Entomologia Experimentalis et Applicata 85: 211–219, 1997. 211c 1997 Kluwer Academic Publishers. Printed in Belgium.

Costs and benefits of acclimation to elevated temperature in Trichogrammacarverae

Megan Scott, David Berrigan1 & Ary A. Hoffmann�School of Genetics and Human Variation, La Trobe University, Bundoora 3083, Australia; 1Current address:Department of Zoology Box 351800, University of Washington, Seattle, WA 98195, USA; �Author forcorrespondence

Accepted: August 26, 1997

Key words: acclimation, stress resistance, parasitoids, survival, fecundity, Trichogramma

Abstract

The consequences of acclimation for survival and other fitness components in the parasitoid wasp, Trichogrammacarverae (Oatman and Pinto), were examined. Heat hardening adult wasps at 33 �C or 35 �C for one to two hincreased survivorship at 40 �C. This benefit was apparent for several hours after heat-hardening and occurredin both males and females. Heat hardening at 33 �C during development also resulted in significant increases insurvivorship of adults after exposure to 40 �C. However, this developmental hardening reduced longevity of adultmale and female wasps and also reduced parastism rate. This suggests costs and benefits of exposure to non-lethaltemperature increases. Acclimating wasps by rearing them under constant temperatures (14, 25 or 30�C) influencedparasitism rates at these temperatures at the adult stage; only females reared at 14 �C parasitised eggs at 14 �C,while parasitism at 25 �C and 30 �C was not significantly influenced by rearing temperature. Acclimation may beuseful for increasing the survival or fecundity of mass-reared Trichogramma in inundative releases, but any benefitscould be offset by fitness costs of the acclimation process.

Introduction

Insects frequently experience fluctuating temperatureregimes in nature and these fluctuations may result inexposure to stressful conditions. As a consequence,insects may exhibit fixed differences in resistance totemperature extremes or they may show acclimationresponses to increased or decreased temperature (Lev-ins, 1969; Hoffmann & Parsons, 1991). Acclima-tion occurring over short intervals is often called heator cold hardening (Zachariassen, 1985; Cossins &Bowler, 1987), and hardening in diverse ectotherms(i.e., animals whose body temperature depends largelyon external environmental conditions) has been indir-ectly linked with the production of heat shock proteins(Parsell & Lindquist, 1993).

Acclimation to increased temperature has beenstudied in greatest detail in Drosophila and to a less-er extent in other insects (e.g., Cloudsley-Thompson,1971; Bultmann, 1986; Carvalho & Rebello, 1987;

Fittinghoff & Riddiford, 1990). Drosophila speciesdiffer in the magnitude of hardening (Levins, 1969;Yamamoto & Ohba, 1984) but there is little evid-ence for differences among populations of the samespecies for hardening effects (Hoffmann & Watson,1993; Loeschcke et al., 1994). Drosophila studies havealso shown that acclimation can occur at different lifestages, and that artificial selection for stress resist-ance can influence hardening responses (Loeschcke &Krebs, 1996; Hoffmann et al., 1997).

Force (1967) and Huey & Berrigan (1996) suggestthat acclimation treatments could be used to improvethe quality of beneficial insects. However, few studieshave explored acclimation in beneficial insects, despitethe fact that climatic factors, particularly temperatureextremes, can influence the success or failure of pestcontrol with beneficial insects (Force, 1967; De Bach,1974).

We have therefore investigated heat acclimationin the egg parasitoid Trichogramma carverae (Oat-

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man and Pinto). Like several other species of Tricho-gramma, T. carverae is reared commercially for usein inundative releases to control moth pests. Tricho-gramma carverae is particularly effective in con-trolling the eggs of Epiphyas postvittana (lightbrownapple moth) in grapevines (Glenn & Hoffmann, 1997).However, the success of field releases with this speciescan be variable as in the case of releases with otherTrichogramma species (e.g., Twine & Lloyd, 1982;Bigler & Brunetti, 1986; Smith et al., 1987; Newton& Odenaal, 1990; Li, 1994). Reasons for such vari-ability include the quality of release material (Keller& Lewis, 1985; Bigler et al., 1987; Smith, 1996) andweather conditions at the time of releases (Yu et al.,1984; Smith, 1994; Burgio & Maini, 1995). It is pos-sible that wasps reared under constant conditions incommercial insectaries are of a low quality becausethey fail to survive extreme temperature fluctuationsencountered in the field.

Understanding the overall effects of acclimationon insects requires measurements of the costs of theacclimation responses as well as their potential bene-fits. Several studies in Drosophila have addressed thisissue with both phenotypic and genetic approaches. InD. melanogaster, heat-hardening at 36 �C increasessurvival after exposure to 39 �C for 100 min, howeverhardened females have reduced fecundity (Krebs &Loeschcke, 1994). Berrigan & Hoffmann (unpubl.)studied correlates of acclimation ability among hybridsbetween two species of Drosophila. They report acorrelation between acclimation for knockdown heatresistance and resistance to heat as measured usinga mortality based assay. Expression of a heat shockprotein (hsp70) in Drosophila cells in culture reducescell growth rate in the absence of stress (Feder et al.,1992). This suggests that there may be growth as wellas mortality and stress resistance costs to heat harden-ing. Few studies address the cost of acclimation inorganisms besides Drosophila (Hoffmann, 1996).

We have three main objectives in examininghardening and associated costs in Trichogramma.Firstly, we test for the presence of acclimation totemperature increases in adult T. carverae followinghardening of adult or pre-adult stages. We are unawareof other work showing heat hardening in parasitoidsalthough direct effects of heat exposure have previ-ously been demonstrated for development, longevity,viability, parasitism rate and sex ratio (e.g., Wilkes,1959; Chihrane et al., 1991). Secondly, we test forcosts by examining the effects of acclimation on lifehistory traits in the absence of heat stress. Thirdly,

we consider the effects of acclimation involving con-tinuous rearing at different temperatures; we examineeffects on parasitism rates over the same temperaturerange. This tests if wasps acclimated to particular con-ditions have an increased fitness under those conditions(c.f. Zamudio et al., 1995; Huey & Berrigan, 1996).

Materials and methods

Strains. A mass culture of T. carverae was estab-lished by pooling representatives of seven isofemalelines collected in December 1995 from vineyardsaround Mildura (Victoria, Australia). Strains were alsomaintained as separate cultures. The T. carverae wereheld in plastic containers in an incubator at 25 �C, on aL16:D8 cycle. Adult wasps were reared on eggs of theangoumis grain moth, Sitotroga cerealella, followingGlenn et al. (1997) and Glenn & Hoffmann (1997).

Adult acclimation. Individual adult T. carverae lessthan 24 h old were placed in a glass vial sealed with astopper containing a 3% agar and honey mixture. Vialswere then divided randomly into control and acclima-tion treatments. We assessed the consequences of heathardening for resistance to thermal stress in wasps bycomparing survival after heat shock for 120 min at40 �C. Male and female wasps were acclimated for120 min at 33 �C and left for 1, 2 or 4 h prior tobeing stressed. Sample sizes ranged from 16 to 52per treatment and sex depending on the availabilityof wasps. This experiment was replicated three timesand all treatments were considered in each replicatetrial. Wasps were scored as alive if they were able towalk 24 h after the test. Mortality in unstressed andunhardened controls was negligible (<2%).

The data from this experiment and the immatureacclimation experiment (below) were analyzed usinganalyses of variance (ANOVAs). Survival of the 16–52 wasps was treated as a single data point and angu-lar transformed prior to analysis. These experimentsallowed us to assess effects due to the duration of theacclimation response after a heat treatment, as well asthe presence or absence of acclimation in the two sexes.Posthoc comparisons of treatment means were under-taken using the Tukey B procedure. A trial effect wasincluded in all ANOVAs as a random factor becausethe mean level of heat resistance could vary betweentrials as a results of slight differences in the heat stressimposed or minor uncontrolled changes in culture con-ditions. Trial effects highlight the importance of having

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all treatments including controls in each trial, as well asthe difficulty of directly comparing the survival of con-trols across experiments. Interactions between the trialterm and other factors were included in the ANOVAsbut were not significant and are not presented. Becausethe data involved survival, analyses were undertakenon angular transformed proportions.

Immature acclimation. Trichogramma are small andadults can have relatively short lifespans, particularlyin the field. Thus it would be particularly useful ifbeneficial acclimation treatments could be applied topre-adult wasps. We tested for the presence of benefi-cial acclimation as a consequence of hardening duringdevelopment by recording resistance to heat shock inadults. To determine if there were negative as wellas positive effects of hardening during development,we measured longevity and fecundity of wasps thathad received hardening treatments. These experimentswere performed on wasps reared at 25 �C and hardenedat 33 �C for varying lengths of time.

We generated wasps for these experiments by col-lecting parasitised eggs over four successive days. Onthe fifth day we incubated the eggs for several hoursper day at 33 �C until eclosion of the adult wasps. Thisprocess resulted in adult wasps that had received 8.5,13, 25.5, and 32 h exposure to 33 �C during devel-opment. Adults (�24 h old) from these treatmentsand from untreated controls were stressed at 40 �Cfor 120 min and then scored for survival 24 h later.For each treatment-sex combination we scored surviv-al based on 20 to 120 wasps depending on availability,although numbers were usually around 45. This exper-iment was replicated three times.

Additional wasps from each of the acclimationtreatments were placed in containers, one male andone female per container, and offered egg cards foroviposition over a 24 h period at either 25 �C or 30 �C.After this oviposition period, the wasps were returnedto 25 �C and held until they died. Longevity wasscored for individual male and female wasps. Deadmales or females were not replaced. For the longevityexperiment, we tested 20–21 males and 20–21 femalesfrom each hardening treatment and 80 wasps of eachsex from the unhardened controls. For the oviposi-tion experiment, a similar number of females from thehardening (20) and control (80) treatments were tested.

Developmental effects on parasitism. This experi-ment tests the effects of culturing Trichogramma at 14,25 and 30 �C on parasitism rates over the same temper-

ature range. We used all seven strains of T. carveraein this experiment rather than the mass bred culture.Wasps were allowed to parasitise Sitotroga eggs for24 h and eggs were then placed at 14, 25 or 30 �C.Eggs at these temperatures were set up at differenttimes to ensure that adults emerged at the same time.After emergence, adults were provided with honey andwasps were tested for parasitism. This was done by pla-cing males and females in pairs into a glass vial withat least 30 eggs on adhesive paper. These were placedat 14, 25 or 30 �C and left for 24 h. We tested 20–30replicate pairs of each strain at each temperature.

Results

Adult acclimation. Acclimation significantly increas-ed survivorship after heat stress (Treatment effect,F(3;14) = 18:64, P < 0:001); about 40% of thecontrols survived the heat stress whereas 50 to 90%of the hardened wasps survived, depending on thelag phase (Figure 1). Posthoc tests indicated signi-ficant differences between the control treatment andall acclimation treatments. The hardening effect seemsto be at a maximum after 2 h and may decline sub-sequently. However, posthoc tests did not indicate sig-nificant differences between acclimation treatments.The improved survival due to hardening appearedgreater in females than in males, although the treatmentby sex interaction term in the ANOVA was not signific-ant (Sex effect, F3;14 = 1:27, P = 0:28). Survival didnot differ significantly among the trials (F2;14 = 0:48,P = 0:63). In other experiments, we have found thathardening can also be induced by higher temperatures;for instance, in one trial, exposure of adults to 35 �Cfor 60 min increased survival of a 40 �C shock from20% to around 70%. Adult wasps are therefore likelyto be hardened by a range of non-lethal temperaturesand this effect lasts for at least a few hours.

Immature acclimation. Heat hardening of immaturewasps significantly influenced survival after heat stress(F4;34 = 54:40, P < 0:001) while there was a sug-gestion that the sexes differed overall (F1;34 = 3:96,P = 0:056). Posthoc tested indicated improvement insurvival in both sexes only after acclimation for 13 hand 25.5 h but not after acclimation for 8.5 h or 32 h(Figure 2). Control wasps showed about 20% survival,and wasps hardened for intermediate times showed 50–60% survival. The interaction between sex and treat-ment was significant (F(4;34) = 4:07, P = 0:008),

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Figure 1. Survival of T. carverae after exposure to 40 �C for 120 min. Control wasps were not acclimated and treated wasps were exposed to33 �C for 120 min. Wasps were tested 1, 2 and 4 h after the acclimation treatment. Means and standard errors (bars) are based on three trials.

reflecting the fact that acclimation was at a maximumin females exposed to 25.5 h and in males exposed to13 h. Survival did not differ significantly between trials(F(2;34) = 1:04, P = 0:36).

We recorded longevity in wasps that had receivedacclimation treatments during development. Treat-ments had a significant influence on longevity (F4;314 =

64:69, P < 0:001). Acclimation for 32 h reducedlongevity from 9–11 days to 3–5 days in both sexes(Figure 3). Control males survived somewhat longer(11.4 days) than females (10.1 days) and there wassuggestive evidence for an overall difference betweenthe sexes (F1;314 = 3:30, P = 0:07) and interactionbetween treatment and sex (F(3;314 = 2:30, P = 0:06).Males receiving acclimation treatments shorter than32 h showed some reduction in longevity whereas thiswas not evident in the females. However, posthoc tests(Tukey B) indicate the same pattern for both sexes: thecontrols and the 8.5, 13 and 25.5 h treatments formed ahomogeneous group, and all these treatments differedsignificantly from the 32 h treatment.

We also measured parasitism rates at two temper-atures (25 and 30 �C) for females that had receivedacclimation treatments. Acclimation for 13 h or morereduced the number of eggs parasitised per female.This reduction occurred at both oviposition temper-atures but was less pronounced at 30 �C (Figure 4).At 25 �C, the average number of eggs parasitised wasreduced from around 14 in controls to five or fewer infemales hardened for 13 h or more. At 30 �C fecund-ity was lower; controls parasitised a mean of around

6 eggs and females hardened for at least 13 h parasit-ised from 1 to 4 eggs. Because a substantial numberof females did not parasitise any eggs at both tem-peratures, producing a skewed distribution, we com-pared treatments with non-parametric Kruskal-Wallistests. Differences among treatments are significant forboth the 25 �C (�2

= 39:54, df = 4, P < 0:001)and 30 �C (�2

= 12:61, df = 4, P = 0:01) data.Multiple comparisons using Dunn’s test indicated twohomogeneous groups for the 25 �C data. The firstconsisted of the controls and 8.5 h treatment, andthe second consisted of the 13, 25.5 and 32 h treat-ments. For the 30 �C data, there were significant dif-ferences between the controls and the 32 h treatment,but other differences were not significant. We obtainedsimilar results when we excluded females that failedto parasitise any eggs and compared treatments withANOVAs; treatment effects were highly significant forboth the 25 �C (F(4;106) = 32:97, P < 0:001) and30 �C (F(4;85) = 3:69, P = 0:008) data, and pos-thoc Tukey B comparisons indicated the same groupsas above. The similar conclusions from these differ-ent tests reflect the fact that acclimation influenced thenumber of eggs parasitised by females rather than thenumber of females parasitising Sitotroga eggs.

Developmental acclimation and parasitism. Rearingtemperature had a marked effect on parasitism rateat 14 �C (Figure 5). Only those wasps reared at14 �C successfully parasitised eggs. If the mean ofeach strain is considered as independent data point

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Figure 2. Effect on survival of exposure to 33 �C for varying times during development (see text). Survival in adult wasps was tested byexposing control and acclimated individuals to 40 �C for 120 min. Means and standard error bars are based on three trials.

Figure 3. Effect on longevity of exposure to 33 �C for varying times (see text) during immature development. Longevity was recorded forfemales and males given access to honey and water ad libitum. Means and standard deviations (bars) are based on 20–21 (hardened) or 80(control) individuals.

Figure 4. Effect on oviposition rate of exposure to 33 �C for varying times (details in text) during immature development. Oviposition successwas scored for 24 h at 25 �C or 30 �C. These data include individuals who laid no eggs. Error bars are standard deviations based on 20(hardened) or 80 (control) individuals.

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for comparing rearing temperatures, this difference ishighly significant by a Kruskal Wallis test (�2

= 18:97,df = 2, P < 0:001). In contrast, differences at 25 �C(�2

= 0:02) and 30 �C (�2= 0:91) are not significant.

There was evidence that strains did not behave consist-ently. While there were no significant strain differenceswhen wasps were tested at 14 �C, these were evidentin all other combinations of rearing and acclimationtemperatures. When wasps were reared at 25 �C and30 �C, three of the strains (3, 5 and 6) failed to para-sitise any eggs in contrast to their parasitism successwhen reared at 14 �C (Figure 5). These strains differedfrom all others at the 5% level by Dunn’s posthoc com-parisons. In addition, strain differences were evidentwhen wasps were raised at 14 �C and tested at both25 �C (strain 5 differed from 1, 2, 3, 4, 7) and 30 �C(2 from 1, 3, 5, 7 and 4 from 3, 5, 7). These data there-fore indicate genotype x rearing interactions which areevident at some temperatures.

Discussion

We found strong evidence for improvements in surviv-al after heat stress when Trichogramma carverae werehardened. Hardening was elicited as a consequence ofexposures to elevated temperatures in both adult andimmature wasps. Longer exposures to elevated temper-ature were required in immatures than adults, possiblyas a consequence of the increased delay prior to test-ing. In adults, improvements were present in waspstested from one to four h after acclimation and wereelicited by one or two h exposures to 35 or 33 �C. Inimmature wasps, 13 h or more of exposure to elevatedtemperature was required to increase resistance to heatshock, although there was no acclimation after 32 h.This could be a consequence of injury (see below) orsome alternative physiological response to temperat-ure increase during development. Our results for adultsare consistent with evidence from other insects indic-ating that short exposures to high temperatures canincrease subsequent resistance levels. For example, inDrosophila, 60–75 min exposures to 36 �C resulted inmaximal increases in resistance to elevated (39.3 �C)temperature (Krebs & Loeschcke, 1994). Comparableresults on the pre-adult stages of Drosophila or otherinsects are not available because most previous studiesof acclimation have focused on adults and on resistanceand acclimation within a life-cycle stage.

Acclimation of immatures had negative as well aspositive effects. Acclimation increased survivorship

after a heat stress but decreased longevity and para-sitism in the absence of a heat stress. The life his-tory traits had different sensitivities to developmentalexposures to elevated temperature; fecundity (as reflec-ted by parasitism rate) was decreased by over 50% at25 �C after 8.5 h of acclimation. At 30 �C, fecunditywas decreased by 30% after 8.5 to 25.5 h and by 80%after 32 h of acclimation. In contrast, longevity at 25 �Cwas only influenced after 32 h. Deleterious effectsof heat exposure are known from studies with otherTrichogramma (e.g., Wilkes, 1959; Chihrane et al.,1991).

These negative effects can be interpreted in twoways. First, they could be costs of acclimation. Forexample if increased resistance to stress is causedlargely by the production of heat shock proteins(Parsell & Lindquist, 1993), then the energetic costsof producing these proteins could be responsible forreduced fecundity. Adult acclimation experiments onDrosophila (Krebs & Loeschcke, 1994) resulted indecreased fecundity, and these authors argue that thisdecrease represents a cost of acclimation itself. Altern-atively, exposure to elevated temperature may injurethe organism directly or result in energetic costs unre-lated to the acclimation process. Exposure to elevatedtemperature results in diverse physiological changes inectotherms (Cossins & Bowler, 1987) some of whichcould be injurious. Separating the costs of acclimationfrom injury due to the acclimation process requiresexperimental or comparative work in addition to simplecomparisons of treated and untreated insects.

Because of these negative effects, it is not clear ifhardening treatments in adult or immature wasps wouldbe a useful tool for improving the quality of Tricho-gramma reared for release. Acclimation treatments ofsufficient duration to increase survivorship after heatstress decreased fecundity by 50% or more, and longerduration treatments reduced longevity as well. On theother hand if production levels are limited and met-eorological conditions indicated a period of high tem-peratures, the potential negative effects of acclimationmay be outweighed by their benefits. This could beparticularly important for releases of T. carverae ingrapevines against flights of lightbrown apple moth insummer when daytime temperatures can exceed 40 �C(Glenn & Hoffmann, 1997).

Results from the rearing experiment suggest thatwasps may have a higher fitness under conditions theyhave encountered at an earlier developmental stage.Wasps reared at 14 �C parasitised eggs at this temper-ature, while wasps reared at 25 �C and 30 �C were

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Figure 5. Effect of rearing conditions on parasitism at 14, 25 and 30 �C for seven strains of T. carverae. Parasitism of female-male pairs wasmeasured over 24 h. Means and standard deviations (bars) are based on 20–30 replicates.

unable to parasitise at 14 �C. Individuals may there-fore have a relatively higher fitness under conditionsthey have previously encountered. This is consistentwith the ‘beneficial acclimation hypothesis’ outlined inZamudio et al. (1995), where acclimation has a direct

benefit to an organism experiencing similar conditions.Evidence for this hypothesis is quite weak, and sever-al laboratory studies appear to contradict it (see Huey& Berrigan, 1996). Trichogramma may be a usefulorganism for investigating this hypothesis under field

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conditions, because the field fitness of Trichogrammacan be readily determined from egg parasitism rates.

Our laboratory results suggest that development-al acclimation could help to increase parasitism ratesunder cool conditions. Releases of T. carverae arenot particularly effective in cool climates, presumablybecause this species normally occurs in warm areas(Glenn et al., 1997). Rearing wasps under cool con-ditions (rather than temperatures of around 25 �C ascurrently used) could help to generate wasps capable ofparasitising lightbrown apple moth under a wide rangeof field temperatures.

Acknowledgements

D. Berrigan acknowledges NSF (INT-942409), NSF-IBN-9514205 to R. B. Huey and the USDA (96-35302-3739). A. A. Hoffmann acknowledges financial sup-port from the Australian Research Council and Grapeand Wine Research and Development Corporation. Weare grateful to D. Glenn for supplying the Tricho-gramma used in this study.

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