NORTH-WESTERN JOURNAL OF ZOOLOGY 8 (2): 353-357 ©NwjZ, Oradea, Romania, 2012 Article No.: 121203 http://biozoojournals.3x.ro/nwjz/index.html

A new neogregarine pathogen of Rhizophagus grandis

(Coleoptera: Monotomidae)

Mustafa YAMAN1*, Renate RADEK2 and Andreas LINDE3 1. Department of Biology, Faculty of Arts and Sciences, Karadeniz Technical University, 61080, Trabzon, Turkey.

2. Institute of Biology/Zoology, Free University of Berlin, Königin-Luise-Str. 1-3, 14195 Berlin, Germany. 3. University of Applied Sciences Eberswalde, Applied Ecology and Zoology, Alfred-Möller Str. 1, 16225 Eberswalde, Germany.

*Corresponding author, e-mail address: yaman@ktu.edu.tr (M. Yaman)

Received: 09. January 2012 / Accepted: 24. February 2012 / Available online: 27. May 2012 / Printed: December 2012

Abstract. Here we provide the first description of a natural infection of members of the beetle family Monotomidae with neogregarines and specifically the first finding of such a pathogen in the predatory beetle Rhizophagus grandis. The fat body of the beetle is the site of infection, and the typical navicular oocysts are 11.87 ± 0.67 μm in length and 6.96 ± 0.43 μm in width (n = 60). Polar plugs are recognisable using light and electron microscopy. The oocyst wall is quite thick, measuring 400 to 500 nm. Oocysts are formed pairwise within a gamontocyst, and each oocyst has eight sporozoites. The described neogregarine pathogen in R. grandis has the typical characteristics of members of the genus Mattesia (family Lipotrophidae) within the order Neogregarinida.

Keywords: Bark beetles, Biological control, Dendroctonus micans, Neogregarine, Mattesia, Rhizophagus grandis.

The great spruce bark beetle Dendroctonus micans (Kugelann) (Coleoptera: Scolytinae) causes severe damage to spruce stands in the Black Sea area and the Caucasus, resulting in significant economical losses. All efforts and resources dedicated to con-trolling this dangerous pest have been inadequate; it is still causing epidemics in the eastern Black Sea region of Turkey. The development of more effi-cient, environmentally safe and sustainable meth-ods for controlling this pest has thus become a priority and necessity. As a result, studies in search of means of biological control of this pest were conducted. The predatory beetle Rhizophagus grandis is a proficient natural suppressing factor of D. micans. This very efficient and voracious hunter is mass-reared using D. micans larvae as food. Un-fortunately, the conditions during cultivation in the bark have been shown to allow a transmission of pathogens from prey to predator (Yaman & Radek 2007, Yaman 2008), which is an undesirable situation in that R. grandis is the most important factor in controlling D. micans populations. Patho-gens infecting the predator would certainly de-crease the efficiency of the beetle as a biocontrol agent.

Yaman & Radek (2005) reported a pathogen identified as Helicosporidium sp. in D. micans, and have confirmed the same infection in R. grandis populations (Yaman & Radek 2007). Furthermore, in 2008, Yaman and Radek recorded a neogre-garine pathogen of D. micans in Turkey. This ob-servation stimulated us to look for such an infec-

tion in R. grandis. A new neogregarine pathogen of R. grandis is reported in this study.

After Yaman’s & Radek’s (2008) observations on the neogregarine record in D. micans in 2005 and 2006, 226 male and 301 female specimens of R. grandis were obtained from the R. grandis rearing laboratory in Giresun in July 2007. Samples from other laboratories could not be obtained in the same time period, because each laboratory starts the mass-rearing procedures at different times of the year. However, all laboratories have the same specific conditions. Each insect was dissected in insect Ringer’s solution, and wet smears were prepared and examined for presence of neogregarine cysts under a light microscope at a magnification of 400–1000x. When an infection was found, neogregarine oocysts were fixed with methanol and dyed with Giemsa solution, measured, and then photographed using an Olympus BX51 microscope equipped with a DP-25 digital camera and a DP2-BSW Soft Imaging System. The cysts were also studied with SEM and TEM microscopes, using a previously reported technique (Yaman & Radek 2005, Yaman et al. 2008, 2010). The results were statistically analysed using SPSS 11.0.

Only oocyst stages of the pathogen could be observed in adult R. grandis. Form and size of the oocysts were very uniform, navicular in shape and with plugs at the two poles (Figs. 1, 2, 3). Further-more, oocyst size did not vary significantly (Fig. 3): within any given beetle as well as between dif-ferent beetles, the oocysts (fixed in methanol and stained with Giemsa) were 11.87 ± 0.67 (10.21-13.48) μm in length and 6.96 ± 0.43 (6.16-7.74) μm in width (n = 60). They are formed in pairs within a gamontocyst (Fig. 2). A polar plug heavily

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Figures 1-3. Fresh (Figs. 1 and 2) and Giemsa-stained oocysts (Fig. 3) of Mattesia sp. from R. gran-dis. Note oocyst pairs within a gamontocyst (Fig. 2) and heavily Giemsa-stained polar plugs at each cell pole of the oocyst (Fig. 3). Bar: 10 µm.

Figures 4-5. Oocysts of Mattesia sp., SEM. Note the navicular oocysts (Fig. 4) and the protrud-ing polar plug in some oocysts (Fig. 5). Bars: 5 µm (Fig. 4) and 2.5 µm (Fig. 5).

stained with Giemsa solution is present at each cell pole of the oocyst (Fig. 3). The polar plugs are also clearly discernable using electron microscopy (Figs. 4, 5, 8) and are 925 to 1120 nm thick (Fig. 8). Some oocysts have protruded polar plugs (Fig. 5). The spore wall is quite thick, measuring 400 to 500 nm (Figs. 6, 7, 8). Each oocyst includes eight sporozoites (Fig. 6).

Other life cycle stages of the neogregarine pathogen could not be observed in the wet or stained smear preparations. However, the de-scribed results of light microscopic observations

combined with electron microscopy revealed that the neogregarine pathogen has the typical charac-teristics of the family Lipotrophidae within the or-der Neogregarinida. The characteristics of the Li-potrophidae are navicular oocysts with pro-nounced polar thickenings and containing eight sporozoites (see Figs 2, 7) (Perkins 2000). Members of the other five families are distinct from the neogregarine described here (Perkins 2000). In comparison, gamontocysts of the Gigaductidae are enclosed in a thick gelatinous capsule that is not present in the neogregarine found in R. grandis. In

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the Caulleryellidae, one or eight oocysts are formed within a gamontocyst, while in the Ophryocystidae only a single one is found. The oocysts of the Syncystidae have three or four spines extending from the poles and about 30-150 oocysts are formed within a single gamontocyst. With their spindle-shaped oocysts containing eight sporozoites, members of the Schizocystidae resemble our pathogen. However, the genera characteristics are different from the neogregarine described here from R. grandis. The members of the included genus Schizocystis have no oocysts with prominent polar thickenings; the genus Machadoella forms 3-6 or 24 oocysts (with polar thickenings), and the genus Lymphotropha pro-duces more than two (4-16) oocysts per gamonto-cyst. Therefore, only the family Lipotrophidae matches our pathogen in all details of the gamon-

tocyst/oocyst characteristics. There are five genera within this family: Farinocystis, Lipocystis, Lipotro-pha, Mattesia, and Menzbieria. Only the genus Mat-tesia is reported to generate as few as one or two oocysts, while members of the other genera have more than two oocysts in the gamontocyst. The neogregarine from R. grandis we describe here, characterized by two spores with eight sporozoites within one gamontocyst (Figs 2, 7), thus un-equivocally belongs to the genus Mattesia (Levine 1988, Kleespies et al. 1997, Perkins 2000).

Levine (1988) listed nine described species in the genus Mattesia. These species have been de-scribed in the fat body tissue, Malpighian tubules or intestine of the insect taxa Coleoptera, Hymen-optera, Lepidoptera and Siphonaptera. Five spe-cies were recorded from the order Coleoptera: M. dispora, M. grandis, M. oryzaephili, M. schwenkei and

Figures 6-8. Mature oocysts of Mattesia sp., TEM. Longitudinal (Fig. 6) and cross (Fig. 7) sections of an oocyst including eight sporozoites. Thick polar plugs are seen (Fig. 8). Bars: 2 µm (Figs 6 and 7) 2.5 µm (Fig. 8).

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Table 1. Mattesia species described in the order Coleoptera.

Mattesia species Spore size Infected organ

Host References

Mattesia dispora 12.2 x 6.7 µm unknown Laemophloeus ferrugineus, L. minutes

Finlayson 1950

Mattesia grandis 9-10.9 x 4.9-11.3 µm Fat body Anthonomus grandis McLaughlin 1965 Mattesia schwenkei 17.5-20.4 x 7.5-9.2 µm (native)

15.5-18.5 x 6.2-7.8 µm (stained) Fat body Dryocoetes autographus Purrini 1977

Mattesia schwenkei 16.3-21.2 x 6.3-10.5 µm (native), 15.5-18.5 x 6.2-7.8 µm (stained)

Fat body Hylurgops glabratus Purrini 1978

Mattesia trogoderma -- Fat body Trogoderma granaria Canning 1964 Mattesia oryzaephili 12 x 7 µm (native),

10 x 6 µm (stained) Fat body Oryzaephilus surinamensis Ormières 1971

Mattesia sp. 14 x 5, tubular type, 13-13.5 x 7, navicular type

Fat body Ips typographus Žižka et al. 1997; Händel et al. 2003

Mattesia sp. 20-22 x 8.5-10 µm Fat body Pityogenes chalcographus Händel et al. 2003 Mattesia sp. 11 x 6 μm (stained) Fat body Dendroctonus micans Yaman & Radek 2008 Mattesia sp. 11.87 x 6.96 (stained) Fat body Rhizophagus grandis This study

M. trogodermae (Table 1). The only named species found to infect bark beetles is Mattesia schwenkei (15.5-18.5 x 6.2-7.5 μm in size) from Dryocoetes autographus (Purrini 1977). Źižka et al. (1997) found two yet unnamed types of Mattesia in the bark beetle Ips typographus; the tubular type meas-ures 14 x 5 μm and the navicular type 13-13.5 x 7 μm. Händel et al. (2003) found a similar infection in Ips typographus and Pityogenes chalcographus. Re-cently, Yaman and Radek (2008) recorded a Matte-sia sp. (11 x 6 μm) infection in Dendroctonus micans, the prey of Rhizophagus grandis (Table 1). As seen in Table 1, the neogregarine pathogen in R. grandis has a slightly larger spore size than Mattesia sp. in D. micans. Until now, the only recorded pathogen from R. grandis is the entomopathogenic alga Heli-cosporidium sp., described by Yaman and Radek (2007). The pathogen described here is the first neogregarine reported from R. grandis. It only in-fects the fat body of its host. Merogony and sporogony of many Mattesia species are known to take place in the fat body, whereby the tissue is lysed (Kleespies et al. 1997, Perkins 2000). Thus an infection with Mattesia is pathogenic and may re-duce the lifespan and reproduction of its host, leading to a decreased potential as a biological control agent.

As mentioned above, Yaman & Radek (2008) recorded a neogregarine infection (Mattesia sp.) from D. micans, the prey of R. grandis. This preda-tory beetle is mass-reared in laboratories for the biological control of D. micans in several countries where this insect is a threat to spruce forests. The mass-rearing procedure includes collecting D. micans larvae from infested spruce stands and us-

ing these as nutrition for parent R. grandis and their new progeny. Under such conditions, a transmission of neogregarines from D. micans to the predator, R. grandis could be possible. Such a transmission was described for Helicosporidium sp. from D. micans to R. grandis by Yaman & Radek (2007). To prevent a disease transmission from prey to predator and thus to improve the produc-tion of this biological control agent, we recom-mend that cultures of D. micans, serving as prey beetles, are free of neogregarine infection. There-fore, D. micans larvae destined for use as food for R. grandis should be only collected from areas in which no infections of the prey by either Helico-sporidium sp. or Mattesia sp. have been detected. Acknowledgements. The study was financially supported as a research project by the Scientific and Technical Council of Turkey (107T166). Dr. Mustafa Yaman was awarded with a grant by the DAAD (German Academic Exchange Service) (316- A/09/03438). The authors wish to express their thanks to Prof. Dr. Klaus Hausmann, Berlin for making this study possible. References Canning, E. U. (1964): Observations on the life history of Mattesia

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