those of Coccinella quin-quepunctata and Coccinella magnifica. In Britain and northern France C. quin-quepunctata is restricted to shingle habitats near water (e.g. Rye & Sharp 1865,Majerus & Fowles 1989, Mann et al. 1993) whereas further west in, for example,Germany and the Czech republic C. quinquepunctata is found in a diversity ofnon shingle habitats, that are also not necessarily associated with water (e.g.Honek 1985, Klausnitzer & Klausnitzer 1997, Nedved 1999). Similarly althoughC. magnifica is an obligate myrmecophile associated with Formica rufa group antsin north-west- and central Europe, in the south and east it seems probable thatit is only facultatively myrmecophilous (Sloggett et al. 2002). Interestingly, asone moves south and east aphids generally remain abundant for longer leadingto longer breeding seasons for ladybirds and a greater number of ladybird gener-ations per year. As noted above, A. bipunctata are primarily univoltine and breedfrom mid April to early July in Britain, but are fully bivoltine and breed frommid April to early August in Bayreuth. Under the latter circumstances the ben-efits to a specialist of remaining in their particular habitat for the entire breed-ing season are decreased, as aphids remain to be exploited outside of this habitatfor longer. Thus in southern and eastern areas, when the benefit of resourcelongevity in the specialist habitat compared to other habitats is lost, so is thehabitat preference.

Thus far only resources that are intrinsically long-lived have been discussed.In other cases adaptation in the specialist facilitates longer exploitation ofresources that are not intrinsically long-lived (e.g. Adalia decempunctata on decid-uous trees). This is strongly linked to dietary specialization and to body sizetrade-offs and foraging adaptations: both facilitate more efficient location andcapture of prey and thus exploitation of lower aphid densities; in its turn theexploitation of lower aphid densities means that aphid resources can be exploit-ed earlier and for longer.

A hypothetical example serves to show how such specialization could evolve:a medium sized generalist feeds on several aphids of different sizes. When feed-ing on a small aphid species, the largest individuals of this ladybird require thehighest aphid densities to sustain egg laying and thus are associated with thisaphid for the shortest period of time, while the smallest can continue to repro-duce at lower aphid densities and remain associated with this aphid for longer.Over time these smaller ladybirds evolve a habitat preference that maintains theassociation with the smaller aphids and become a separate species specialized onthe small aphids. The new specialist species is smaller than the ancestral gener-alist and can utilize the same resource for longer. Such a scenario could explain


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the evolution of the habitat preferences of Adalia decempunctata for trees andshrubs, from an ancestor with broader A. bipunctata-like habitat preferences.

Aphid resource longevity can explain the evolution of habitat specialization:specialists specialize on long lived resources, or adapt to exploit the sameresources for longer. The importance of resource longevity again probablyreflects the fact that ladybird adults eat aphids: because adult ladybirds are for-aging for aphids for much of the year, exploiting these aphids for breeding isfavoured. Again in this respect, ladybirds differ from some other aphidophagousinsects that feed primarily on aphids as larvae or use a resting stage to limit thelength of the breeding season to allow specialization on particularly abundantprey.

Habitat-related trade-offs: why don’t habitat specialistsoccur elsewhereIt is apparent in many cases of closely related dietary and habitat specializationthat specialists may not move to other habitats because the aphids in these habi-tats would be difficult to exploit. However, in the cases of many dietary gener-alist habitat specialists the question of why they are not found elsewhere for atleast part of the season is a problematic one. It is easy to envisage why Coccinellamagnifica is found with ant-tended aphids when aphids elsewhere are scarce, butless so when other prey are common, particularly as untended or non-tendedaphids form a substantial part of its diet even in the presence of ants (Sloggettet al. 2002, Sloggett & Majerus 2003). Furthermore it is of a similar size to thegeneralist Coccinella septempunctata, so is unlikely to have problems exploitingother aphid prey: why therefore does it remain where it does?

We do not yet have any clear answers to such a problem, although some ideasare promising. One possibility is that adaptation to other factors such as micro-climate may make such species less fit elsewhere. For example Anisostictanovemdecimpunctata and Hippodamia tredecimpunctata which both live near wateruse evaporative cooling to reduce body temperature under warm conditions(Pekin 1996). In less humid habitats, such a mechanism might prove costly.Different ladybird species do appear to possess characteristic microclimatic pref-erences (Honek 1985): thus microclimatic trade-offs seem of likely considerableimportance in maintaining habitat associations.

It has been suggested that dispersal might in itself be costly and that special-ist ladybirds remain in the same habitat to avoid such costs (Sloggett & Majerus2000a). This was suggested for C. magnifica, which is an extremely sedentaryspecies, with ladybirds breeding round the same ant colonies all season andacross multiple seasons (Sloggett & Majerus 2000b). It does seem likely that incases such as this, relocating suitable habitat when aphids become scarce mightprove problematic if ladybirds have strayed a long way from their vicinity.However, an assessment of the full costs of dispersal is likely to prove difficultto achieve and currently such a hypothesis remains open.

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Another possibility, related to neural processing and information, is that gen-eralists are less efficient than specialists in locating suitable habitats as a conse-quence of the habitat diversity they face. This idea, that animals may be con-strained in their habitat preferences by the amount of information from theenvironment that they are able to process, has been most frequently discussed inthe context of phytophagous insects choosing host plants (Levins & MacArthur1969, Fox & Lalonde 1993, Bernays 2001) but may be equally applicable to preda-ceous insects (e.g. see Vet & Dicke 1992, Steidle & van Loon 2003). Further workis needed to examine the relative efficiencies of specialist and generalist lady-birds in detecting aphids. However, it is worth noting in this context that habi-tat specialists appear to use rather simple chemical habitat cues: for example, theconifer specialist Anatis mali uses pine volatiles (Kesten 1969) and the myrme-cophile Coccinella magnifica uses ant trail pheromones (Godeau et al. 2003). Bycontrast the cues used by generalists appear to be much more variable (e.g.Blackman 1967b, Sengonca & Liu 1994, Mondor & Warren 2000, Pettersson et al.2005).

Thus, a number of potentially fruitful lines of enquiry exist for the future,but at the moment it is impossible to assess their relative importance. In all theexamples given here, even fundamental knowledge is limited: for example,although our understanding of how ladybirds locate habitats and prey hasimproved in recent years it is still far from complete. Thus we are still some con-siderable way from understanding fully the trade-offs involved in habitat (asopposed to prey) specialization.

CONCLUSIONSSince the last review of specialization in ladybirds was published (Sloggett &Majerus 2000a), a considerable amount of relevant work has been carried out,which has done much to clarify thinking on the matter. Nonetheless, as indicat-ed in many of the sections above, firm conclusions in many areas remain diffi-cult to make and additional work on most aspects of ladybird specialization isrequired. As is also apparent, much of the work that has been carried out hasbeen concentrated on a rather narrow range of systems, and the basis of ourknowledge of ladybird specialization needs to be expanded: there remain someexceedingly suitable, but under-researched systems in this respect (e.g. Sasaji1980).

An underlying theme of this paper has been the differences between lady-birds and other aphidophagous insects, which may affect the evolution of spe-cialization. The comments on the limitations of currently available work on spe-cialization in ladybirds also apply to other aphidophages and a fuller understand-ing of the differences between these groups is likely to be informative. In gener-al aphidophagous insects provide good systems with which to study predatorspecialization due to the simplified prey range involved and the large body of


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existing research, in part linked to the capacity of these groups for aphid biocon-trol.

Such research may not just expand our knowledge base, but may have valuein an applied context. Most ladybirds used in aphid biocontrol are generalistspecies: their success has been patchy at best (Dixon 2000) and has had undesir-able side-effects (e.g. Koch 2003, Harmon et al. 2007). Specialized ladybirds maybe more useful and effective as biocontrol agents in agroecosystems where theyoccur (Sloggett 2005): current evidence indicates that this is indeed the case (e.g.Goidanich 1943, Cecilio & Ilharco 1997, Leather & Kidd 1998, Berthiaume et al.2007). Thus ultimately further work on specialized ladybirds and other aphi-dophagous insects may be of value beyond that to the interested naturalist oracademic.

REFERENCESAlbuquerque, G.S., Tauber, M.J. & Tauber, C.A. 1997. Life-history adaptations and

reproductive costs associated with specialization in predacious insects. J. Anim. Ecol.66: 307-317.

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Katherine Parker, Peter Roessingh & Steph B.J. MenkenUniversity of Amsterdam, Institute for Biodiversity and Ecosystem Dynamics, POBox 94062, 1090 GB Amsterdam, The Netherlands, E-mail: [email protected]

Many male Lepidoptera transfer a large ejaculate, containing nutrientsand/or secondary plant compounds. In principle this ‘nuptial gift’could help to increase female survival and longevity. Here we investi-gated the effects of both multiple mating and extra nutrition in theadult stage on males and females in two species of Yponomeuta. Matedfemales had significantly shorter live spans than unmated females andsimilar fecundity. Females therefore do not appear to benefit from re-mating, which suggests that the considerable weight transfer of themales to the females during mating does not provide nutritional value.Females of Y. cagnagellus, which received honey throughout their adultlifetime, lived significantly longer and laid larger egg batches thanfemales, that did not receive honey. In Y. padellus this effect was notfound. It is concluded that neither species benefits from multiple mat-ing and that in Yponomeuta cagnagellus adult nutrition rather then mat-ing increases female reproductive output and longevity, which con-trasts with many studies of Macrolepidoptera.

Keywords: nuptial gift, honey, reproductive output, Macrolepidoptera

Female butterflies and moths require significant amounts of energy forpheromone production, calling, courtship, and oviposition. Many empiricalstudies in Macrolepidoptera have found that, in addition to adult nutrition, mul-tiple mating can have a positive effect on female longevity and fecundity. A sin-gle mating can provide a female with enough sperm to fertilize all her eggs(Lederhouse 1981). Remating is therefore not needed for fertilisation. However,males can transfer an ejaculate to the female that contains a ‘nuptial gift’, acces-sory substances, which provide nourishment to the female, increasing her fecun-dity (Cook & Wedell 1996).

In addition to direct nutritional effects that increase fecundity, nuptial giftsmight also increase female lifespan. This benefits the female, presuming that thelonger a female lives the more opportunities she has to produce offspring and findsuitable partners that can provide a genetic benefit to her offspring in the form of

Effects of multiple mating and adult nutritionon longevity and fecundity in two Yponomeutaspecies


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genetic variation, bet hedging for imperfect mate assessment, and higher levels ofsperm competition (Tores-Vila & Walker 1980, Halliday 1983, Watson 1991).

Most studies look at the reproductive behavior of butterflies, few havefocused on moths. An exception is the study by De Jong (1988) who found insmall ermine moths that females who do not receive honey before mating hadstunted growth, small egg number, produce less attractive pheromones, and yetwere more successful in mating repeatedly. We therefore designed an experi-ment in Yponomeuta moths with the following questions in mind:

(1) How do multiple mating and adult nutrition affect female longevity, mat-ing frequency, and egg production in two closely related species of Yponomeuta?

(2) Can nutritionally stressed females increase their longevity and reproduc-tive output by mating more frequently?

Both species feed on their own specific host-plant: Yponomueta padellus can befound feeding on plants from various species of Rosaceae including Prunus spin-osa while Yponomeuta cagnagellus exclusively feeds on Euonymus europaeus belong-ing to the Celastraceae (Menken 1992). In late spring females deposit up to 150eggs in clusters near a bud or side branch of their food plant and eggs hatch threeweeks later (Povel 1984).

We tested the potential benefits of nuptial gifts on fecundity and longevityby mating females with one or two males or by providing continuous access tomales. In addition, the effect of honey was investigated. We quantified the totalnumber of matings, the insect’s total lifespan, and the total surface of all eggbatches laid over a female’s complete lifetime.


Mating and feeding treatmentsA total of 389 Y. cagnagellus and 396 Y. padellus males and females were assignedto unmated (‘virgin’), once-mated, twice-mated, and unlimited mating groups.Half of the insects were fed honey throughout their lifetime and half were not.

Both mating and courtship were recorded once per hour between 9:00 and18:00 hours. A mating was recorded when the male and female remained in cop-ula for more than 1.5 hours. Females that did not want to mate were suppliedwith a new virgin male twice per week.

Egg production analysis & longevityMost studies examining the importance of male investment for female repro-duction assume that the mass of the male ejaculate is positively correlated withfemale output. Spermatophore size is however not the best measurement of maleinvestment, since it is the content and not the size of the spermatophore thatdetermines its true value (Marshall1989, Delisle & Bouchard 1995). For this rea-son we measured lifetime female fecundity rather than spermatophore size. Ahost-plant stem was placed with every female in a Petri-dish and was checked


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3-5 times per week for egg batches. Batches were photographed, and the eggbatch surface was measured using the program ‘ImageJ’ (Rasband 1997).Longevity was measured from the moment a moth enclosed till its death for allmoths in both experiments.

RESULTS

Adult NutritionThe lifespan for Y. cagnagellus was approximately one month and for Y. padellusit was approximately two months. Yponomeuta cagnagellus females did utilizehoney to increase their lifespan (Fig. 1; ANOVA: df=1, F=15.3, p<0.001). Honeydid not increase the lifespan of the shorter lived Y. padellus (Fig. 2; ANOVA:df=1, F=1.82, p=0.18).

Yponomeuta cagnagellus females with access to honey laid significantly moreegg batches and also produced a significant larger egg batch surface (Fig. 3;

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Figure 1. Effect of mating frequency onlongevity of Yponomeuta cagnagellus forunmated (n=61), once-mated (n=47),twice-mated (n=43), and free-mating (3+)females (n=48). Approximately half thenumber of females assigned to the fourmating groups (0-3+) received honeywhilst the other half did not.

Figure 2. Effect of mating frequency onlongevity in Yponomeuta padellus forunmated (n=60), once-mated (n=49),twice-mated (n=38), and free-mating (3+)females (n=49). Approximately half thenumber of females assigned to the fourmating groups (0-3+) received honeywhilst the other half did not.

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ANOVA: df=1, F=7.78, p<0.01) than females without this extra source of energy.Yponomeuta padellus females on the other hand did not benefit significantly fromtheir access to honey and both groups produced about equal egg batch surfaces(Fig. 4; ANOVA: df=1, F=0.14, p=0.91).

Multiple MatingMultiple mating did not increase female longevity in either species ofYponomeuta. Mating not only failed to extend female lifespan but in fact signif-icantly reduced it (Figs. 1 and 2; ANOVA, df=1, Fcag=17.49 and Fpad=43.05,P<0.001).

Total egg production (both egg batch number and batch surface) did notchange significantly when female were mated multiple times (Figs. 5 and 6;ANOVA, df=1, Fcag=0.08 and Fpad=3.13, P=ns for both species). Unmated femalesof both species in all cases laid only very few (unfertilized) eggs.

DISCUSSIONOur results show that contrary to expectation (based on literature data forMacrolepidoptera), multiple mating has a negative effect on longevity andfemales did not convert the content of the male spermatophore into eggs.


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Figure 3. Effect of honey on total eggbatch surface for mated Yponomeutacagnagellus females that received (n=26) ordid not receive honey (n=22) and for vir-gin females that received (n=31) or did notreceive honey (n=30).

Figure 4. Effect of honey on total eggbatch surface for mated Yponomeuta padel-lus females that received (n=24) or did notreceive honey (n=25) and for virginfemales that received (n=30) or did notreceive honey (n=30).

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Lederhouse et. al (1990) observed that Papilio glaucus ingest nectar, sodium, andother electrolytes and transfer this extra source of nutrition to females duringmating in order to increase female reproductive output. Arnqvist & Nilsson(2000) suggested that multiple mating may increase female fitness by stimulat-ing egg production, replenishing sperm supplies, and offering accessory sub-stances that contribute to female survival and/ or reproduction. Here we foundno support for these ideas as mating multiply had a significant negative effecton female longevity and did not affect reproductive output. It should be notedhowever that the lack of effect of the nuptial gift as an energy source does notrule out that it might contain secondary plant compounds that increase the sur-vival of the female and/or the offspring.

In contrast to the lack of a nutritional benefit from multiple mating, honeycan be used as an energy source by Y. cagnagellus to extend lifespan and producelarger eggs batches. Yponomeuta padellus on the other hand did not profit fromhoney. This is possibly due to time constraints related to its shorter lifespan. Itseems that Y. cagnagellus has the ability to use external carbohydrate sources inits adult life, while Y. padellus females are relying exclusively on their larvalresources.

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Figure 5. Effect of mating on total eggbatch surface produced by Yponomeutacagnagellus for unmated (n=61), once-mated (n=47), twice-mated (n=43), andfree-mating(3+) females (n=48).Approximately half the number offemales assigned to the four matinggroups (0-3+) received honey whilst theother half did not.

Figure 6. Effect of mating on total eggbatch surface produced by in Yponomeutapadellus for unmated (N=60), once-mated(N=49), twice-mated (N=38), and free-mating (3+) females (N=49).Approximately half the number offemales assigned to the four matinggroups (0-3+) received honey whilst theother half did not.

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Sopher Ondiaka, Tullu Bukhari, Marit Farenhorst, Willem Takken & BartG.J. KnolsLaboratory of Entomology, Wageningen University & Research Centre, PO Box 8031,6700 EH Wageningen, The Netherlands, E-mail: [email protected]

Malaria remains a key hindrance to the improvement of health inAfrica. Transmission rates and the risk of the disease can be greatlyreduced by vector control. At present, control of adult mosquitoes isalmost exclusively based on chemical insecticides. However, develop-ment of resistance to chemicals is of great concern for sustainablemalaria control. Entomopathogenic fungi are effective against adultvectors and can be used as an alternative to insecticides. As slow-killing agents, fungi are expected to impose limited evolutionary pres-sure for resistance formation in exposed populations. The host-seekingresponse, feeding propensity, blood meal size (quantified throughhaematin analysis), and fecundity was evaluated by exposing mosqui-toes infected with the fungus Metarhizium anisopliae to human volun-teers. It was found that fungal infection reduces feeding propensity butblood meal size and fecundity remained unaffected. The implicationsof these findings with regard to potential resistance developmentagainst fungal infection are discussed.

Keywords: Anopheles gambiae, feeding propensity, blood meal size,fecundity, Metarhizium anisopliae, resistance

Malaria remains a major global problem, exerting an unacceptable toll on thehealth and economic welfare of the world’s poorest communities (WHO 2005,Breman et al. 2007). The burden of disease is greatest in Africa where childrenunder the age of five and pregnant women are most vulnerable due to their lowerlevel of malaria immunity (WHO 2006). Each year, over one million deathsfrom the direct effects of the disease occur in the continent (World Bank 2007)and it is therefore regarded as the leading cause of morbidity and mortality inthe sub-Saharan region. Malaria is caused by protozoan parasites of the genusPlasmodium and is transmitted through bites of mosquitoes belonging to thegenus Anopheles. Females of An. gambiae s.l. are the principal vectors of malariain Africa, besides An. funestus. Their dominance as malaria vectors is largely due

Effects of fungal infection on the host-seekingbehaviour and fecundity of the malariamosquito Anopheles gambiae Giles


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to a preference for human blood, high vector competence, and high daily sur-vival rates (Day 2005, Besansky et al. 2004).

The transmission rates and risks of the disease can be greatly reduced by vec-tor control (WHO 2006). Contemporary adult mosquito control is almost exclu-sively based on indoor application of chemical insecticides in the form ofimpregnated bed nets or as indoor residual spraying (of walls and ceilings).However, sustainable use of chemicals is undermined by problems of insecticideresistance in mosquito populations, environmental contamination and risks tohuman health. Growing concern to these problems has increased interest in thesearch for alternative approaches (Zaim & Guillet 2002). Biological control isone option, and several biological control agents, like Bacillus thuringiensis israe-lensis have been used successfully to control mosquito larvae (Fillinger et al. 2003,2006). Entomopathogenic fungi are effective against adult vectors and are cur-rently being developed as biopesticides (Scholte et al. 2004, 2005, Blanford et al.2005, Knols & Thomas 2006, Thomas & Read 2007). As slow-killing agents,fungi are expected to impose limited risks for resistance formation in malariamosquitoes (Thomas & Read 2007). Fungal resistance is not considered animmediate risk in mosquito populations based on their multiple modes of action.Fungi use an array of weapons to attack the insect, such as chitinases, proteasesand release of toxins (Hajek & St. Leger 1994). Compared to insecticides, fungihave low virulence as they kill an insect in 6-14 days after infection dependingon the fungal species and isolate used. Within this period, the females are like-ly to be able to mate and reproduce. Therefore, the slow killing mechanism ofthe fungus imposes a limited selection pressure on the mosquitoes thus reducingthe likelihood of anti-fungal resistance (Knols & Thomas 2006).

To curb malaria transmission, understanding behavioural consequences offungal infections in mosquito populations is vital. The propensity to selecthumans for blood feeding is arguably the most important component of mosqui-to vectorial capacity (Zwiebel & Takken 2004). This aspect further determinesthe success of mating, blood feeding and oviposition. A laboratory study inwhich infected female mosquitoes were blood fed by arm in small cups revealedthat fungal infection reduces (but not eliminates) feeding propensity and fecun-dity (Scholte et al. 2006). However, it remains unknown if under more realisticconditions, whereby mosquitoes have to perform a host-seeking response, simi-lar results are obtained.

Here we report findings of the impact of progressive fungal infections on thefeeding propensity, blood-meal size and subsequent number of eggs laid byfemale An. gambiae one, three and five days after infection with spores of M.anisopliae. The experiment was conducted under simulated room conditions inthe laboratory where insects had a choice to locate and bite a host instead ofmaking the host directly available to them when placed in cups.

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MATERIALS AND METHODS

MosquitoesAnopheles gambiae (Suakoko strain; courtesy Prof. M. Coluzzi) were maintainedat 27 ± 1°C, 80 ± 5% relative humidity (RH) and a photoperiod of 12:12 light:dark.Adults were held in 30 × 30 × 30 cm gauze cages and had ad libitum access to a 6%glucose solution on filter paper. They were fed on a human arm twice a week.Eggs were laid on wet filter paper and transferred to water trays. Larvae werereared in tap water in plastic trays and were fed daily on Tetramin® fish food.Pupae were collected daily and placed in adult cages for emergence. A cone ofdamp white filter paper held in pint-sized cups was introduced in the cageswhere mated females oviposited following a bloodmeal.

Fungus application in clay potsFour Ghanaian clay water storage pots (two for control and two for infectionwith fungus) were used in the study according to protocols described byFarenhorst et al. (2008). These pots have previously been shown to be highlyattractive resting sites for anopheline mosquitoes in Western Kenya (Odiere etal. 2007). Each of the control pots was sprayed with 50 ml of Ondina oil (Shell,The Netherlands) while each of pots for infection was sprayed with 35 mlOndina oil. Two hours later, pots for infection were each sprayed with 17 ml ofM. anisopliae (IC30 isolate) formulated in Ondina oil at a concentration of 4.0 ×1010 spores/m2. Both control and fungus-treated pots were left to dry for 15 hours.

Mosquito infectionA wet cotton pad soaked in 6% glucose solution was placed at the mouth of eachpot and covered with a cylindrical nylon paper firmly held with a rubber band.Groups of 800 female adults, 3-5 days old, were randomly collected from rearingcages with a mouth aspirator and were introduced in clay pots through a roundhole at the base of the pot. The holes were sealed with a stopper to prevent mos-quito escape. Each control and fungus-treated pot had 150 and 250 adults, respec-tively. Six hours later, both infected and control mosquitoes were transferredinto separate rearing cages and were provided with 6% glucose solution (sup-plied on filter paper wicks). For the human volunteer experiments mosquitoesof one, three and five days post infection were used. Approximately 2 hoursbefore each experiment, two groups of 30 mosquitoes, from control and fungus-treated mosquito cages were removed at random and released in a large nettingcage (3 × 3 × 3 m) fitted in an experimental room maintained at 26 ± 1°C, 75 ± 5%RH. For mosquitoes one and three days post infection, the experiment was repli-cated twice, for mosquitoes 5 days post infection thrice.

Assessing feeding propensityUpon release, the insects were given the option to respond, locate and bite ahuman host and take a blood meal for a fixed period of 30 minutes. The volun-

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teer entered the cage into which the mosquitoes had been released, and laid downon a bed with exposed arms and legs to facilitate biting. Both blood fed andunfed mosquitoes were collected individually into 30 ml cylindrical plastic tubes(9 × 2.5 cm) covered with hollow plastic caps. The tubes were lined with a stripof thin filter paper firmly held with a paper clip for mosquitoes to rest on. Thecaps had several holes to allow for feeding. Cotton pads soaked in 6% glucosewater were placed in each cap. Thereafter, the tubes were assigned numbers(both for blood-fed and non blood-fed). They were then arranged in holdingracks and maintained at 27 ± 3°C, 70 ± 10% RH and 12:12 L:D photoperiod forhematin (excreted during the post-diuresis phase) collection. Cotton pads werereplaced daily. After two days, blood-fed mosquitoes were transferred intooviposition tubes fitted with wet filter paper that served as oviposition substrate.The tubes were assigned numbers corresponding to labels on the hematin tubes.Hematin within the tubes was quantified using a standard curve to provide anestimate of blood meal size (see below). Two days later, filter paper in oviposi-tion tubes containing eggs were removed and the number of eggs per individualrecorded by counting under a stereomicroscope. Dead individuals in hematin oroviposition tubes including the non-bloodfed insects were collected and platedon petri dishes containing wet filter paper to allow growth of fungus on thecadaver. Petri dishes were placed in an incubator for three days at 26 ± 2°C topromote fungal growth. Mosquitoes without fungal growth were assumed tobelong to the control treatment. The procedure described above was repeatedwith mosquitoes 3 and 5 days post infection.

Estimation of blood meal sizeThe amount of hematin excreted was determined by the method of Briegel(1980). The excreta in holding tubes were dissolved in 1 ml of 1% lithium carbon-ate (LiCO3) solution. The absorbance of the resulting solution was read at 387nm and compared to a standard curve made from bovine haematin (Hogg &Hurd 1997, Hurd et al. 1995).

Statistical analysisIndividuals that took a blood meal and died in the hematin or oviposition tubeswere excluded from the analysis. Feeding propensity was expressed as mean per-centage (± SE) of the total number of mosquitoes that took a blood meal in boththe control and infected groups while blood meal size and number of eggsoviposited were expressed as means (± SE) per individual mosquito. Thesemeans were compared using χ2 square analysis.

RESULTSReduction in feeding propensity was significant (P=0.006) for mosquitoes thatwere three days old after fungus infection when compared against uninfected

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mosquitoes of that age. However, this difference was not significant betweenthe fungus-infected and control groups one (P=0.68) and five day(s) (P=0.33)post infection (Fig. 1A). The amount of blood consumed by fungus-treated mos-quitoes was not significantly different from the amounts taken by mosquitoesfrom the control groups for all periods post infection (Fig. 1B). Unfortunately, aconsiderable number of mosquitoes in both treatments died before oviposition.Nevertheless, the number of eggs laid by the few surviving individuals was notaffected by fungal infection (Fig. 1C).


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Figure 1. A: Proportion (mean ± SE) of M. anisopliae-infected or control female An. gam-biae blood feeding 1, 3 and 5 days post infection; B: Blood meal size (mean ± SE) ofmosquitoes surviving to oviposition, and C: Number of eggs laid by surviving femalesthat blood fed 1, 3 and 5 days post fungus infection. Numbers inside bars indicate num-ber of mosquitoes tested (n).

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DISCUSSIONResults from the current study show that feeding propensity decreased in adultfemale An. gambiae mosquitoes that were three days old after infection with fun-gus M. anisopliae, but not one or five days post infection. Such an impact onbehaviour will result in a reduction of female lifetime vectorial capacity andhence malaria transmission risk. Scholte et al. (2006) observed virtually similareffects 2, 3 and 4 days post infection where the 4-day treatment yielded margin-al significance (P=0.048) in feeding propensity reduction compared to controlmosquitoes. Interestingly, therefore, it appears as if mosquito feeding appetitedecreases 2-3 days after infection, but that this effect is no longer apparent oneor two days later. From the perspective of resistance developing against fungalinfections, this finding is important in the sense that mosquitoes with infectionsdo still engage in host-seeking behaviour and are willing to consume blood mealsin similar proportions as their uninfected counterparts.

We further observed that fungal infection had no effect on blood meal sizeand fecundity. Scholte et al. (2006) reported a significant reduction in blood-mealsize for mosquitoes four, but not two and three days post infection. Our findingsdiffer only for the groups four days post infection, which consumed similaramounts of blood as control mosquitoes. Again, considering that blood meal sizeremains unaffected by fungal infection in the first five days, it is likely thatthese mosquitoes will engage in egg development and completion of at least onegonotrophic cycle, thereby enabling reproduction and thus reduction of thepotential for resistance development. Lifetime fecundity in An. gambiae, follow-ing infection with M. anisopliae, was also reported to reduce significantly(Scholte et al. 2006), though we did not observe this effect in the present study.Such findings have also been reported for other insects infected with fungus. Forinstance, Ekesi and Maniania (2000) reported a reduction in fecundity in thripsMegalurothrips sjoistedti upon infection with M. anisopliae.

According to Blanford et al. (2005), fungus interferes with blood meal intakein An. stephensi 8-14 days after infection. Assuming that this is the same for An.gambiae then our focus on insects 1-5 days post infection would not reveal sucheffect. Generally, upon contact with a mosquito, the fungal spores begin toinvade and develop inside the mosquito, after which the fungus multiplies andkills its host within two weeks; the approximate time a malaria parasite takes todevelop into its infective form (sporozoites). This slow-kill approach by fungi isan advantage given that mosquitoes cannot transmit sporozoites until about twoweeks after an infectious blood feed (Kanzok & Lorena 2006). Besides, Blanfordet al. (2005), when evaluating blood meal intake and using a mouse malariamodel system established that fungal infection has a negative effect onPlasmodium development in the mosquito. Putting the effects of blood mealintake, which also directly influences fecundity and Plasmodium developmenttogether, M. anisopliae could reduce malaria transmission by approximately 80

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times. Nonetheless, mouse malaria may have different characteristics fromhuman malaria and many different factors can come into play when applyingresearch findings in the field. The factors include fungal specificity and the pos-sibility of insects developing resistance to fungi. Mosquitoes might evolve waysto prevent the fungus from entering their body or limit its growth if theybecome infected but it seems unlikely that they would intensify Plasmodiumtransmission or virulence (Michalakis & Renaud 2005).

The future of using M. anisopliae as a novel vector control tool is increasingas pressure mounts on the search for alternative public health insecticides.Studies by Jenny Stevenson (Stevenson et al., unpubl. data) have shown thatfungus is effective against a multiple insecticide-resistant strain of An. stephensi,fuelling hope to solve problems of insecticide resistance (Knols & Thomas 2006).However, the big challenge towards sustainable use of fungus-based measuresfor vector control is the possible resistance development by mosquitoes. So far,this has not been reported in mosquitoes or any other insect, and our currentfindings support the idea that the evolutionary pressure exerted on populationswill remain small. Nevertheless, we intend to conduct similar studies undersemi-field and field conditions before drawing final conclusions.

Ackowledgements We thank Niels Verhulst for serving as a volunteer in thisstudy and Leo Koopman, Frans van Aggelen and Andre Gidding for supplying theexperimental mosquitoes. This study was supported by a Wageningen University sand-wich PhD fellowship (PE&RC project # 07045) awarded to SO. BGJK is supported by aVIDI grant (#864.03.004) from the Dutch Scientific Organisation.

REFERENCESBesansky, N.J., Hill, C.A. & Costantini, C. 2004. No accounting for taste: host prefer-

ence in malaria vectors. Trends Parasitol. 20: 249-251.Blanford, S., Chan, B.H.K., Jenkins, N., Sim, D., Turner, R.J., Read, A.F. & Thomas,

M.B. 2005. Fungal pathogen reduces potential for malaria transmission. Science 308:1638–1641.

Breman, J.G., Alilio, M.S. & White, N.J. 2007. Defining and Defeating the InteolerableBurden of Malaria. III. Progress and Perspectives. Objectives andAcknowledgements. Am. J. Trop. Med. Hyg. 77: i-ii.

Briegel, H. 1980. Determination of uric acid and hematin in a single sample of excretafrom blood-fed insects. Experientia 36: 1428.

Day, F.J. 2005. Host-seeking strategies of mosquito disease vectors. J. Am. Mosq. Cont.Assoc. 21: 17–22.

Ekesi, S. & Maniania, N.K. 2000. Susceptibility of Megalurothrips sjostedti developmen-tal stages to Metarhizium anisopliae and the effects of infection on feeding, adultfecundity, egg fertility and longevity. Ent. Exp. Appl. 94: 229-236.

Farenhorst M., Farina D., Scholte E.-J., Takken, W., Hunt, R.H., Coetzee M. & KnolsB.G.J. 2007. African water storage pots for the delivery of the entomopathogenicfungus Metarhizium anisopliae to the malaria vectors Anopheles gambiae s.s. and An.


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funestus. Am. J. Trop. Med. Hyg. Submitted.Fillinger, U., Knols, B.G.J. & Becker, N. 2003. Efficacy and efficiency of new Bacillus

thuringiensis var. israelensis and B. sphaericus formulations against the malaria vectorAnopheles gambiae in Western Kenya. Trop. Med. Int. Health. 8: 37-48.

Fillinger, U. & Lindsay, S.W. 2006. Suppression of exposure to malaria vectors by anorder of magnitude using microbial larvicides in rural Kenya. Trop. Med. Int. Health11: 1629-1642.

Hajek, A.E. & St. Leger, R.J. 1994. Interactions between fungal pathogens and insecthosts. Annu. Rev. Entomol. 39: 293-321.

Hogg, J.C. & Hurd, H. 1997. The effects of natural Plasmodium falciparum infection onthe fecundity and mortality of Anopheles gambiae s.l. in north east Tanzania.Parasitology 114: 325-331.

Hurd, H., Hogg, J.C. & Renshaw, M. 1995. Interactions between bloodfeeding, fecundi-ty and infection in mosquitoes. Parasitol. Today 11: 411-416.

Kanzok, S.M. & Lorena, M.J. 2006. Entomopathogenic fungi as biological insecticides tocontrol malaria. Trends Parasitol. 22: 49-51.

Knols, B.G.J. & Thomas, M.B. 2006. Fungal entomopathogens for adult mosquito con-trol-a look at the prospects. Outl. Pest Manag. 17: 257-259.

Michalakis, Y. & Renaud, F. 2005. Fungal allies enlisted. Nature 435: 891.Odiere, M., Bayoh, M.N., Gimnig, J., Vulule, J., Irungu, L. & Walker, E. 2007. Sampling

outdoor, resting Anopheles gambiae and other mosquitoes (Diptera: Culicidae) inWestern Kenya with clay pots. J. Med. Entomol. 44: 14-22.

Scholte, E.-J., Knols, B.G.J., Samson, R.A. & Takken, W. 2004. Entomopathogenic fungifor mosquito control: a review. J. Insect Sci. 4: 19.

Scholte, E.-J., Ng’abi, K., Kihonda, J., Takken, W., Paaijmans, K., Abdulla, S., Killeen,G.F. & Knols, B.G.J. 2005. An entomopathogenic fungus for control of adult Africanmalaria mosquitoes. Science 308: 1641–1642.

Scholte, E.-J., Knols, B.G.J. & Takken,W. 2006. Infection of Anopheles gambiae with theentomopathogenic fungus Metarhizium anisopliae reduces blood feeding and fecundi-ty. J. Invertebr. Pathol.91: 43–49.

Thomas, M.B. & Read, A.F. 2007. Can fungal biopesticides control malaria? NatureMicrobiol. Rev. 5: 377-383.

World Bank, 2007. World Bank Malaria Implementation Resource Team. The WorldBank Booster Program for Malaria Control in Africa. October edition.

World Health Organization. The Africa Malaria Report 2006.World Health Organization. Malaria Vector Control and Personal Protection 2006..

WHO Technical Report Series, No. 936. Geneva, Switzerland.World Health Organization/UNICEF. The World Malaria Report 2005.Zaim, M. & Guillet, P. 2002. Alternative insecticides: an urgent need. Trends Parasitol.

18: 161-163.Zwiebel, L.J. & Takken, W. 2004. Olfactory regulation of mosquito–host interactions.

Ins. Biochem. Mol. Biol. 34: 645–652.

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Niels O. Verhulst, Willem Takken & Renate C. SmallegangeLaboratory of Entomology, Wageningen University and Research Centre, PO Box8031, 6700 EH Wageningen, The Netherlands, E-mail: [email protected]

Host-seeking of the malaria mosquito Anopheles gambiae sensu stricto ismediated by human odours. Dual-choice olfactometers are used toidentify attractive odour components. We examined whether an olfac-tometer equipped with a funnel as trap entry would catch more mos-quitoes than when with a baffle. When tested directly against eachother using a human skin extract as stimulus, the funnel caught moremosquitoes in the first of the two experiments performed. In the sec-ond experiment, worn socks were used as an attractive odour sourceinstead of human skin washings. This experiment showed that thetotal response of mosquitoes was significantly higher when funnelswere used in both trapping devices than when baffles were used.Increasing the total number of mosquitoes trapped by using a funnel,can make it easier to distinguish which odour is more attractive whenodours are tested that only differ little in their attractiveness.

Keywords: Anopheles gambiae sensu stricto, baffle, funnel, skin extract,olfaction, flight behaviour

Odours play an important role in the host seeking of mosquitoes (Takken &Knols 1999). The host-seeking flight of a mosquito can roughly be divided intolong range, medium and short range attraction. Long distance attraction is deter-mined by odour stimuli, medium attraction by odours and CO2 and attraction onshort distance by odours, CO2 and non-olfactory cues, such as heat, body mois-ture and visual cues (Gillies & Wilkes 1969, Takken 1991). The malaria mosqui-to Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) (henceforth termedAn. gambiae), however is nocturnal and therefore visual cues are of minor impor-tance. An. gambiae is highly attracted to odours emitted from the human skin(Takken & Knols 1999). Identifying odours that are used by mosquitoes to locatetheir host can be important in the development of odour-baited traps. Wind tun-nels and olfactometers have long been used to test the behavioural response ofmosquitoes to odours (Acree et al. 1968, Eiras & Jepson 1991, Geier & Boeckh1997). In 1994, Knols et al. developed a dual-choice olfactometer to test the behav-

Structural design affects entry response ofmosquitoes in olfactometers


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ioural response of An. gambiae (Fig. 1), which was the first effective 2-choice sys-tem available for studying the behavioural response of this mosquito species.Since then, numerous experiments have shown the effectiveness of the setup intesting the olfactory response of An. gambiae to individual odours (Smallegangeet al. 2005), blends of odours (Knols & Meijerink 1997, Smallegange et al. 2005)and human skin emanations (Pates 2002, Qiu et al. 2004, Qiu et al. 2006).However, since 1994 the olfactometer has not been changed in order to increasethe effectiveness of the system, or to test why it is effective. Increasing the dis-criminative power of the system will increase the efficiency of experiments,especially when tested odours differ only slightly in their attractiveness. It wastested if the use of a funnel instead of a baffle as trap entry in the olfactometerwould increase the efficiency of the trapping system. Human skin washing sam-ples and worn socks have shown to be an attractive odour source to An. gambiaeand were used as a standard odour source (Pates et al. 2001, Pates 2002, Qiu 2005,van Agtmaal 2006).


InsectsThe An. gambiae colony at Wageningen University, The Netherlands, originat-ed from Suakoko, Liberia. The mosquitoes have been cultured in the laboratorysince 1988 with blood meals from a human arm twice a week. The adult mosqui-toes were maintained in 30 x 30 x 30 cm gauze-covered cages in a climate-con-trolled room (27 ± 1°C, 80 ± 5% RH, LD 12:12). They had access to a 6% (v/v) glu-cose solution on filter paper. Eggs were laid on wet filter paper, emerged in tapwater in plastic trays and fed daily with Tetramin® baby fish food. Pupae werecollected daily and placed in adult cages for emergence.

OlfactometerA dual-port olfactometer (Fig. 1) (Knols et al. 1994, Braks & Takken 1999) wasused for the experiments. Pressurized air was charcoal filtered, humidified andled through two glass mosquito trapping devices, which were linked to two ports(diameter 5 cm, 30 cm apart). Trapping devices were equipped with either a baf-fle or a funnel (Fig. 2), depending on the experiment. The air entered the flightchamber (1.60 x 0.66 x 0.43 m) with a speed of 0.21 ± 0.01 m/s, temperature of 28.5± 0.8°C and relative humidity above 80%. The experimental room was main-tained at a temperature of 26.6 ± 0.7°C and a relative humidity of 63.6 ± 2.7%.

Experiments were prepared and performed according to the methods des-cribed by Smallegange et al. (2005). For each experiment thirty female mosqui-toes of 5-8 days old, which had not received a blood meal, were selected 14-18hours before and placed in a cylindrical release cage (d=8, h=10 cm) with accessto tap water from damp cotton wool. The experiments were performed duringthe last 4 h of the dark period, when An. gambiae females are known to be more

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responsive to host-odours (Maxwell et al. 1998, Killeen et al. 2006). In each trialtest odours were released in the air stream before a group of mosquitoes was setfree from a cage which was placed at the downwind end of the flight chamber,1.60 m from the two ports. Mosquitoes were left in the flight chamber for 15 min.The female mosquitoes that had entered either trapping device were counted atthe end of the experiments, after anaesthetization with 100% CO2. Mosquitoesremaining in the flight chamber were removed with a vacuum cleaner. Each trialstarted with new mosquitoes, clean trapping devices and new stimuli. Thesequence of treatments was randomized on the same day and between days.Treatments were alternated between right and left ports in different replicates

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Figure 1. A diagram of the dual-choice olfactometer (modified after Pates et al., 2001).

Figure 2. Schematic drawing and picture of a trapping device for the olfactometerequipped with a baffle or funnel. All dimensions are in centimeters.

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to rule out any positional effects. Surgical gloves were worn by the experimenterto avoid contamination of the equipment with human volatiles.

Odour sourcesPrevious research showed that human skin washing with ethanol is a reliablemosquito attractant (Pates 2002). Human skin washing samples were collected,since it has the advantage that it can be used in the behavioural bioassay as astandard attractive odour source. Cotton wool (approx. 5 x 5 cm) pads withethanol were used to rub both hands, under the arm pits and feet of a human sub-ject (female, 39 years old) for two minutes. Twenty five cotton wool pads con-taining human skin emanations were packed in a glass column (1 m long) andeluted with 550 ml absolute ethanol (Merck). The ethanol elution was concen-trated from 190 ml to 27 ml using a rotating vacuum evaporator, 55°C and 625mBar. The concentrated skin washing samples were decimally diluted in ethanoland stored at -20 °C until use.

Before each olfactometer experiment 100μl of cold-stored diluted (1:1000)human skin washing sample was pipetted onto a sand blasted glass slide (7.6 x2.6 cm) (Braks & Takken 1999). After evaporation of the ethanol, slides wereplaced in both trapping devices and mosquitoes released. Previous experimentsshowed that a dilution of the skin washing sample of 1:1000 was most attractivefor An. gambiae in the same olfactometer (van Agtmaal 2006).

Worn socks were also used as an attractant. Nylon socks (Hema, TheNetherlands) were worn for 24 hours by a human volunteer (Male, 28 years old).The volunteer refrained from alcohol, garlic and spicy food for 24 hours. The lastshower was without soap and the volunteer did not use any soap, deodorant orperfume when wearing the socks. Socks were stored at -20°C until use.

Experiment 1It was examined whether using a funnel instead of a baffle would increase thenumber of mosquitoes caught in the trapping device. The trapping devices in theolfactometer were equipped with either a baffle or funnel at the point of entry.Baffles and funnels were tested directly against each other in the olfactometer tocompare trap entry response. Also tests with only baffles or only funnels weredone to compare the total trap entry response. Skin washing samples with a dilu-tion of 1:1000 were used as an attractant. As a control, a worn sock was testedagainst a clean sock in trapping devices with baffles. Clean air experiments weredone to determine trap entry response when no odour was present. Experimentswere repeated six times on different days.

Experiment 2The experimental setup was equal to that of experiment 1, with the exceptionthat worn socks were used as an attractant instead of skin washing samples.Experiments with baffles and worn socks or funnels and worn socks were per-

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formed twice each day, for five days. Experiments, during which a baffle and afunnel were tested directly against each other, were performed only once a dayfor five days, as were the control experiments. Experiments were performed forfive days.

StatisticsFor each two-choice test in the olfactometer a χ2-test was used to analyzewhether the total (i.e. sum of all replicates) number of mosquitoes that wastrapped in the treatment trapping device and the total number that was trappedin the control trapping device differed from a 1:1 distribution. A GeneralizedLinear Model with binomial function (GLM; Genstat for Windows, release 9.2)was used to investigate the effect of treatments on the trap entry response; theeffect of a treatment on the number of female mosquitoes caught in both trap-ping devices as percentage of mosquitoes that flew out of the release cage.

RESULTSIn the first experiment, when a baffle and funnel were tested directly against eachother, the trapping device with a funnel caught significantly more mosquitoesthan the one with a baffle (χ2-test, P<0.001) (Fig. 3). A worn sock caught signifi-cantly more mosquitoes than a clean sock (χ2-test, P<0.001). The total responsewas significantly higher when a sock was used compared to the other treatments(GLM, P<0.05). No significant effect was found of skin washings, baffles or fun-nels on the total response (Fig. 3). The experiments with clean air only showedthat the trapping system was symmetrical with a total response of 21-24%.


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Figure 3. Average response of An. gambiae when trapping devices were equipped withbaffles or funnels. Skin washings (SW) or worn socks were used as an attractant.Error bars represent standard errors of the mean; ***: χ2-test P<0.001. R=Total responsein both trapping devices, data not sharing the same superscript letter differ significant-ly (GLM, P<0.05).

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In the second experiment, no significant difference between a baffle and afunnel was found when they were tested directly against each other (χ2-test,P=0.1967) (Fig. 4). Again a worn sock caught significantly more mosquitoes thana clean sock (χ2-test, P<0.001). The total response was significantly higher whenfunnels were used in both trapping devices than when baffles were used (GLM,P<0.05) (Fig. 4).

DISCUSSIONWhen baffles and funnels were tested directly against each other, the two exper-iments showed conflicting results. In the first experiment, the trapping devicewith a funnel caught significantly more mosquitoes than the trapping deviceequipped with a baffle. In the second experiment no significant difference wasfound between the two types of trap entrance. The only difference between theexperiments was the use of skin washings in the first experiment and wornsocks in the second experiment. Worn socks were highly attractive, skin wash-ings were not. Maybe this shows the discriminative power of a funnel when anodour is only slightly attractive. Previous experiments have shown that skinwashing samples can be attractive to An. gambiae (Pates 2002, van Agtmaal2006), whereas our results did not show any attractiveness of the samples.Although van Agtmaal (2006) showed that a dilution of 1:1000 of the originalsample showed maximum attractiveness, storage might have had a negativeinfluence on the concentration or composition of the original sample. Furtherresearch should indicate what affected the skin washing samples and what madethem scarcely attractive to An. gambiae.

Because the first experiment showed that skin washing samples were not

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Figure 4. Average response of An. gambiae when trapping devices were equipped withbaffles or funnels. Worn socks were used as an attractant. Error bars represent stan-dard errors of the mean; ***: χ2-test P<0.001. R=Total response in both trappingdevices, data not sharing the same superscript letter differ significantly (GLM, P<0.05).

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attractive, worn socks were used in the second experiment to increase the totalnumber of mosquitoes caught. In this second experiment, the total number ofmosquitoes caught when using a funnel was significantly higher than whenusing a baffle. Thus, trapping devices equipped with a funnel-like entrance aremore efficient than traps equipped with a baffle. When odours are tested whichonly differ little in their attractiveness, increasing the total number of mosqui-toes trapped by using a funnel can make it easier to distinguish which odour ismore attractive. Another advantage of the funnel is that it is easier to fit in thetunnel and less breakable than the glass baffle.

The aim of research on odours that may play a role the host seeking behav-iour of mosquitoes is to develop a trap which can be used to catch and/or mon-itor mosquitoes in the field. The results presented here should be taken intoaccount when developing new traps.

Acknowledgements We are grateful to F.K.M. van Aggelen, A.J. Gidding and L.Koopman for rearing mosquitoes and Yu Tong Qiu for providing skin washing samples.This project was funded by the Bill & Melinda Gates Foundation (GCGH#121).

REFERENCESAcree, F., Jr., R.B. Turner, H.K. Gouck, M. Beroza & N. Smith, 1968. L-Lactic Acid: A

mosquito attractant isolated from humans. Science 161: 1346-1347.Agtmaal van, M., 2006. Masking the attractiveness of human odours to mosquitoes: A

dual-port olfactometer study which investigated the putative allomonal effects ofseveral selected human volatiles on Anopheles gambiae s.s. host seeking behaviour.Msc-thesis, Laboratory of Entomology, Wageningen University, 71 pp.

Braks, M.A.H. & W. Takken, 1999. Incubated human sweat but not fresh sweat attractsthe malaria mosquito Anopheles gambiae sensu stricto. Journal of Chemical Ecology 25:663-672.

Eiras, A.E. & P.C. Jepson, 1991. Host location by Aedes aegypti (Diptera Culicidae): awind tunnel study of chemical cues. Bulletin of Entomological Research 81: 151-160.

Geier, M. & J. Boeckh, 1997. A new Y-tube olfactometer for mosquitoes to measure theattractiveness of host odours. Entomologia Experimentalis et Applicata 92: 9-19.

Gillies, M.T. & T.J. Wilkes, 1969. A comparison of range of attraction of animal baitsand of carbon dioxide for some West African mosquitoes. Bulletin of EntomologicalResearch 59: 441-456.

Killeen, G., J. Kihonda, E. Lyimo, F. Oketch, M. Kotas, E. Mathenge, J. Schellenberg, C.Lengeler, T. Smith & C. Drakeley, 2006. Quantifying behavioural interactionsbetween humans and mosquitoes: Evaluating the protective efficacy of insecticidalnets against malaria transmission in rural Tanzania. BMC Infectious Diseases 6:161.

Knols, B.G.J., R. de Jong & W. Takken, 1994. Trapping system for testing olfactoryresponses of the malaria mosquito Anopheles gambiae in a wind tunnel. Medical andVeterinary Entomology 8: 386-388.

Knols, B.G.J. & J. Meijerink, 1997. Odors influence mosquito behavior. Science and


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Medicine 56-63.Maxwell, C.A., J. Wakibara, S. Tho & C.F. Curtis, 1998. Malaria-infective biting at dif-

ferent hours of the night. Medical and Veterinary Entomology 12: 325-327.Pates, H., 2002. Zoophilic and anthropophilic behaviour in the Anopheles gambiae com-

plex. In: University of London, London.Pates, H.V., W. Takken, K. Stuke & C.F. Curtis, 2001. Differential behaviour of

Anopheles gambiae sensu stricto (Diptera: Culicidae) to human and cow odours in thelaboratory. Bulletin of Entomological Research 91: 289-296.

Qiu, Y.T., R.C. Smallegange, S. Hoppe, J.J.A. van Loon, E.J. Bakker & W. Takken,2004. Behavioural and electrophysiological responses of the malaria mosquitoAnopheles gambiae Giles sensu stricto (Diptera: Culicidae) to human skin emanations.Medical and Veterinary Entomology 18: 429-438.

Qiu, Y.T., 2005. Sensory and behavioural responses of the malaria mosquito Anophelesgambiae to human odours. Phd Thesis, Wageningen University, 207 pp.

Qiu, Y.T., R.C. Smallegange, J.J.A. Van Loon, C.J.F. Ter Braak & W. Takken, 2006.Interindividual variation in the attractiveness of human odours to the malaria mos-quito Anopheles gambiae s.s. Medical and Veterinary Entomology 20: 280-287.

Smallegange, R.C., Y.T. Qiu, J.J.A. van Loon & W. Takken, 2005. Synergism betweenammonia, lactic acid and carboxylic acids as kairomones in the host-seeking behav-iour of the malaria mosquito Anopheles gambiae sensu stricto (Diptera: Culicidae).Chemical Senses 30: 145-152.

Takken, W., 1991. The role of olfaction in host-seeking of mosquitoes: a review. InsectScience and its Application 12: 287-295.

Takken, W. & B.G.J. Knols, 1999. Odor-mediated behavior of afrotropical malaria mos-quitoes. Annual Review of Entomology 44: 131-157.

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J. Beeuwkes, J. Spitzen, C.W. Spoor, J.L. van Leeuwen & W. TakkenLaboratory of Entomology, Wageningen University, PO Box 8031, 6700 EHWageningen, The Netherlands, E-mail: [email protected]

A wind tunnel and a novel auto detecting digital video system wereused to study the flight behaviour of the nocturnal malaria mosquitoAnopheles gambiae Giles sensu stricto. Females of this anthropophilicmosquito need to locate a host to obtain a blood meal in order to devel-op eggs. This host-seeking behaviour is mainly guided by olfactorystimuli, but little is known to what extent these stimuli affect flightand landing parameters.In this study single female mosquitoes were exposed to natural andsynthetic human odours in a wind tunnel. Flight behaviour was digi-tally recorded and with the use of computer programmes the 3-dimen-sional (3-D) flight track of the mosquito was reconstructed. From theobtained tracks, the amount of plume contact, average flight speed andflight angles were calculated.Mosquitoes were found to significantly reduce their flight speed, track-and course angle when they were inside a plume of odours. Drift anglewas not different inside and outside the odour plume. The flight speedwas reduced when the mosquitoes came nearer to the odour source.The observed responses to host-stimuli can be explained by the noctur-nal activity pattern of this mosquito. Reducing flight speed and courseangle may be an efficient strategy to stay inside the trail of odour mol-ecules when flying in the dark. It is important to know more about theflight behaviour of this malaria vector in response to odours, because itmay help in the development or improvement of odour-baited mosqui-to traps.

Keywords: Anopheles gambiae, 3-D, flight behaviour, odour plumes, autodetection

Appetitive female haematophagous mosquitoes (Diptera: Culicidae) are exposedto a wide variety of visual, olfactory and physical stimuli. These stimuli can beused by the mosquito during host-seeking in order to obtain a blood meal.Olfactory stimuli have been shown to be most important at long ranges from ahost (>5 m) (Dekker et al. 2005 and references therein). At smaller distances (<1

3-D flight behaviour of the malaria mosquitoAnopheles gambiae s.s. inside an odour plume


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m), heat and water vapour also become important to get into contact with thehost (Healy et al. 2002, Kellogg & Wright 1962, Takken et al. 1997).

Host seeking of a mosquito can be imagined as a journey through an odourtrail. But the trail of odour molecules, mostly referred to as plume, is not a cone-shape cloud with straight edges. In a natural windy situation, an odour plume ishighly heterogeneous and not perfectly cone-shaped, because of turbulent diffu-sion. Turbulence separates both odour filaments bundles and filaments within abundle by regions of clean air (Murlis et al. 1992). Therefore instantaneous winddirections do not necessarily provide reliable information about the directiontoward an odour source (Brady et al. 1989).

The source size and shape together with the wind speed and direction willinfluence the degree of dilution of the odour. Overall it can be said that gaps ofclean air will expand as the plume travels away with distance from the source(Murlis et al. 2000). The temporal and spatial fluctuating odour concentrationswithin the plume body are referred to as fine-scale plume structure and havebeen shown to be important in instantaneous responses of insects (Dekker et al.2001, Mafra-Neto & Cardé 1994, 1995b, Murlis et al. 1992).

In order to find an odour source, an insect needs to orientate itself. Kennedy(1939) showed that the mosquito Aedes aegypti (L.) responds well to visual stim-uli. From this and many other studies it is believed that flying insects appraisetheir movement visually by comparing the course heading with the apparentdirection of ground movement (Bell & Cardé 1984 and references therein). Thevisual control of flight speed and course angle by reaction on the flow of groundimages is called optomotor anemotaxis.

Flights can be described by looking at specific flight parameters such as flightspeed and flight angles. Therefore a flight is simplified as a sequence of coordi-nates, where each flight coordinate is a vector (motion consisting of both a mag-nitude and direction) directed to the next coordinate (see Fig. 1). The flight trackis the result of the connections between all the courses steered by the mosquitoand the wind-drift at any instant.

Flight behaviour has mainly been studied in male moths attracted topheromone sources (Justus & Cardé 2002, Marsh et al. 1978, Schofield et al. 2003).Flight behaviour of these relatively large insects is generally projected in onlytwo dimensions and thus the flight misses the Z-component in the analysis.However, current techniques facilitates complicated analysis methods and 3-Dflight behaviour of even very small insects is reported increasingly: mosquitoes(Cooperband & Cardé 2006, Dekker et al. 2005), aphids (El-Sayed et al. 2000) andfruit-flies (Budick & Dickinson 2006). This paper examines the flight behaviourof the mosquito Anopheles gambiae Giles sensu stricto (henceforth termed An. gam-biae). Its response to natural human odours and the synthetic components lacticacid (LA) and ammonia (NH3) is tested. Flight parameters inside and outside anodour plume are compared to assess behavioural effects of plume contact.

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MATERIAL AND METHODSFemales of the An. gambiae colony at Wageningen University, The Netherlands,of 5-8-days old were used for the experiments. The mosquitoes were non-blood-fed and not previously exposed to host odours in a bioassay set-up. SeeSmallegange et al. (2005) for mosquito rearing details. Individual mosquitoeswere collected randomly from a cage with a suction tube and placed in cylindri-cal plastic cups (diameter 5 cm, height 3 cm) at least 16 hours prior to testing.Side-walls of the cups were perforated with 12 holes and damp cotton wool wasoffered to avoid dehydration.

A Lexan polycarbonate wind tunnel (180 x 60 x 60 cm) depicted in Figure 2was used as flight arena. The side walls and bottom of the flight chamber (120 x60 x 60 cm) were covered with black polycarbonate. The experimental room wasmaintained in darkness, but infrared (IR) lights placed at the downwind-side ofthe wind tunnel were used for illumination.

Air was withdrawn from the experimental room by a fan, led through acti-vated charcoal and a nylon tube (diameter 15 cm) before it entered the wind tun-nel. Subsequently, the air-flow passed a humidifying cloth, an aluminium lami-nation screen and black-painted stainless steel mesh screen. The wind speed was0.2 m/s, measured right in front of the mesh screen. Immediately behind thescreen in upwind direction, a glass funnel was placed, which was used to gener-ate an odour plume. For each treatment, different odour stimuli were placed inthe funnel. A worn sock, which is a known attractive natural human odoursource to An. gambiae (Pates et al. 2001), was used as a positive control. Ammoniaand lactic acid (LA) were tested separately and in combination with each other,where NH3 was tested in two concentrations (low: 136 and high: 1,363 ppm).Ammonia was transferred from a gas bag to the funnel by silicon tubes using a

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Figure 1. Construction of the triangle of velocities between consecutive points of a track(t, t + 1) in the XY plane after Marsh et al. (1978). Thick arrows indicate known vec-tors (track, wind). The broken arrow indicates the inferred vector (course). Arcs indi-cate course (γ), drift (δ), and track (τ) angles (Kerguelen & Cardé 1997).

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flow of 230 ml/min. Liquid LA was transferred from a wash bottle to the funnelby a flow of pressurized air (15 ml/min) through silicon tubes. All treatmentswere run in the presence of an artificial heat source (34°C) in the funnel to sim-ulate heat from a human skin (Clements 1999, Healy et al. 2002).

Tests were performed in the last 3.5 hours of the dark period. Single mosqui-toes were released from a release platform 110 cm downwind from the source inthe centreline of the wind tunnel. If the mosquito did not take off within twominutes, the experiment was aborted.

The flights were recorded with two monochrome CCD video cameras thatwere placed in the longitudinal axis of the wind tunnel (parallel to the air-stream) with an angle of 40° relative to each other. The effective stereoscopicview was ± 60 x 60 x 60 cm.

The video files were saved as MPEG-2 and 2-D tracks were subtracted usingan auto detecting computer program (Ethovision 3.1, Noldus IT, Wageningen,The Netherlands). Subsequently the two track files were exported to a pro-gramme written in Matlab 7.0 (Mathworks) that calculated 3-D coordinates ofthe mosquito. Several parameters were calculated by the programme; flightspeed was computed as product of the motion in three dimensions and flightangles were calculated in the XY-plane. Smoke was used to assess the dimen-sions of the odour plume, and these dimensions were inserted in the Matlab pro-gram. In this way, the program calculated for each coordinate of the mosquito’sflight whether it was inside or outside the odour plume.

Statistics were performed with SPSS 12.0.1. Flight analyses were performedover a distance of 50 cm. Upwind flight angles and flight speed inside the plumewere compared with the same parameters when flying outside the plume.

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Figure 2. Wind tunnel set-up – Air inlet (AI), humidifying cloth (HC), laminationscreen; (LS), glass funnel containing heat element (F), mesh screen (S), release cup(RC), cameras (C1,2), IR lights type 1 (IR1), IR lights type 2 (IR2).

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Therefore all upwind directed parameters within or outside the plume bound-aries were averaged per mosquito. Differences between the averages werechecked for normality and tested in a one sample T-test. In this way each mos-quito contributed equally to the sample means.

RESULTS AND DISCUSSIONOf the 245 mosquitoes tested in the wind tunnel, 156 (64%) took off and wereobserved in one of the camera views. From the plume analyses in Matlab it turnedout that 156 of the recorded mosquitoes, only 37 had been in contact with the plume.The proportion of plume contact within a flight varied from 0.1-49% between mos-quitoes. Flight paths of the mosquitoes were shown to be performed in all threedimensions. When mosquitoes never contacted the plume, flight paths were ratherstraight. Mosquitoes that flew a considerable proportion of their flight within theplume boundaries, showed longer and more complicated flight paths (Fig. 3).

When mosquitoes were only a short period of time in contact with theplume, they did not seem to show a behavioural response to offered odour. Inassessing the effect of plume contact, possible effects could be masked by theseweakly or not responding mosquitoes. Therefore the geometric mean of plumecontact proportions was calculated (5.6%) and was used as a threshold for analy-sis. A total of 14 mosquitoes had been within the plume dimensions >5.6% oftime. Because these flights were divided over 5 different odour treatments, theywere pooled to have sufficient replicates. Of these 14 flight tracks, 7 mosquitoeswere exposed to a worn sock, 3 to NH3 at low concentration, 2 to NH3 at highconcentration, 1 to LA and 1 to NH3 at low concentration + LA.

Figure 4 shows that the flight speed inside a plume of odours was significant-ly smaller than outside the plume (P<0.01). Track angle and course angle insidethe plume were also significantly smaller than outside the plume (P<0.05 and


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Figure 3. Two examples of 2D-projected flight tracks of mosquitoes exposed to odoursfrom a worn sock. Flight path of a mosquito that had no contact with the plume (a.),flight in which the plume was repeatedly contacted (b.).

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P<0.01, respectively). Drift angle inside the plume was not significantly differentfrom outside the plume (P=0.52). From a study with a different mosquito, Ae.aegypti, it was also shown that track angle was decreased inside a plume. Flightspeed of this mosquito, however, remained fairly constant (Dekker et al. 2005).This difference may be explained by the difference in activity pattern. Anophelesgambiae searches a host during the night and mostly indoors, while Ae. aegypti ismainly active during day-time. Aedes aegypti responds well to visual cues(Kennedy 1939) and visual cues are used for orientation during host seeking(Bidlingmayer 1994, Muir et al. 1992). Anopheles gambiae also seems to use visualcues to orientate (Gibson 1995, Gibson & Torr 1999), even under low light condi-tions, but possibly to less extent than Ae. aegypti. Flying towards a host in dark-

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Figure 4. Average (+SE) upwind flight speed (A) and flight angles (B) inside and out-side the odour plume (* P<0.05; ** P<0.01).

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ness may be more efficient for An. gambiae when flight speed is lowered and theflight is more aimed at the source upon plume contact and vice versa upon loss ofcontact. However, the adaptive advantage of a longer and thus more costly pathupon plume contact remains unclear. In male moths it was also shown that flightangles became smaller when the plume was contacted (Mafra-Neto & Cardé 1994,1995a, 1995b, Vickers & Baker 1996). But contrary to An. gambiae mosquitoes, flightspeed was increased inside an odour plume (Mafra-Neto & Cardé 1998). Thisvariation in behavioural responses of insects with different ecology and phyloge-ny may indicate differences in the underlying processes at the neural level.

To analyse differences in flight parameters in relation to the mosquito’s dis-tance from the odour/heat source; the plume was divided in four distance class-es with a length of 12.5 cm. When a mosquito had >20 upwind coordinates in eachdistance class, parameter values were averaged and compared between distanceclasses. This applied for 7 of the 37 mosquitoes of which 4 were exposed to asock, 2 to NH3 at high concentration and 1 to the combination of NH3 at low con-centration + LA. With decreasing distance to the source, flight speed decreased,and the difference was significant between class 1 and 3 and between 1 and 4(P<0.05; see Fig. 5). P-values in comparisons of the other classes were: 1 and 2(P=0.42); 2 and 3 (P=0.13); 2 and 4 (P<0.16); 3 and 4 (P=0.62). Track angle seemedfairly constant over distance. Only between class 2 and 3 a significant difference


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Figure 5. Average (+SE) upwind flight speed (A) and track angle (B) inside the odourplume at different distance classes from the source. Class definitions: 1. 0-12.5 cm; 2.12.6-25 cm; 3. 25.1-37.5 cm; 4. 37.6-50 cm. Bars that share no letters above differ signifi-cantly at P<0.05.

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was found (P<0.05). P-values in comparisons of the other classes were: 1 and 2(P=0.80); 1 and 3 (P=0.35); 1 and 4 (P<0.44); 2 and 4 (P=0.16); 3 and 4 (P=0.54). Thereduction in flight speed closer to the source may be caused by the sensing ofheat emitted by the heat element and considered a response in preparation of alanding. Healy et al. (2002) found that significantly more An. gambiae mosqui-toes landed on heated cylinders when they were near an object with a tempera-ture similar to that of human skin. The behavioural change may also be causedby a change of plume structure.

As mentioned before, plume dimensions were assessed by artificial smoke. Itwas seen that the plume increased in turbulence with distance. The observedincrease in speed with distance may also be a response to the larger size of air-gaps between odour filaments. Changing speed may provide continuous stimu-lation of olfactory neurons. In a moth species, Cadra cautella, it is also suggestedthat an extreme flickering signal may be perceived as a fused signal (Justus &Cardé 2002, Justus et al. 2002).

This study shows interesting flight behavioural responses of a malaria mos-quito by using novel auto-detecting software. Increased knowledge on the flightbehaviour of this insect may help us to develop or improve odour-baited traps.When it becomes clearer how mosquitoes navigate into an odour-baited trap,design of those traps may improve, although attractants that can compete withhuman odours still need to be discovered. The current set-up may also be also bea useful tool for studying different insects, for instance parasitic wasps usingherbivore-damaged plant volatiles to find a host.

Acknowledgments We thank Frans van Aggelen, André Gidding and LeoKoopman for rearing of the mosquitoes. Lucas Noldus and Fabrizio Grieco are acknowl-edged for their effort in developing a 3-D tracking system.

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Marsh, D., Kennedy, J.S., & Ludlow, A.R. (1978) Analysis of anemotactic zigzaggingflight in male moths stimulated by pheromone. Physiological Entomology, 3, 221-240.

Muir, L.E., Kay, B.H., & Thorne, M.J. (1992) Aedes-Aegypti (Diptera, Culicidae) Vision– Response to stimuli from the optical environment. Journal of Medical Entomology,29, 445-450.

Murlis, J., Elkinton, J.S., & Cardé, R.T. (1992) Odor plumes and how insects use them.Annual Review of Entomology, 37, 505-532.

Murlis, J., Willis, M.A., & Cardé, R.T. (2000) Spatial and temporal structures ofpheromone plumes in fields and forests. Physiological Entomology, 25, 211-222.


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Pates, H.V., Takken, W., Stuke, K., & Curtis, C.F. (2001) Differential behaviour ofAnopheles gambiae sensu stricto (Diptera: Culicidae) to human and cow odours in thelaboratory. Bulletin of Entomological Research, 91, 289-296.

Schofield, S.W., Justus, K.A., Mafra-Neto, A., & Cardé, R.T. (2003) Flight of maleCadra cautella along plumes of air and pheromone superimposed on backgrounds ofpheromone. Entomologia Experimentalis et Applicata, 109, 173–181.

Smallegange, R.C., Qiu, Y.T., , Loon van, J.J.A., & Takken, W. (2005) Synergismbetween ammonia, lactic acid and carboxylic acids as kairomones in the host-seek-ing behaviour of the malaria mosquito Anopheles gambiae sensu stricto (Diptera:Culicidae). Chemical Senses, 30, 145–152.

Takken, W., Knols, B.G.J., & Otten, H. (1997) Interactions between physical and olfac-tory cues in the host-seeking behaviour of mosquitoes: The role of relative humidi-ty. Annals of Tropical Medicine and Parasitology, 91, S119-S120.

Vickers, N.J. & Baker, T.C. (1996) Latencies of behavioral response to interception of fil-aments of sex pheromone and clean air influence flight track shape in Heliothisvirescens (F) males. Journal of Comparative Physiology A-Sensory Neural and BehavioralPhysiology, 178, 831-847.

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Lia Hemerik & Egbert H. van Nes*Biometris, Department of Mathematical and Statistical Methods, WageningenUniversity, PO Box 100, 6700 AC Wageningen, The Netherlands, E-mail:[email protected]; *Department of Aquatic Ecology and Water QualityManagement, Wageningen University, PO Box 47, 6700 AA Wageningen, TheNetherlands

For many practical purposes it is useful to predict the development rateof insects, for instance because not all stages of a pest insect are equal-ly damaging crops. As insects are poikilothermous, their developmen-tal rate is strongly influenced by temperature. In the literature theduration of the development of egg, larval and pupal stages of insectsis mostly reported at constant temperatures. In an orchard or in cropfields however, fruit growers might like to predict when some stages ofpest insect can be expected based on the current temperature. Measuresto eradicate such a pest species can then be taken at the right time.Here, we present a new version of the age-structured insect simulationmodel INSIM that was originally developed by Diederik and Mols(1996). The model is re-implemented in MATLAB and can now simu-late multiple seasons. It calculates the occurrence of insect stages dur-ing the year based on daily minimum and maximum temperatures.The model needs detailed information on sex ratio, relative mortalityand developmental times at different temperatures. Furthermore, it isnecessary to know the threshold value for the development, and thethreshold value for the reproduction of adults. If more than one gener-ation is simulated, similar information of reproductive capacity ofadults is needed. The model can also take into account that there areindividual differences in development time, resulting in a dispersion ofstages. We describe the input tables for the program in detail and givethe outcome for the western corn root borer Diabrotica virgifera vir-gifera.

Keywords: degree-day, phenology, prediction, dynamics

In orchards earwigs Forficula auricularia (L.) are polyphagous predators of pestspecies, like several apple aphid species and pear psyllid (Solomon et al. 2000).These species are important for integrated pest management in such environ-

A new release of INSIM: A temperature-dependent model for insect development


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ments, but both the amounts of earwigs and the time at which they appear showa large variation over the years. The timing of the appearance of adults is relat-ed to the temperature, and there are rules of thumb that relate this to the tem-perature sum above a threshold temperature (Helsen et al. 1998).

Mols et al. (1998) describe one of the first studies that used a more precise esti-mation of temperature-dependent development for discovering optimummoments for monitoring and control. They studied three Orthosia species.Larval emergence and development were predicted, with the Quick Basic com-puter program INSIM. Results of the simulations with the model (Diederik &Mols 1996) were in close agreement with field observations. The heat sum forembryonic development was determined by the model as 72 degree days above adevelopmental threshold of 7.0°C. This model was also successfully used to pre-dict the emergence of Dasineura tetensi adults (Cross & Crook 1999) and thereproduction of the western corn rootworm, Diabrotica (virgifera) virgifera(Hemerik et al. 2004).

The INSIM program that was used in these examples, uses the developmentrate values as measured under laboratory conditions. The model calculates dailydevelopment amounts from daily maximum and minimum air temperatures andthe temperature sum as an accumulation of these amounts. The model uses box-car trains (see Goudriaan & van Roermund 1989) to simulate developmental dis-persion. When we tried to use the Quick Basic implementation of INSIM, wediscovered several shortcomings of the program: (1) the necessary data was noteasily imported into INSIM, although the help function told us that space-delimited text files in a certain format should work, (2) the program could notrun for a longer time span than a year, which is not enough for simulating thelife cycle of, for instance, click beetles (Elateridae) that can live 3-7 years as wire-worms in the soil (Kabanov 1975), and (3) if the program was used withoutreproduction and with constant developmental rate for a few stages, some neweggs were introduced due to numerical errors after a while. These shortcomingsof the original INSIM program made it hard to start simulating insects forwhich temperature data could be found in the literature. Therefore, we decidedto develop a new implementation of INSIM. Here we report the results of somesimple tests and the results for the development of D. virgifera (Hemerik et al.2004).


Structure of the INSIM modelINSIM applies the boxcar train technique (Goudriaan & van Roermund 1989) tosimulate the distribution of individuals over the various life stages. In thisapproach each particular life stage (called the ‘boxcar train’) is subdivided in sev-eral substages (classes or ‘boxcars’). It uses the ‘escalator boxcar train’ for stages

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that have a negligible variance in development rates and the ‘fractional boxcartrain’ to apply an individual variance in development. In this latter case, thenumber of boxcars cannot take any number. The new implementation ofINSIM calculates the optimal number of boxcars and the optimal step size of theEuler integration (see Goudriaan & van Roermund 1989). The average develop-ment time, the dispersion in development time and the mortality rate at eachtime step in the simulation are determined by linear interpolation of tables inwhich these values are given for different temperatures.

Simulation example

Life cycle and feeding habits of the western corn rootwormThe species D. virgifera is uni-voltine and thus completes one generation peryear (Fig. 1). In the soil, during summer, female deposit their eggs. These hiber-nate and hatch in late May or in early June. D. virgifera’s first larval stage feedson fine root hairs and burrows into the root tips of corn whereas later stages. Thewhite ‘worms’, i.e. larval stages two and three, feed on primary roots. Larvae ofthe third instar pupate in the soil and emerge as adults in July and August. Malesemerge before the females. Mating takes place soon after emergence. Femalesstart ovipositing after two weeks. The adult beetles are approximately 5-6 mmlong and feed on corn silk or leaf tissue. The duration of each stage (egg, larvalinstars L1-L3 and pupae) depends mainly on temperature.

Necessary inputsThe implementation of the model for D. virgifera in INSIM requires informa-tion concerning (a) the sex ratio, (b) the life cycle, (c) the temperature-depend-ent development in each life stage, (d) the threshold values for development inthese life stages, (e) the temperature-dependent mortality, (f) a threshold valuefor oviposition and (g) the age-dependent number of eggs laid per female per dayat certain temperatures. All this information is entered in INSIM as a tab-delim-ited text file, with labeled (square brackets) tables. We explain all tables that areneeded as input for INSIM in detail.

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Figure 1. The life cycle of the uni-voltine western corn rootworm in the course of oneyear.

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In the table [oovveerrvviieeww] the name (not obligatory) and symbol (obligatory,preferably short) for each stage in the life cycle of the insect are denoted (see Table1). The sequence of developmental stages is defined in another column where it isstated from which of the other stages the input stems for the current stage. Itshould be noted that ‘NaN’ (or ‘not a number’) stands for no developmental inputfor the egg stage. This stage gets its input from the reproduction by adults (seebelow, where we describe the table [rreepprroodduuccttiioonn]). Note that the input for the L1female states that it is 0.5*Diapeggs. Here we assumed a 1:1 sex ratio to assure thatwe are only modeling female stages. The last column gives the number of classesinto which the stage should be subdivided in the computer program.

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Table 1. In table [overview] the coupling of the life stages is defined.

[overview]Name Symbol Input Classes egg egg NaN 10Diapause eggs Diapeggs egg 5L1 Female FL1 0.5*Diapeggs 5L2 Female FL2 FL1 10L3 Female FL3 FL2 10Pupal Female FPup FL3 10Preoviposition Preovi FPup 5Adult female FAdult Preovi 5

Table 2. An example of a table that contains the temperature-dependent developmentand mortality data (for the female second larval instar [FL2])).

[fl2]temp Development time Standard deviation Relative mortality rate0 NaN NaN NaN5.5 43.1 1.8 0.00737.8 30.4 1.7 0.010368.3 28.6 1.7 0.011029.6 24.7 1.7 0.012759.7 24.4 1.7 0.012889.8 24.2 1.7 0.0130110.5 22.6 1.6 0.0139411.2 21.2 1.6 0.0148715 15.8 1.5 0.0199218 7.1 0.5 0.0196121 5.4 0.6 0.0154424 4.9 0.9 0.0237827 4.1 0.6 0.017730 3.4 0.6 0.0409631.5 3.5 0.5 0.16566

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After having defined the life cycle as described above tables for each stageshould be defined separately, e.g. [ttaabbllee] FFLL22 (Table 2). For each temperature forwhich data on development time and mortality are known from the literature arow is added to the table for the stage. The first row below the headers ‘temper-ature’, ‘development time’, ‘standard deviation’ and ‘relative mortality rate’should hold the threshold temperature for development followed by NaNrepeated three times. The example given in Table 2 includes ‘development time’and the ‘standard deviation’ both in days. It should be noted that in INSIM forMatLab it is also possible to define a table with row headers ‘temperature’,‘development rate’, ‘standard deviation’ and ‘relative mortality rate’. In that case‘development rate’ and its ‘standard deviation’ are both presumed to be given asrate per day. The ‘relative mortality rate’ has also the dimension per day. At atemperature of for instance 31.5°C the relative mortality rate is 0.16566 per day(Table 2). This implies that during the mean development time of 3.5 days a frac-tion 1−exp(−0.16566*3.5)=0.44 of the individuals have died. So for calculating theinput for INSIM if we know that a fraction f has died during the total develop-ment time D, then we can compute the relative mortality rate m as (−ln(1−f))/D.

This far we have defined the life cycle and the temperature-dependent devel-opment and mortality in a total of nine tables (one for the overview and one foreach stage). We however, have not dealt with reproduction yet. The table for thereproducing stage, here the adult females of D. virgifera, FAdult, is defined astable [rreepprroodduuccttiioonn] FFAAdduulltt →→ eegggg (see Table 3). The number of classes that hasto be defined for oviposition in table [rreepprroodduuccttiioonn] is the same number as thenumber of classes for the reproducing stage, which was defined in the[oovveerrvviieeww] table, here 5. The number of eggs per day per class of adults is readfrom Table 3 (with linear interpolation). For instance in the last row of Table 3it states that at a temperature of 30°C the adult females have an egg productionof 12.3809 per day during the first 1/5th part of their average development timeof their adult life. During the second 1/5th of their adult life the egg production


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Table 3. Age-dependent and temperature-dependent reproduction (from Elliott et al.1990). At each temperature, the maximum longevity is divided into five classes ofequal length. Reproduction is given as the number of eggs laid per day in these classes.

[REPRODUCTION] FAdult → eggTemp. Class 1 Class 2 Class 3 Class 4 Class 515 0.0000 0.0000 0.0000 0.0000 0.000016 1.1429 4.5714 4.2857 4.2857 0.571419.5 2.8571 10.3571 3.9286 1.4285 1.071423 9.6428 7.8571 2.8571 0.9523 0.000026.5 15.2380 11.4285 1.9047 0.9523 0.000030 12.3809 10.0000 7.1400 3.5714 0.0000

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is 10 eggs per day and so on. In the INSIM program the total egg production iscalculated using a simple matrix multiplication.

The last required input table is the file with daily minimum and maximumtemperature for a period of one year or longer e.g. ddeebbiillttddiiaabb..ttxxtt (see Table 4 forthe first rows). In the temperature file three different columns have to bedefined: ‘day number’, ‘the minimum temperature on that day Tmin’ and ‘themaximum temperature on that day Tmax’. In the simulation INSIM uses equa-tion 1 for determining the current temperature from the minimum and maxi-mum temperatures on that day. The day starts at midnight with the minimumtemperature from the file and at noon the maximum temperature is reached.Thereafter, the temperature starts falling again up to the next midnight, wherethe next day starts with the new minimum temperature.

(1)

How to use INSIM for MatLabAfter having made a Tab-delimited text file, say ffiilleennaammee..ttxxtt, with all necessaryinformation on the life cycle, the temperature-dependent development in eachlife stage including threshold values for development, the temperature-depend-ent mortality, and the age-dependent number of eggs laid per female per day atcertain temperatures together with a threshold value for oviposition firstINSIM for MatLab has to be installed. For the installation procedure, we referto http://www.aew.wur.nl/uk/insim/ where INSIM for MatLab will be freelyavailable as part of the package GRIND for MatLab. After the installationINSIM for MatLab can be started in Matlab by typing: ‘insim ffiilleennaammee..ttxxtt[return]’, and ‘time [return]’. If the text file is correct, the simulation results forthe time span of one year will appear in two figures: one figure with the mini-mum and maximum temperatures in the course of the year and the second fig-ure with the appearance and disappearance of life cycle stages throughout the

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Table 4. An example of the start of a climate file that includes the daily minimum(Tmin) and maximum temperature (Tmax).

debiltdiab.txtt Tmin Tmax1 -1.4 1.22 -4.1 03 -5.3 -0.14 -6 -0.15 -5.5 1.9... … …

( )max min max min( ) cos 2 tT T T T

T t π+ −⎛ ⎞ ⎛ ⎞= −⎜ ⎟ ⎜ ⎟⎝ ⎠ ⎝ ⎠2 2

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simulated year. For insects with a life cycle that lasts more than one year thesimulation time can be set at the preferred length, say 1000, by typing ‘simtime0 1000’ in MatLab after the ‘insim ffiilleennaammee..ttxxtt [return]’ command line. It shouldbe noted that we also plan to have at least one fully worked out example on thewebsite and a more detailed manual.

RESULTSThe first test of INSIM for MatLab was comparing the results of a single stagewith a constant death rate at different temperature with an exponential decayfunction. This first test was easily passed and the deviation between the expo-nential model and the simulation was smaller than in the original Quick Basicimplementation of INSIM.

In a second test we tested a single stage with a fixed development time of 100days and a standard deviation of 20 days. The average development time and dis-persion of last substage closely resembled a gaussian curve with a standard devi-ation of 20 days.

Finally we modelled the development of D. virgifera using the filesddaayy__ddiiaabb..ttxxtt (with the [oovveerrvviieeww] table, the tables for the different stages, theinitial values in the stages and the reference to the name of the temperature file)and the temperature file debiltdiab.txt to get the resulting time plots. From thesethe one with the occurrence of the life cycle stages throughout one year is repro-duced in Figure 2. We compared the resulting occurrences with the data provid-ed by EPPO (1995) (Table 5). A rather good similarity exists.


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Figure 2. The occurrence of the different female stages of D. virgifera in the course ofone year. From the left to the right the diapause eggs are starting to develop fromJanuary 1 onwards, and only around day 150 the first larval stage appears, the pupaeappear around day 150 and both the ovipositing females (Fadult) and the eggs start toappear at day 210.

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CONCLUSIONThe advantages of the current implementation of the INSIM model are that

(1) temperature-dependent development and survival can be easily provided tothe MatLab programme in a Tab-delimited text file,

(2) the program is suitable for insects with egg to egg development times thatexceed one calendar year,

(3) the program is suitable for insect with life stages that have different develop-mental thresholds

(4) the program is faster and more flexible than the original Quick Basic pro-gramme,

(5) the new program is user friendly, and last but not least:

(6) it will be freely available from the internet (http://www.aew.wur.nl/uk/insim/).

Acknowledgements We thank Dirk Diederik for explaining the main parts of theoriginal Quick Basic program to us and for his enthusiasm in doing so. Joop vanLenteren from the Laboratory of Entomology at Wageningen University, upon request,allowed us to develop a new version of the Quick Basic program in MatLab.

REFERENCESBernardi M (2001) Linkages between FAO agroclimatic data resources and the develop-

ment of GIS models for control of vector-borne diseases. Acta Tropica, 79: 21-34.Elliott NC, Lance DR and Hanson SL (1990) Quantitative description of the influence

of fluctuating temperatures on the reproductive biology and survival of the westerncorn-rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera, Chrysomelidae).Canadian Entomologist 122: 59-68.

EPPO (1995) Report of the 1st international Meeting on Diabrotica virgifera virgiferaLeConte, Graz, Austria, 6 pp. http://www.eppo.org.

Goudriaan J and Roermund HJW van (1989) Modelling of ageing, development, delaysand dispersion. In: Rabbinge SA, Ward SA and Laar HH van (eds) Simulation and

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Table 5. Simulated and observed occurrences of the stages of the western corn root-worm, D. v. virgifera.

Occurrence interval in INSIM Occurrence near Zhagreb1

First larval instar 24 May – 29 June Late May to end JuneSecond larval instar 5 June – 7 JulyThird larval instar 14 June – 25 JulyPupae 4 July – 4 AugustPre-oviposition female 18 July – 15 August Start end JuneOvipositing adult female 31 July – 1 Oct Stop 9 Oct1 according to EPPO report (1995)

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systems management in crop protection, pp 115-157. Pudoc, Wageningen.Helsen H, Vaal F and Blommers L (1998) Phenology of the common earwig, Forficula

auricularia L. (Dermaptera: Forficulidae) in an apple orchard. International Journalof Pest Management 44: 75-79.

Hemerik L, Busstra C and Mols P (2004) Predicting the temperature-dependent naturalpopulation expansion of the Western Corn Rootworm, Diabrotica virgifera.Entomologia Experimentalis et Applicata 111: pp. 59-69.

Kabanov VA (1975). The occurrence and development of Agriotes lineatus (Coleoptera,Elateridae) in the European part of the USSR. Pedobiologia 15: 98–105.

Mols PJM and Diederik D (1996) INSIM a simulation environment for pest forecastingand simulation of pest natural enemy interaction. Acta Horticultura 416: 255-262.

Mols PJM, van den Ende E and Blommers LHM (1998) Embryonic and larval develop-ment of Orthosia (Lep., Noctuidae) species used for optimizing timing of monitor-ing and control in apple orchards. Journal of Applied Entomology 122: 431-439.

Cross JV and Crook DJ (1999) Predicting spring emergence of blackcurrant leaf midge(Dasineura tetensi) from air temperatures. Entomologia Experimentalis et Applicata,91: 421-430.

Solomon, MG, Cross JV, Fitzgerald JD, Campbell CAM, Jolly RL, Olszak RW,Niemczyk E and Vogt H (2000) Biocontrol of pests of apples and pears in northernand central Europe: 3. Predators. Biocontrol Science and Technology 10: 91-128.


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Barry J. Pieters, Daniël Bosman-Meijerman, Erik Steenbergen, Evert-Janvan den Brandhof, Patrick van Beelen, Esther van der Grinten, WilkoVerweij & Michiel H.S. Kraak*National Institute for Public Health and the Environment, PO Box 1, 3720 BABilthoven, The Netherlands, E-mail: [email protected]; *Department of AquaticEcology and Ecotoxicology, IBED, University of Amsterdam, Kruislaan 320, 1098 SMAmsterdam, The Netherlands

Routine chemical monitoring gives insight in the presence of contam-inants in surface waters, but not in their joint ecological effects.Therefore ecological water quality is assessed with bioassays. Recently,a new bioassay using the chydorid Chydorus sphaericus has been devel-oped. Working with smaller volumes, materials and being less timeconsuming than the traditional Daphnia magna test regarding the cul-ture and experimental design, the ‘Chydotox-test’ shows a comparablesensitivity. The new Chydotox-test is a promising alternative for theexisting Daphnia sp. acute immobilisation test (OECD 1984).

Keywords: ecological water quality, bioassay, Chydorus sphaericus,Daphnia magna, toxicity

Routine chemical monitoring gives insight in the presence of contaminants insurface waters. Yet, for many compounds no reliable analytical method is avail-able and many known compounds are not measured. The biggest limitation ofchemical monitoring however is the lack of insight in the bioavailability of thepresent toxicants and the joint effects of mixtures of (un)known compounds onbiota (Hendriks et al. 1994). Therefore, bioassays are deployed as a complemen-tary tool, giving insight in biological effects, but not in their causes.

When bioassays are incorporated in ecological quality assessments, generallya battery of tests is deployed, because of species and compound specific sensitiv-ities. The battery of short-term bioassays used by the RIVM (in cooperationwith RIZA) consists of an algal photosynthetic-efficiency test (PAM-test) a bac-terial test (Microtox), and three zooplankton tests, using the daphnid Daphniamagna (Daphnia IQ), the rotifer Brachionus calyciflorus (Rotox) and the crus-tacean Thamnocephalus platyurus (Thamnotox). Though the Daphnia IQ test issensitive, it involves the use of chemicals for fluorescence measurements and the

Ecological quality assessment of Dutchsurface waters using a new bioassay with thecladoceran Chydorus sphaericus


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use of relatively large test volumes due to the size of the animal. Moreover D.magna is not a common representative species for Dutch surface water. Thereforean additional surface water bioassay was developed and applied using the benth-ic Cladoceran Chydorus sphaericus, a useful and sensitive test species (Koivisto etal. 1992, Dekker et al. 2002, Bossuyt & Janssen 2005, Dekker et al. 2006).

Chydorus sphaericus is one of the most common cladocerans in The Nether-lands (Fig. 1). It is a water column inhabiting and sediment dwelling speciesoccurring in a variety of habitats (Duigan & Kovach 1991, Fryer 1995, Van deBund & Spaas 1996), feeding mainly on detritus. The abundance of chydorids inlittoral regions of freshwater lakes makes them an important component of theaquatic ecosystem (Williams 1982). They hold a key position in the food web byconverting organic material into their own body mass that becomes in turnavailable for predators such as juvenile fish. Being cladocerans, chydorids havethe same advantages as daphnids for use in experiments, such as ease of handlingand parthenogenetic reproduction. Therefore C. sphaericus was considered a suit-able species for standardized laboratory toxicity testing and recently a sedimenttoxicity test using this species has been developed and applied (Dekker et al.2006). Departing from this sediment test, the aim of the present study was todevelop and apply a surface-water toxicity test using C. sphaericus. One of themain objectives was to reduce the test volume to 250 μl, which would be anadvantage when testing concentrates, a common practice in assessing ecologicalwater quality using the aforementioned test battery (Struijs & de Zwart 2003).The C. sphaericus laboratory culture was further optimized and the developedtest was validated by determining the EC50 for the OECD reference compoundpotassium dichromate (K2Cr2O7) used in standard D. magna tests. This allowedus to compare the sensitivity of C. sphaericus with that of Daphnia magna, as pre-viously performed for copper, cadmium and ammonia (Dekker et al. 2006).

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Figure 1. The benthic CladoceranChydorus sphaericus.

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Finally, the bioassay was put into practice by testing series of surface water sam-ples and it was attempted to relate the observed effects to measured toxicantconcentrations.


Chydorus sphaericus laboratory cultureThe clone of C. sphaericus used in this study originated from one gravid femaleretrieved from a culture from the University of Amsterdam which was original-ly collected in the summer of 1998 in the Drontermeer, a eutrophic, sandy lakein The Netherlands. The animals were kept in polyethylene plastic containersfilled with 100 ml of Dutch Standard Water (DSW) and about 1 g of combusted(3 h at 550°C) quartz sand (grain size 100-400 mm). Twice a week, the animalswere fed 2 ml of a food suspension consisting of dried, ground nettle powder(Urtica dioica) from BV De Tuinen, Beverwijk, and the living diatom Nitzschiaperminuta. This suspension was made by adding 1 g of nettle powder to 80 mL ofDSW and 20 mL of concentrated N. perminuta culture originating from a batchculture. N. perminuta was batch-cultured in modified WC medium (Van derGrinten et al. 2005), where 6 ml of a stock solution (14.21 g/l Na2 SiO3.9H2O) wasadditionally added to 3 L of WC medium. The N. perminuta culture was main-tained at a temperature of 15°C, lightintensity of ±35 μmol/m/s and a light:darkregime of 16:8 h.

Every week, around 70% of the C. sphaericus culture medium was renewed, bydecanting most of the medium from the container. Along with the medium,some of the animals in the culture were also removed. This partial removal pre-vented crowding which assured that the females continued to reproduceparthenogenetically and did not form ephippia. Every month each container wasreplaced by a new one that was inoculated by decanting part of the contents ofan older container into it. The temperature in the culture room was maintainedat 20°C and a light:dark regime of 16:8 h was applied.

Chydotox-testThe 48-h acute C. sphaericus test developed in this study was based on theDaphnia sp. acute immobilisation test (OECD 1984) and adjusted to small testvolumes and to the life-history characteristics of C. sphaericus.

One day before the start of the experiment, adult females containingparthenogenetic eggs were collected from the culture by a mesh filter with adiameter of 250 μm and transferred into glass jars containing 200 ml of DSWmedium. The jars were placed overnight in a climate room under the same cul-ture conditions (see above). The next day, newborn neonates (<24 h) were col-lected by a mesh filter with a diameter of 250 μm and used for the experiments.The tests were carried out in small 2 mL HPLC vials to which 250 μl of testmedium and five neonates were added. Potassium dichromate (CAS: 7778-50-9)

B.J. PIETERS, D. BOSMAN-MEIJERMAN, E. STEENBERGEN, E.-J. VAN DEN BRANDHOF, P.VAN BEELEN, E. VAN DER GRINTEN, W. VERWEIJ & M.H.S. KRAAK

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was chosen as model toxicant because it is recommended as a reference com-pound by the OECD for the Daphnia sp. acute immobilization test (OECD 1984)and consequently, a large set of toxicity data is available for this compound.Exposure consisted of nine different nominal concentrations in triplicate: con-trol, 0.032, 0.1, 0.32, 0.56, 1, 1.8, 3.2 and 10.0 mg/L. Next, five <24 h neonates wereadded to a single droplet of DSW with a diameter of approximately 5 mm andsubsequently transferred to the 250 μl test medium in the vials with a pipette. Apilot experiment with 45 vials showed that the neonates containing dropletincreased the 250 μl test solution by 5.8% (SD = 1.6) and therefore slightlydecreased the nominal exposure concentrations. The vials were covered with alid to prevent evaporation and incubated for 48 h under the same conditions asthe culture (see above). After 48 h the vials were placed under a reverse dissect-ing microscope and immobilization was determined by activation of the animalsby slightly shaking the vial and monitoring them for 30 sec. Immobilization wasplotted against the nominal total potassium dichromate concentration in thewater. From this dose-response relationship, the EC50 value and its correspon-ding 95% confidence limits were calculated using the log-logistic curve fittingprocedure of Haanstra et al. (1985).

Surface water testsIn 2005 routine chemical and biological monitoring was performed by the RIZAat different locations in the river Meuse. At each location 334 physical and chem-ical parameters (e.g. nutrients, metals, pesticides, PAC’s and PCB’s) were meas-ured. In addition, the effect parameter acetylcholinesterase-inhibition was deter-mined by measuring enzymatic responses of combinations of coumpounds (e.g.carbamates and organofosfates) with specific mechanisms of action. Water sam-ples from the location ‘Eijsden’ were used to investigate the applicability of theChydotox-test for water quality assessment. Since toxicants in Dutch surfacewaters rarely reach lethal concentrations, a concentration technique (Collombon1997) for the water samples was adopted by the RIVM. Following this procedure,water samples were concentrated a thousand-fold by extracting all (organic)micro-pollutants with XAD-resins and subsequently resolving the pollutants ina smaller water volume. This XAD-concentrate can be diluted afterwards todesired test concentrations. Due to the concentration technique, confoundingabiotic environmental factors such as ammonium, pH, humic acids and salinityare also circumvented.

RESULTSFigure 2 presents the 48 h dose-response relationship for C. sphaericus exposed topotassium dichromate. No mortality occurred in the controls and a clear dose-response relationship was obtained, resulting in an EC50 value (R2 = 0.88) of 780μg/l (95% CI: 580-980).

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The toxicity of the concentrated water samples from the Meuse measuredwith the Chydotox-tests is shown in Figure 3. Toxicity is expressed as the con-centration factor (cf) of the surface water at which 50% of the exposed animalsshow an effect. Thus the lower the cf needed to obtain 50% effect, the higher thetoxicity of the samples. Acute effects were demonstrated in the water samplesfrom the location Eijsden in the cf range between 1 and 10. In july and octoberEC50 values even reached the cf value of 1.

Analytical-chemical measurements performed by RIZA showed that at loca-tion Eijsden the Maximum Permissible Concentrations (MPC) threshold levelsfor acetylcholinesterase-inhibition were exceeded in March, July and October.Plotting these data together with the results of the Chydotox-test (Fig. 4) clear-ly shows that when concentrations of acetylcholinesterase-inhibiters are high,toxicity to the chydorids was also high.


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Figure 2. 48 h dose-response relationship for Chydorus sphaericus exposed to potassiumdichromate, using immobilisation as the effect parameter.

Figure 3. Time series of the toxicity of water samples from the river Meuse at Eijsdenin 2005. Toxicity is expressed as the concentration factor (cf) of the surface water atwhich 50% of the exposed animals show an effect: cf = 1000 is concentrated a thousand-fold. The lower the cf the more toxic the sample.

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DISCUSSIONThis study reports on the development and application of a newly developedacute toxicity test for surface water using the benthic cladoceran C. sphaericus, asan alternative for the standard Daphnia sp. acute immobilization test. A C.sphaericus laboratory culture was started and its performance under control con-ditions was optimized. The test was firstly validated by determining the dose-response relationship for potassium dichromate, showing a 48 h EC50 value forC. sphaericus (780 μg/l) within the range of toxicity data found in literature forDaphnia magna (average: 400 μg/l; range: 20-2700 μg/l; n = 38 (http://www.epa.gov)). It is concluded that for this OECD recommended reference compound C.sphaericus and D. magna are equally sensitive.

Using the Chydotox-test we monitored the toxicity of surface waters overtime (Fig. 3). This was only possible due to the extremely small test volumesused by the Chydotox-test (250 μl), as the concentration technique results invery small sample volumes. Standard Daphnia tests require much larger volumeshampering the assessment of toxicity of such concentrated samples. Figure 4also demonstrated that, despite the markedly improved water quality of Dutchsurface waters in the past decade, fully diluted concentrates from the riverMeuse may occasionally still be acutely toxic to macroinvertebrates.

Routine chemical monitoring gives insight in the presence of some contami-nants in surface waters, but not in the joint ecotoxicological effects of all con-taminants present in the water. The benefit of using bioassays for testing of sur-face waters is that the biological effects of (un)known (mixtures of) compoundsare assessed. However, only 11% of the toxicity of XAD-concentrates from fieldsamples could be attributed to the measured compounds, for which EC50 values

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Figure 4. Results of the Chydotox-test and acetylcholinesterase-inhibition at locationEijsden in the river Meuse in 2005.

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were obtained from databases (Hendriks et al. 1994). This lack of causality isexpected to be caused by identified compounds for which no effect concentra-tions are available and from compounds that could not be identified (Lahr et al.2003). In the present study, however, toxicity to C. sphaericus could be partlyattributed to acetylcholinesterase-inhibition, assuming all cladocerans like D.magna show acetylcholinesterase-inhibition (Guilhermino et al. 2000). The spe-cific compounds still need to be identified. Based on the results of the presentstudy it is concluded that the newly developed Chydotox-test is a promisingalternative for the existing Daphnia sp. acute immobilisation test (OECD 1984).

Acknowledgements We acknowledge the help of RIZA for providing water sam-ples and are very grateful for performing analytical measurements.

REFERENCESBossuyt, B.T.A. & Janssen, C.R., 2005. Copper toxicity to different field collected clado-

ceran species: intra- and inter-species sensitivity. Environ. Pollut. 136: 145-154.Collombon, M., Kamp R. & Van de Struijs, J., 1997. Procedures for extracting organic

micro-pollutants from water samples to monitor toxicological stress. RIVM rapport607042008.

Dekker, T., Greve, G.D., Ter Laak, T.L., Boivin, M.E., Veuger, B., Gortzak, G.,Dumfries, S., Lücker, S.M.G., Kraak, M.H.S., Admiraal W. & Van der Geest, H.G.,2006. Development and application of a sediment toxicity test using the benthiccladoceran Chydorus sphaericus. Environ. Pollut. 140: 231-238

Dekker, T., Krips, O.E. & Admiraal, W., 2002. Life history changes in the benthic clado-ceran Chydorus piger induced by low concentrations of sediment-bound cadmium.Aquat. Toxicol. 56: 93-101.

Duigan, C.A. & Kovach, W.L., 1991. A study of the distribution and ecology of littoralfreshwater chydorid (Crustacia, Cladocera) communities in Ireland using multi-variate analysis. J. Biogeogr. 18: 267-280.

Fryer, G., 1995. Crustacean diversity in relation to the size of water bodies: some factsand problems. Freshw. Biol. 15: 347-361.

Guilhermino, L., Lacerda, M.N., Nogueira A..J.A. & Soares, A.M.V.M., 2000. In vitroand in vivo inhibition of Daphnia magna acetylcholinesterase by surfactant agents:possible implications for contamination biomonitoring. Sci. Total. Environ. 247: 137-141.

Haanstra, L., Doelman, P. & Oude Voshaar, J.H., 1985. The use of sigmoidal dose res-ponse curves in soil ecotoxicological research. Plant and Soil 84: 293-297.

Hendriks, A.J., Maas-Diepenveen, J.L., Noordsij A. & Van der Gaag, M.A., 1994.Monitoring response of XAD-concentrated water in the Rhine Delta: a major partof the toxic compounds remains unidentified. Wat. Res. 28: 581-598

Koivisto, S., Ketola, M. & Walls, M., 1992. Comparison of five cladoceran species inshort- and long-term copper exposure. Hydrobiologia 248: 125-136.

Lahr, J., Maas-Diepeveen, J.L., Stuijfzand, S.C., Leonards, P.E.G., Drüke, J.M., Lücker,S., Espeldoorn, A., Kerkum, L.C.M., van Steed, L.L.P. & Hendriks, A.J., 2003.Responses in sediment bioassays used in the Netherlands: can observed toxicity be


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explained by routinely monitored priority pollutants? Wat. Res. 37: 1691–1710.OECD, Organization for Economic Cooperation and Development, 1984. OECD guide-

lines for testing of chemicals: Daphnia magna acute immobilisation test, 202.Struijs J. & De Zwart, D., 2003. Evaluation of pT/Measuring toxic pressure in surface

water. RIVM rapport 860703001.Tessier, A.J. & Consolatti, N.L., 1989. Variation in offspring size in Daphnia and conse-

quences for individual fitness. Oikos 56: 269-276.Van de Bund, W.J. & Spaas, S.J.H., 1996. Benthic communities of exposed littoral sand-

flats in eighteen Dutch lakes. J. Aquat. Ecol. 30: 15-20.Van der Grinten, E., Janssen, A.P.H.M., De Mutsert, K., Barranguet C. & Admiraal,

W., 2005. Temperature and light dependent performance of the cyanobacteriumLeptolyngbya foveolarum and the diatom Nitzschia perminuta in mixed biofilms.Hydrobiologia 548: 267-278.

Williams, J.B., 1982. Temporal and spatial patterns of abundance of the Chydoridae(Cladocera) in Lake Itasca, Minnesota. Ecology 63: 345-353.

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Brigitta Wessels-Berk1 & Ernst-Jan Scholte1,21Dutch Plant Protection Service, PO Box 9102, 6707 EA Wageningen, TheNetherlands, E-mail: [email protected]; 2National Institute for PublicHealth and the Environment (RIVM), PO Box 1, 3720 BA Bilthoven, the Netherlands

The emerald ash-borer, Agrilus planipennis constitutes a major risk toash (Fraxinus) trees in Europe. This beetle, originating from EasternAsia, has become a pest in northern America, killing over 20 millionFraxinus trees. The larvae destroy ash trees by boring serpentine tun-nels in the phloem, just beneath the bark, cutting off food. No controlmethod exists other than destroying the larvae by cutting and chippinghost trees. The potential impact if this beetle invades Europe will behuge.

Keywords: Agrilus planipennis, Fraxinus, phytosanitary risk, Europe

The emerald ash-borer, Agrilus planipennis, has the dubious honour to be one ofthe most feared beetles on earth (Fig. 1). It has the highest quarantaine status(IAI), and National Plant Protection Organizations (NPPO’s) around the worldcontinuously inspect their indigenous Fraxinus trees and imported wood prod-ucts on the presence of this beetle because its presence usually results in thedestruction of Fraxinus trees (Nomura 2002).

Agrilus planipennis belongs to the family of the Buprestidae, also called jewelbeetles or metallic wood-boring beetles. Buprestidae are relatively small, elon-

One beetle too many: The emerald ash-borer,Agrilus planipennis (Coleoptera: Buprestidae),threatens Fraxinus trees in Europe


Figure 1. Habitus and close-up of emerald ash borer, Agrilus planipennis. Source:www.padil.gov.au

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gated beetles. Adult beetles measure between 3 and 100 mm, although mostspecies are smaller than 20mm, with bright, iridescent colours. The larvae of allspecies of this genus bore tunnels in plant material, including leafs, logs, stems,and roots. A. planipennis measures approximately 7.5-15 mm and its larvae (1.5-3cm, white colour) bore into wood. Females lay eggs on the bark of a host tree(Fraxinus) in May-June. One female lays about 65-90 eggs during her lifetime.Eggs hatch and the larvae bore to the phloem area just behind the bark, wherethey tunnel serpentine tunnels. The development through four larval stagestakes approximately 1-2 years in temperate zones. Fourth instar larvae excavatechambers either in the bark or slightly in the sapwood, where they become pre-pupa in September-October. Most of the population overwinters in this stage,although some individuals overwinter as earlier-instar larvae. In these cham-bers, they moult into pupae during spring, and emerge as adults in May/June.The adults live approximately 2-3 weeks, during which they disperse no morethan several kilometres (Nomura 2002)

Host plants of A. planipennis are almost exclusively Fraxinus trees: in north-ern America they include F. americana, F. chinensis, F. japonica, F. lanuginose, andF. nigra, but the species was also found in Ulmus davidiana var. japonica, Ulmuspropinqua, Juglans mandshurica var. sieboldiana, and Pterocarya rhoifolia, and inEurope F. excelsior, F. pennsylvanica, and possibly F. angustifolia (Lyons et al. 2007,Volkovich 2007).

The original distribution of A. planipennis includes Mongolia, central, eastern,and north-eastern China, Taiwan, both Koreas and Japan (EPPO 2006). In July2002 the species was found in Northern America: specimens were identified insouth-eastern Michigan, USA, but evidence suggests that it may have beenestablished in the state five years earlier (Haack et al. 2002). It is currently pres-ent in seven US states (CEAP 2008), including Michigan (2002), Ohio (2003),Indiana (2004), Illinois (2006), Maryland (2006), Pennsylvania (2007), and inOntario, Canada. It is believed that the species entered the USA at Detroit, indunnage from cargo ships. In Europe, the species was found in Moscow, Russiain 2005, and seems to be spreading (Volkovich 2007). The pathway of this intro-duction is not known. The species has not been found in other areas of Europe.

The species damages infested trees because the larvae bore serpentine tunnelsin the cambial layer and the inner bark (phloem), disrupting, or even complete-ly cutting off the nutrient flow from the leaves to the roots. Nomura (2002)states that, ‘during the early stage of an infestation, when A. planipennis popula-tion is low, the initial damage is low. However, after 2 to 3 years of continuousinfestation, the population builds up, and eventually the tree’s nutrient andwater transport system is disrupted, culminating in wilting and eventual treemortality. A. planipennis will kill apparently healthy trees during high beetle pop-ulation levels which are probably triggered by a few years of hot and dry climat-ic conditions. A. planipennis can cause severe damage to ash stands over 8 years

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of age that are not crown-closed, with good sun light penetration, and that arecomprised of trees with bark fractures. After 1 to 2 years of infestation, the barkoften falls off in pieces from damaged trees thereby exposing the tunnel-riddensapwood. In the most severe cases, entire stands may be destroyed’.

In its natural area, A. planipennis population dynamics are balanced due tonatural enemies: parasitoids, predators, and entomopathogenic fungi. In a searchfor natural enemies in China, only two species were found: a Spathius sp.(Braconidae), and a Tetrastychus nov. sp. (Eulophidae), but with relatively highinfection levels, ranging between 0 and 50%, with averages of 6.3 and 6.6%respectively (Liu et al. 2003). In Northern America, natural enemies were foundas well, but infection levels were always lower than 1% (Bauer et al. 2004).Apparently, the absence of most of these natural enemies in the ‘new’ areas hasan enormous impact on the populationdynamics, allowing build-up of A. pla-nipennis populations to such high levels as to become a true pest. As a result,more than 20 million Fraxinus trees were killed by this species in southernMichigan. In the Moscow area, where A. planipennis was first found in 2005,many Fraxinus trees in city squares or along railway tracks declining or dying.In some places, 70-80% of the Fraxinus trees have lost most of their foliage(EPPO 2007).

Other symptoms that may indicate an A. planipennis infestation and may beseen from the outside, include crown dieback/chlorosis, epicormic shoots,increased woodpecker and squirrel feeding, bark deformities, foliage feeding,and exit holes. The exit holes are D-shaped of 3.5-4.1 mm in diameter. A clearsign of Agrilus sp. infestation that can not be seen from the outside, is the pres-ence of typically serpentine shaped larval galleries, mostly filled with frass.They can easily be found by peeling away the bark (Lyons et al. 2007, Moraal andWessels-Berk 2007). It should be noted, however, that once signs and symptomsbecome apparent, trees are often severely infested and infestation may havespread to surrounding areas where infestation signs are not apparent yet. Basedon the infestation symptoms, examination techniques applied by inspectorsshould include visual inspection, crown survey, and bark peeling.

Control of larvae is difficult. Only systemic chemical insecticides can reachthe well-hidden larvae. This can be done by tree-injection, soil drenching, or soilinjections with imidacloprid (Smitley & McCullough 2004). Adults can be killedby using trunk and foliage spraying with cyfluthrin. Ongoing studies suggest thatbiological control using the entomopathogenic fungus Beauveria bassiana sprayedon tree trunks is another option for control of adults, causing up to 50% infectionrates. Currently, the most effective way to control and try to eradicate an out-break of A. planipennis is by cutting down and chipping infested trees in the infest-ed and surrounding area. In northern America, domestic phytosanitary measureshave been imposed to restrict the movement of ash trees, firewood, branches, andlogs from infested to non-infested areas (Haack et al. 2002, EPPO 2005)

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Having witnessed the spread and damage by A. planipennis in the USA, thefact that indigenous Fraxinus trees (F. excelsior, F. angustifolia, F. ornus) andimported F. pennsylvanica are very common in Europe (Pliura & Heuertz 2003),and the fact that the species has already entered eastern Europe, it is clear whythis species poses a serious phytosanitary risk for Europe. Since Fraxinus excelsioris very common in the Netherlands, it is recommended that the Dutch NPPOdevelops a contingency plan for this species.

REFERENCESBauer LS, Liu HP, Haack RA, Petrice TR & Miller DL. 2004. Natural enemies of emer-

ald ash borer in southeastern Michigan, pp. 33-34. In: Emerald Ash Borer Researchand Technology Development Meeting, Port Huron, MI, 30 Sept-1Oct 2003. USDAForest Service, Fort Collins, CO. FHTET/2004-02.

CEAP (Cooperative Emerald Ash Borer Project). 2008 www.emeraldashborer.infoWebsite last visited 11 Jan. 2008.

EPPO. 2006. Distribution map of Agrilus planipennis. http://www.eppo.org/QUARAN-TINE/ sects/grilus_planipennis/AGRLPL_map.htm Website last visited 11 Jan.2008.

EPPO. 2005. Agrilus planipennis Data sheet. OEPP/EPPO Bulletin 35: 436-438.Haack RA, Jendek E, Liu H, Marchant KR, Petrice TR, Poland TM & Ye H. 2002. The

emerald ash-borer: A new exotic pest in North America. Newsletter of the MichiganEntomological Society 47: 1-5.

Liu H, Bauer LS, Gao R, Zhao T. Petrice TR & Haack RA. 2003. Exploratory survey forthe emerald ash borer, Agrilus planipennis (Coleoptera: Buprestidae), and its natu-ral enemies in China. The Great Lakes Entomologist 36: 191-204.

Moraal L & Wessels-Berk B. 2007. Aziatische essenprachtkever kan ramp veroorzaken.Tuin en Landschap 4: 40-41.

Lyons DB, Caister C, De Groot P, Hamilton B, Marchant K, Scarr T & Turgeon J. 2007.Survey guide for detection of emerald ash borer. Handbook Manual. CanadianForest Service and the Canadian Food Inspection Agency, Ontario, Canada, 52 pp.

Nomura S. 2002. Agrilus planipennis. Canadian Food Inspection Agency ScienceBranch.http://www.inspection.gc.ca/english/sci/surv/data/agrplae.shtml

Pliura A & Heuertz M. 2003. EUFORGEN Technical Guidelines for genetic conserva-tion and use for common ash (Fraxinus excelsior). International Plant GeneticResources Institute, Rome, Italy, 6 pp.

Smitley D & McCullough D. 2004. How homeowners can protect ash trees from theEmerald Ash Borer in southeastern Michigan. www.emeraldashborer.info

Volkovich MG. 2007. Agrilus planipennis, a new and dangerous pest of Fraxinus in theEuropean part of Russia. (in Russian). In: EPPO reporting service (nov. 2007): Firstrecord of Agrilus planipennis in the region of Moscow, Russia.

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169

Author index

Alanen, E.-L. 59

Allsopp, M.H. 41

Beelen, P. van 157

Beeuwkes, J. 137

Boot, W.J. 41, 53

Bosman-Meijerman, D. 157

Brandhof, E.-J. van den 157

Bukhari, T. 121

Calis, J.N.M. 41, 53

Chagas, F. 23

Cortopassi Laurino, M. 23

Farenhorst, M. 121

Grinten, E. van der 157

Hemerik, L. 147

Knols, B.G.J. 121

Kraak, M.H.S. 157

Langoya, L.A. 67

Leeuwen, J.L. van 137

Menken, S.B.J. 115

Michez, D. 31

Nes, E.H. van 147

Noordijk, J. 75

Ondiaka, S. 121

Paalhaar, J. 53

Parker, K. 115

Pieters, B.J. 157

Rijn, P.C.J. van 67

Roessingh, P. 115

Schaffers, A.P. 75

Scholte, E.-J. 165

Sloggett, J.J. 95

Smallegange, R.C. 129

Spitzen, J. 137

Spoor, C.W. 137

Steen, J.J.M. van der 53

Steenbergen, E. 157

Sýkora, K.V. 75

Takken, W. 121, 129, 137

Velthuis, H.H.W. 23

Vereecken, N.J. 9

Verhulst, N.O. 129

Verweij, W. 157

Wessels-Berk, B. 165

171

Subject index

Adult nutrition 115

agricultural landscape 59

Agrilus planipennis 165

ammonia 137

Anopheles gambiae 121, 129, 137

aphidophagous ladybirds 95

aphids 67, 95

Apis mellifera capensis 41

Apis mellifera scutellata 41

Apis mellifera 31, 53

Araneae 75

auto detection 137

Baffle 129

bee nest 23

bees 9

biogeography 31

blood meal size 121

Bombus 59

Brazil 23

Brevicoryne brassicae 67

bumblebee 59

Buprestidae 165

Cabbage aphid 67

Cape honeybee 41

Carabidae 75

Chydorus sphaericus 157

Coccinellidae 95

Colletes cunicularius 9

conservation biological control 67

conservation value 75

corridors 75

cross-pollination 53

Daphnia magna 157

degree-day 147

Diabrotica virgifera virgifera 147

dietary specificity 95

diversification 31

dynamics 147

Ecological water quality 157

ecology of infrastructure 75

emerald ash-borer 165

Episyrphus balteatus 67

Europe 165

FAB 67

fecundity 115, 121

feeding propensity 121

flight behaviour 129, 137

floral evolution 9

floral odours 9

floral resources 67

food plants 59

Formicidae 75

Fraxinus 165

fnctional arobiodiversity 67

fungal infection 121

funnel 129

Habitat 59, 95

highway verges 75

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172

honey 115

honeybee 31

honeydew 67

host seeking 121, 129, 137

host stimuli 137

hoverflies 67

human odour 137

In-hive transfer 53

INSIM 147

Lactic acid 137

longevity 115

Macrolepidoptera 115

malaria mosquito 121, 129, 137

management 75

mate choice 9

Melipona quinquefasciata 23

Melittidae 31

Metarhizium anisopliae 121

monophyletic group 31

multiple mating 115

Natural control 67

nectar 59, 67

nocturnal activity 137

nuptial 115

Odour plume 137

olfaction 129

olfactometer 129

Ophrys 9

orchids 9

Phenology 147

phylogeny 31

phytosanitary risk 165

pollen 53, 59, 67

pollination 9

prediction 147

pseudo-copulation 9

Queens 59

Regulation of physical

conditions 23

reproductive isolation 9

reproductive output 115

resistance 121

revision 31

Salix 59

selection 9, 41

sex pheromones 9

sexual deception 9

skin extract 129

social parasitism 41

specialization 95

stingless bee 23

surface water 157

Taraxacum 59

toxicity 157

trade-offs 95

tree pest 165

SUBJECT INDEX

173

Western corn root borer 147

wind tunnel 137

worker reproduction 41

Yponomeuta 115

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