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Jun 3, 2014 - The effect of overwintering temperature on the body energy reserves and phenoloxidase activity of bumblebee Bombus lucorum queens.
Insect. Soc. (2014) 61:265–272 DOI 10.1007/s00040-014-0351-9

Insectes Sociaux

RESEARCH ARTICLE

The effect of overwintering temperature on the body energy reserves and phenoloxidase activity of bumblebee Bombus lucorum queens S.-R. Vesterlund • T. M. Lilley • T. van Ooik J. Sorvari



Received: 17 October 2013 / Revised: 30 April 2014 / Accepted: 7 May 2014 / Published online: 3 June 2014 Ó International Union for the Study of Social Insects (IUSSI) 2014

Abstract Warming winters and changes in species composition related to the estimated global warming may cause a threat to bumblebees adapted to cold winters. During the overwintering period, their intermediary and respiratory metabolism decreases but metabolism remains responsive to temperature. The effect of temperature on diapause survival, phenoloxidase (PO) activity, and energy expenditure of the white-tailed bumblebee (Bombus lucorum) after a 4-month diapause were studied by manipulating the diapause temperature. Two overwintering temperatures were used, cold (1.8 °C) and warm (9 °C). Body fat content was used as an estimate of the remaining energy resources and PO activity as an immune function parameter of overwintering bumblebee queens. The baseline levels of PO activity were used to measure the differences in B. lucorum queen responses after overwintering in either temperature. We found a 0.4 g pre-diapause threshold weight of survival in B. lucorum. Large queens had more fat left and a higher PO activity compared to small ones after overwintering in warm conditions, but in the cold there was no effect of size on the remaining fat in the fat body of queens or their PO activity. The observed difference in energy usage appears to relate to normal size-dependent metabolism and variation in

S.-R. Vesterlund (&)  T. van Ooik Section of Ecology, Department of Biology, University of Turku, FI-20014 Turku, Finland e-mail: [email protected] T. M. Lilley Section of Biodiversity and Environmental Science, Department of Biology, University of Turku, FI-20014 Turku, Finland J. Sorvari Department of Environmental Science, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland

energy allocation between basic metabolism and immune functions. Keywords Immunity

Diapause  Fat body  Metabolism 

Introduction The life cycle of northern bumblebees begins with a new queen emerging from winter hibernation, finding a suitable nest site, and founding a colony by laying the first group of eggs (Alford, 1978). The colony grows until new queens and drones are produced and they leave the nest for mating (Goulson, 2008). Only new queens survive the winter in the northern hemisphere, where bumblebees usually produce only a single colony each year (Alford, 1978; Goulson, 2010; but see Stelzer et al., 2010). The new queens excavate their hibernating chambers (2–15 cm deep) for example under large trees or into sand banks depending on the species (Alford, 1978). Bumblebee species exhibit a range of inter-specific variance in temperature tolerance with some species adapted to cooler climatic regions or higher altitudes, such as the white-tailed bumblebee Bombus lucorum (Linnaeus, 1761) (Benton, 2006). Others, such as for example the buff-tailed bumblebee (Bombus terrestris), are typically found in a warmer climate or lower altitude (Benton, 2006). The latest Intergovernmental Panel on Climate Change report predicts that the mean global temperature may rise as much as 4.8 °C during the next 100 years (IPCC, 2013). Winter temperatures will most likely be affected the most, which suggests that future winter temperatures in Fennoscandia are going to be similar to current southern European conditions. Overwintering temperature may be higher than

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on average also, if the habitat chosen by individual queens is well protected from freezing (e.g., inside buildings or greenhouse surroundings). Warmer than usual overwintering temperature is likely to have implications on the diapause of the northern populations of cold-adapted bumblebee species. The costs associated with diapause are most likely due to damage from assorted diapause-associated stress, such as desiccation or cold shock, and the depletion of metabolic reserves (Hahn and Denlinger, 2007). During the overwintering period, the metabolism of many insects is significantly decreased (Tauber et al., 1986; Danks, 1987; Leather et al., 1993; Guppy and Withers, 1999). However, metabolism remains responsive to temperature: insects diapausing at lower temperatures show decreased respiration and slower consumption of reserves than insects wintering at warmer conditions (Chaplin and Wells, 1982; Irwin and Lee, 2000; Thompson and Davis, 1981; Williams et al., 2012). Bumblebee queens overwintering in warm conditions remain more active and consume their energy reserves faster than in the cold (Alford, 1978). The survival of B. terrestris queens decreases with increasing diapause duration (Gosterit and Gurel, 2009), but earlier studies by Beekman et al. (1998) have not shown an effect of temperature on bumblebee queen survival during diapause. Weight at the start of the diapause, however, is important in determining B. terrestris queen survival through winter hibernation: queens weighing under 0.6 g prior to diapause did not survive in laboratory conditions irrespective of how long (1, 2, 4, 6, or 8 months) they were kept in diapause (Beekman et al., 1998). The survival of the red wood ant (Formica aquilonia) is lower at an elevated, 7 °C, than the normal overwintering temperature (1 °C) (Sorvari et al., 2011). Additionally, the prepupae of a northerly rose galling wasp, Diplolepis spinosa, exhibit lower survival in an experimentally raised overwintering temperature (comparisons between -22, 0, ?5, or ?10 °C), but in contrast, there was no difference in the survival of pupae or eggs of the lower latitude species Diplolepis variosa (Williams et al., 2003). This indicates that there may be differences in the response of species belonging to the same genus depending on their natural distribution. The fat body is essential to insects for their energy storage and utilization and it also synthesizes most of the circulating metabolites and hemolymph proteins (Arrese and Soulages, 2010). Fatty acid synthesis and the production and storage of triacylglyceride are situated primarily in the insect fat body, and the size of the lipid storage is considered to be important in mitigating the metabolic demands of diapause (Hahn and Denlinger, 2011). Red wood ant workers had less body fat resources left after diapausing in higher overwintering temperature compared to colder

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conditions in a diapause experiment, likely because of the faster consumption of energy reserves (Sorvari et al., 2011). Higher overwintering temperatures also increase metabolic rate and decrease survival of the gall fly Eurosta solidaginis (Irwin and Lee, 2000, 2003). Phenoloxidase (PO) activity of insects has been shown to vary according to temperature, some studies indicating an increase in PO activity with higher than average temperature (Zhuo et al., 2011; Karl et al., 2011), whereas others suggest that higher PO activity is linked with superior thermoregulation capacity in colder environments (e.g. Fedorka et al., 2013). The invertebrate immune system fights against invaders in multiple ways, of which, the encapsulation of foreign particles by covering it with layers of hemocytes, melanization, is often used (Dunn, 1986; Gillespie et al., 1997; Karp, 1990). This process is activated by several enzymes, of which PO has a key role (Sugumaran and Kanost, 1993). It also forms a part of the induced immune response of insects (Gillespie et al., 1997). Phenoloxidase in uninfected, i.e. naı¨ve individuals is always present in the hemocytes as well as the hemolymph in its inactive form, pro-PO (Brookman et al., 1989). Higher baseline PO levels have often been associated with a better defence against pathogens (Tucker and Stevens, 2003; Okado et al., 2009). In Bombus muscorum, levels of PO in workers declined significantly with increasing parasite abundance and were influenced by worker size: larger bees had higher levels of volume-corrected active PO (Whitehorn et al., 2011). Phenoloxidase activity in social insects has been used to study for example covariation between colony social structure and immune defences in ant workers (Castella et al., 2010). Rantala et al. (2003) and Siva-Jothy and Thompson (2002) reported that a short-term nutrient deprivation (only ad libitum water) reduced the hemolymphal PO activities of mealworm beetles (Tenebrio molitor). Therefore, it is plausible that reduced fat storage during diapause would have a decreasing effect on PO activity. The effect of temperature on diapause survival, PO activity, or energy expenditure is yet to be studied in one of the most common northern bumblebee species, the whitetailed bumblebee B. lucorum. In the present experiment, we manipulated the overwintering temperature of B. lucorum to study its effects on diapause survival, body fat content, and baseline PO activity after a 4 month diapause. We used two over-wintering temperatures, cold (0–3 °C) and warm (7–10 °C). Immune system and energy store use are biochemically temperature sensitive. Therefore, we predict that (1) the weight of bumblebee queens at the beginning of the diapause affects their survival, such that queens under a certain threshold weight do not survive until the end of the 4 month diapause period, and (2) at a warm diapause temperature individuals use more of their fat resources than at a cold

Overwintering effect on B. lucorum queens

diapause temperature, and thus, have proportionally less fat left in their fat bodies, and (3) PO activity is higher after overwintering in colder diapause conditions because the queens have more fat left to use as energy resource for metabolic functions.

Materials and methods Bumblebee rearing and hibernation experiment One hundred and fifty queens of native B. lucorum were collected from four locations in southwestern Finland (3 in Turku, 1 in Kemio¨) in late April–early May 2011. The queens were reared in a laboratory under a red light [24–25 °C, 50–60 % r.h., pollen, and Biogluc-sugar solution (Biobest ND) ad libitum]. Of these queens, 130 individuals started laying eggs and 33 nests produced new queens 1–6 queens each. Of all the 83 hatched new queens, 63 mated with drones from other reared nests and were used in the experiment. The queens were divided into two treatment groups in the order of their mating, but taking care that there was an equal number of sister queens in both treatment groups when possible. Both groups received an acclimatizing treatment without feeding at constant darkness at 15 °C for 2 weeks before the start of the experiment. Each queen was placed in a closed 50 ml plastic tube with a cotton ball moistened with 2 ml of distilled water on the bottom and above that a 7 cm layer of vermiculiteperlite-mix, and the queen on the top. Group A was put to diapause at cold (0–3 °C; mean 1.8 °C; SE 1.18) and group B at warm (7–10 °C; mean 9.0 °C; SE 0.14) conditions. The container lid was only lightly closed allowing adequate gas flow. The lid was also opened for a short while when the queens were weighed once a month, including pre- and postdiapause weights. During measurements the queens were removed from the container for *30 s. PO activity and total protein content measurement Hemolymph was needed for the measurement of the PO activity and total protein content. The queens were removed from hibernation and kept at ?20 °C for a couple of hours to fully awaken them. The queens were then chilled on ice before 10 ll of hemolymph per queen was extracted from the thorax using a Hamilton syringe. The extracted hemolymph sample was put into a 1.5 ml microcentrifuge tube containing 50 ll of cold Ringer’s solution, giving a total volume of 60 ll hemolymph solution. Of the solution, 40 ll was further diluted with 30 ll of cold Ringer’s solution, immediately frozen with liquid nitrogen and stored at -80 °C for the measurement of PO activity.

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The hemolymph extract samples (dilution 1 in 10.5 hemolymph/Ringer’s solution) for the measurement of the PO activity were thawed on ice and 5 ll was pipetted in triplicate to a 396-microplate well containing 35 ll of distilled water, 5 ll of phosphate buffer saline (PBS 8.74 g NaCl, 1.78 g Na2HPO42H2O, 1,000 ml distilled water, pH 6.5). At this stage the plate was spun to make sure all reagents were at the bottom of the well before placing the plate into a multilabel counter (EnSpire Multimode Reader, Perkin-Elmer, Turku, Finland), which dispensed 20 ll of L-DOPA solution (4 mg ml-1 mqH2O) into each well read. The reaction proceeded at 30 °C in the multilabel counter for 40 min. Each well was read once a minute at 490 nm and analysed. Enzyme activity was measured using the following equation: m¼

y2  y1 x2  x1

where m is the slope of the reaction curve (DAbs min-1 ml-1) during the linear phase of the reaction, counted from the ratio of the altitude change (y2 - y1; rise) to the horizontal distance (x2 - x1; run) between two points in the reaction curve. The baseline levels of the PO activity were used to measure the differences in B. lucorum queen responses after overwintering in either temperature. Induced by the transformation of L-DOPA to dopachrome by phenoloxidase, the total amount of enzymes is proportional to the increase in the optical density (So¨derha¨ll and Cerenius, 1998). PO activity is often correlated with protein concentration of the hemolymph protein (for example Klemola et al., 2007). Therefore, the total protein concentration in the hemolymph was quantified using the BioRad protein assay kit based on the Bradford method (Bradford, 1976). Lipid extraction Body fat content can be used to quantify the nutritional status of individual insects (e.g. Sundstro¨m, 1995; Hahn, 2006; Sorvari and Hakkarainen, 2009). After the extraction of hemolymph, the abdomen of each queen was carefully cut off the thorax, and the honey stomach was removed. The abdomens were then inserted in individual test tubes and frozen at -21 °C until freezedrying for 48 h (Christ LMC-1 freeze-dryer). The abdomens were weighed on a precision scale after which 8 ml of petroleum ether solvent was added to the tubes and they were placed in an ultrasonic bath for 4 h. Petroleum ether was used instead of chloroform because the latter tends to cause the bristles and hairs to fall off the bumblebee torso, causing measurement errors.

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Petroleum ether functioned better with this species when comparing these solvents. After the ultrasonic bath, the samples were rinsed twice for 10 min with 8 ml petroleum ether. The abdomens were then placed in a drying oven at 45 °C for 48 h and weighed. The difference in dry weight before and after petroleum ether treatment is interpreted as the amount of fat dissolved from the individual during the process (Sundstro¨m, 1995; Sorvari and Hakkarainen, 2009). The fat-free dry weight of the abdomen (hereafter ‘‘queen size’’) is used as a measure of the size of queens in the analyses of body fat and immune parameters. Statistical analyses All statistical analyses were made with statistical software SAS version 9.2 (SAS Institute, Cary, NC, USA). Diapause survival We used a generalised linear model (GLIMMIX procedure) with binomial distribution and logit link function. Survival was used as the binomial dependent variable (survived = 1, deceased = 0) and treatment and the fresh weight of queen before diapause as a fixed effects. The colony of origin was not used as a random factor in the model because the model did not converge with it. However, there are on average 2.1 queens per nest equally distributed into the two temperature treatments, which should level the possible effect of the colony origin in the analysis. Body fat

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Results Diapause survival The fresh weight of queens prior to diapause affected their survival: large queens had better survival compared to smaller queens (queen size: F1,57 = 14.48, P = 0.0003). All queens under the wet weight of 0.4 g at the beginning of the experiment deceased during the 4 month diapause in both warm and cold diapause conditions (Fig. 1). This effect of queen size was independent of overwintering temperature (interaction term queen size 9 treatment: F1,56 = 0.80, P = 0.37). The treatment (warm/cold) did not affect survival (F1,56 = 0.02, P = 0.89). Body fat There was a significant interaction effect between treatment and queen size on the amount of fat left in the abdomen after the diapause (F1,42 = 16.42, P = 0.0002; Fig. 2: data presented as recommended for covariate by treatment interaction models; Littell et al., 2006). Larger queens that overwintered in the warm treatment had more fat in their fat body after the diapause compared to smaller queens (F1,20.9 = 13.07, P = 0.0016), but in the colder treatment, the trend was opposite yet statistically insignificant such that larger queens had less fat in their fat body after the diapause compared to smaller queens (F1,21 = 2.90, P = 0.10; Fig. 2). The amount of body fat was generally lower in queens that overwintered in warmer temperature when the body size was used as a covariate (F1,42 = 20.25, P \ 0.0001). However, because there was a significant

We used a REML-based mixed model with Kenward–Roger approximation of the degrees of freedom. The amount of fat (mg) extracted after overwintering was used as the dependent variable in the analysis, with queen size, treatment, and their interaction as fixed effects. The nest of origin was used as a random factor. PO activity and protein concentration We used a REML-based mixed model with Kenward–Roger approximation of the degrees of freedom. PO activity differences were analysed using treatment, protein concentration, queen size, and their interactions as fixed effects and nest of origin as a random factor. Second, since PO activity is related to the amount of total protein, the effect of treatment on PO activity and protein concentration was analysed further using them as dependent variables in two separate analyses, where treatment, queen size, and their interaction were fixed effects, and nest of origin a random factor.

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Fig. 1 The cumulative survival of B. lucorum queens in relation to their fresh weight (mg) at the start of the overwintering period

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Fig. 4 The estimated marginal means of the hemolymph protein concentration of B. lucorum queens (mg ml-1 ±95 % CL) in the cold (C, mean 1.8 °C) and warm (W, mean 9 °C) temperature treatment

Fig. 2 The estimated marginal mean amount of fat after extraction (mg ±95 % CL) at 90th percentile, upper quartile, median, lower quartile, and 10th percentile of B. lucorum queen size (the fat-free dry weight of the abdomen) in the cold (mean 1.8 °C, open circle) and warm (mean 9 °C, filled circle) temperature treatment

Fig. 3 The estimated marginal means of the body fat of B. lucorum queens (mg ±95 % CL) in the cold (C, mean 1.8 °C) and warm (W, mean 9 °C) temperature treatment

interaction between queen size and treatment the P value should be treated with caution. However, the mean of the other treatment does not overlap with the 95 % confidence limits of the other, which also indicates a significant difference between the two treatments (Fig. 3).

PO activity and protein concentration Queen size did not affect the total protein concentration of the hemolymph in either of the temperature treatments (interaction queen size 9 treatment: F1,42 = 0.61, P = 0.44; size: F1,43 = 0.11, P = 0.74). Queens diapausing in the colder temperature treatment had a higher hemolymph protein concentration than queens diapausing in the warmer treatment (F1,43 = 7.02, P = 0.011; Fig. 4) after the 4 month period. Hemolymph protein concentration affected the PO activity of queens differently between the treatments (protein concentration 9 treatment: F1,42 = 4.91, P = 0.032). An increase in the hemolymph protein concentration increased the PO activity of queens in the cold temperature treatment, whereas in the warm temperature treatment there was no such effect (cold: F1,19.7 = 16.10, P = 0.0007; warm: F1,21 = 0.19, P = 0.67; Fig. 5: data presented as recommended for covariate by treatment interaction models; Littell et al., 2006). Queen size affected the PO activity differently between the treatments (queen size 9 treatment: F1,41.7 = 5.36, P = 0.026): PO activity increased with increasing queen size in the warm (F1,21 = 12.62, P = 0.0019), but in the cold, queen size did not affect PO activity (F1,19.7 = 0.05, P = 0.83; Fig 6: data presented as recommended for covariate by treatment interaction models; Littell et al., 2006).

Discussion During the experimental 4 month diapause period, a fresh weight of 0.4 g prior to overwintering was estimated to be

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Fig. 5 The estimated marginal means of the PO activity (DAbs min-1 ml-1) of B. lucorum queens (±95 % CL) at 90th percentile, upper quartile, median, lower quartile, and 10th percentile of the hemolymph protein concentration (mg ml-1) in the cold (mean 1.8 °C, open circle) and warm (mean 9 °C, filled circle) temperature treatment

Fig. 6 The estimated marginal means of the PO activity (DAbs min-1 ml-1) of B. lucorum queens (±95 % CL) at 10th percentile, lower quartile, median, upper quartile, and 90th percentile of queen size (the fat-free dry weight of the abdomen) in the cold (mean 1.8 °C, open circle) and warm (mean 9 °C, filled circle) temperature treatment

the threshold weight for survival of B. lucorum queens. Queens used more fat in warm conditions (mean 9 °C), but especially large individuals had more fat left after the

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diapause compared to small ones. Larger queens also had a higher PO activity compared to smaller queens in the warm diapause treatment. However, in cold diapause conditions (mean 1.8 °C) the size of the queen affected neither the remaining fat in the fat body nor the PO activity of the queens. In the cold treatment, queens had a higher hemolymph protein concentration, and an increase in hemolymph protein concentration correlated with increased PO activity. The threshold weight for the diapause survival of B. lucorum was 0.2 g lower than previously shown in the southern sister species B. terrestris (Beekman et al., (1998)). The survival of queens was not otherwise analysed, since the diapause experiment lasted only 4 months (instead of 7–8 months typical to B. lucorum in Finland). The experiment was not designed for measuring overwintering survival but instead we wanted to have as many queens alive after the diapause as possible for PO activity and fat analyses. Thus, this result cannot be compared with survival studies that measure survival during longer diapause periods. However, during the overwintering we still observed a similar threshold weight of survival as Beekman et al. (1998) in their experimental research on B. terrestris. We assume the lower survival weight of B. lucorum to be a probable consequence of the overall size difference between the two species, B. terrestris being on average the larger one. As all B. lucorum queens under the threshold weight died early on, it may be that they had already depleted some important part of their energy reserves, such as glycogen or the unsaturated fatty acids (Hahn and Denlinger, 2011). Generally, queens had less fat left in their fat bodies after the diapause in warm conditions, as was expected by our prediction stating that their rate of energy consumption should be higher than that of queens that diapaused in cold conditions. Research indicates that diapause depths and preferred locations are fixed and vary between species rather than between individuals of the same species (Alford, 1978). Thus, it does not seem likely that queens would be able to regain digging behavior to avoid freezing or warming after they have moved into the diapause state. Recently it has been shown that bumblebees are highly flexible in terms of weather and the time of year: in urban Britain the queens of B. terrestris are active throughout mild winters if there are flowering plants available (Stelzer et al., 2010). New queens also excavate in the late summer before the temperature decreases and their survival might be linked to the duration of cold versus warm periods during the whole diapause length. The survival of B. terrestris queens decreased with increasing duration of diapause even when durations less than four months were used (Gosterit and Gurel, 2009). Thus, it is likely that mild and long autumns would lower queen survival and indicate high energy expenditure because the queens are not able to reach full inactive diapause state until the temperature becomes cold enough. Usually the diapause

Overwintering effect on B. lucorum queens

temperature of northern bumblebees stays so low that their metabolism becomes very slow and they remain mostly in a hibernation state (Alford, 1978). In our experimental study, large queens had more fat left compared to small ones after overwintering in the warmer, but in cold conditions (considered as normal overwintering temperature), there was no effect of size on the remaining fat in the fat body. The fat consumption difference between queens of variable sizes disappeared when the diapause temperature stayed close to the normal mean. Thus, the observed difference seems to relate to a normal sizedependent metabolism rather than adaptive traits. Larger female carpenter bees have proportionally larger abdomens and as a consequence, lower mass-specific metabolic rates (Roberts et al., 2004). All B. lucorum queens in our study used more fat in the warm compared to cold temperature but in general, larger queens had more fat left after the 4-month diapause than the smaller queens. This result is also consistent with intraspecific allometries found in social insects such as ants and honeybees (Waters and Harrison, 2012). Though fat reserves are by far the most important energy source during diapause (Hahn and Denlinger, 2007), there is also the possibility that some form of non-fat energy reserve plays a part in bumblebee overwintering, and solely fat consumption may not reveal all of the dynamics of energy metabolism during diapause. The larger the queen, the higher its PO activity after diapause in warmer temperature. In the cold, size did not affect PO activity after diapause. As a vital part of the immune system of insects, PO-activity levels can be used as an indicator of the strength of the immune system (Dunphy, 1991; Gillespie et al., 1997; Reeson et al., 1998). The effect of body size on the PO activity has been earlier noticed by Castella et al. (2010), who suggest that F. selysi ant workers originating from monogyne colonies may be able to mount a comparatively higher immune response for the same level of pathogen exposure than workers originating from polygyne colonies simply because of their larger body size. Karl et al. (2011) investigated the effects of temperature extremes on fitness-related adult traits of the tropical butterfly Bicyclus anynana and concluded that there seems to be competing energetic demands between the immune system and general metabolism. It seems that when the diapause temperature was high, larger queens had significantly more resources left for their immune functions than the smaller ones, possibly because their fat resources lasted longer and were enough to cover for their basic metabolic needs over the whole diapause period. Smaller queens in the same treatment group, however, were not able to allocate as much energy to immune functions (hemolymph protein concentration and PO activity). To conclude, larger queens in the warmer temperature treatment group had higher PO-activity and more fat left

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after the diapause than smaller queens experiencing the same conditions. These differences seem likely to be a consequence of (1) the lower mass-specific metabolic rates of larger queens, and (2) the ability of larger queens to allocate more resources to immune functions without compromising their other metabolic needs during diapause. In the colder temperature there were no size-related differences, most likely because the overall energy consumption was closer to normal diapause conditions where the queens are in full hibernation and their metabolism is very low. Thus, in the cold also smaller queens had enough resources to use for their immune functions and the differences became visible only in the warmer treatment group queens. Overwintering insects that have a tight energy budget are likely to be especially vulnerable to high temperatures associated with climate change (Hahn and Denlinger, 2011). The difference between large and small body size may become more significant if the hibernation temperature is high enough to force the queens to be semi-active rather than in a full diapause state. Large queens seem better able to survive warmer than average diapause periods and have better protection from pathogens the following spring. Long, warm autumns and warm springs with early snowmelt, or both, during one diapause period, would be possible future scenarios in Fennoscandia. When diapause temperature is elevated, even small differences in the rate of metabolism may become crucial for both survival and the amount of energy resources left for nest founding in the spring. Theoretically, also constant changes between full diapause and more active states during hibernation should increase the differences found between different sized individuals, but more research is needed to resolve this question. Acknowledgments We would like to thank MSc Maria Kakko, MSc Marja-Katariina Haatanen, Oskari Ha¨rma¨, and Erkki Kaarnama for their assistance. We would also like to thank two anonymous reviewers for helping to improve the manuscript. This research was funded by Maj and Tor Nessling Foundation, Societas Entomologica Fennica, University of Turku (grants to S.-R. V.), Kone Foundation, and Emil Aaltonen Foundation (grants to J.S.).

References Alford D.V. 1978. The Life of the Bumblebee. Davis-Poynter, London. Arrese E.L. and Soulages J.L. 2010. Insect fat body: energy, metabolism, and regulation. Annu. Rev. Entomol. 55: 207-225 Beekman M. van Stratum P. and Lindeman R. 1998. Diapause survival and post-diapause performance in bumblebee queens (Bombus terrestris). Entomol. Exp. Appl. 89: 207-214 Benton T. 2006. Bumblebees. HarperCollins, UK. Bradford M.M. 1976. A dye binding assay for protein. Anal. Biochem. 72: 248-254 Brookman J.L., Ratcliffe N.A. and Rowley A.F. 1989. Studies on the activation of the prophenoloxidase system of insects by bacterial cell wall components. Insect Biochem. 19: 47-58

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272 Castella G., Christe P. and Chapuisat M. 2010. Covariation between colony social structure and immune defences of workers in the ant Formica selysi. Insect. Soc. 57: 233-238 Chaplin S.B. and Wells P.H. 1982. Energy reserves and metabolic expenditures of monarch butterflies overwintering in southern California. Ecol. Entomol. 7: 249-256 Danks H.V. 1987. Insect Dormancy: An Ecological Perspective. Biological Survey of Canada Press, Canada. Dunn P.E. 1986. Biochemical aspects of insect immunology. Annu. Rev. Entomol. 31: 321-339 Dunphy G.B. 1991. Phenoloxidase activity in the serum of two species of insects, the gypsy moth, Lymantria dispar (Lymantriidae) and the greater wax moth, Galleria mellonella (Pyralidae). Comp. Biochem. Physiol. B 98B: 535-538 Fedorka K.M., Copeland E.K. and Winterhalter W.E. 2013. Seasonality influences cuticle melanization and immune defense in a cricket: support for a temperature-dependent immune investment hypothesis in insects. J. Exp. Biol. 216: 4005-4010 Gillespie J.P., Kanost M.R. and Trenzcek T. 1997. Biological mediators of insect immunity. Annu. Rev. Entomol. 42: 611-643 Gosterit A. and Gurel F. 2009. Effect of different diapause regimes on survival and colony development in the bumble bee, Bombus terrestris. J. Apic. Res. 48: 279-283 Goulson D., Lye G.C. and Darvill B. 2008. Decline and conservation of bumble bees. Ann. Rev. Entomol. 53: 191-208 Goulson D. 2010. Bumblebees: Behaviour and Ecology. 2nd edn. Oxford University Press Inc., New York Guppy M. and Withers P. 1999. Metabolic depression in animals: physiological perspectives and biochemical generalizations. Biol. Rev. 74: 1-40 Hahn D.A. 2006. Two closely related species of desert carpenter ant differ in individual-level allocation to fat storage. Physiol. Biochem. Zool. 79: 847-856 Hahn D.A. and Denlinger D.L. 2007. Meeting the energetic demands of insect diapause: Nutrient storage and utilization. J. Insect Physiol. 53: 760-773 Hahn D.A. and Denlinger D.L. 2011. Energetics of insect diapause. Annu. Rev. Entomol. 56: 103-121 IPCC 2013: Climate Change 2013: The Physical Science Basis. http:// www.ipcc.ch/report/ar5/wg1/#.UkwOoKyAmrA (site visited14. Oct 2013) Irwin J.T. and Lee R.E. 2000. Mild winter temperatures reduce survival and potential fecundity of the goldenrod gall fly, Eurosta solidaginis (Diptera: Tephritidae). J. Insect Physiol. 46: 655-661 Irwin J.T. and Lee R.E. 2003. Cold winter microenvironments conserve energy and improve overwintering survival and potential fecundity of the goldenrod gall fly, Eurosta solidaginis. Oikos 100: 71-78 Karl I., Stocks R., De Block M., Janowitz S. and Fischer K. 2011. Temperature extremes and butterfly fitness conflicting evidence from life history and immune function. Global Change Biol. 17(2): 676-687 Karp R.D. 1990. Cell-mediated immunity in invertebrates. Bioscience 40: 732-737 Klemola N., Klemola T., Rantala M.J. and Ruuhola T. 2007. Natural host-plant quality affects immune defence of an insect herbivore. Entomol. Exp. Appl. 123: 167-176 Leather S.R., Walters K.F.A. and Bale J.S. 1993. The Ecology of Insect Overwintering. Cambridge University Press, Cambridge. Littell R.C., Milliken G.A., Stroup W.W., Wolfinger R.D. and Schabenberger O. 2006. SASÒ for Mixed Models, 2nd edition. SAS institute Inc. Okado K., Shinzawa N., Aonuma H., Nelson B., Fukumoto S., Fujisaki K., Kawazu S.-I. and Kanuka H. 2009. Rapid recruitment of

123

S.-R. Vesterlund et al. innate immunity regulates variation of intracellular pathogen resistance in Drosophila. Biochem. Biophys. Res. Commun. 379: 6-10 Rantala M.J., Kortet R., Kotiaho J.S., Vainikka A. and Suhonen J. 2003. Condition dependence of pheromones and immune function in the grain beetle Tenebrio molitor. Funct. Ecol. 17: 534-540 Reeson A.F., Wilson K., Gunn A., Hails R.S. and Goulson D. 1998. Baculovirus resistance in the noctuid Spodoptera exempta is phenotypically plastic and responds to population density. Proc. R. Soc. Lond. B 265: 1787-1791 Roberts S., Harrison J.F. and Dudley R. 2004. Allometry of kinematics and energetics in carpenter bees (Xylocopa varipuncta) hovering in variable density gases. J. Exp. Biol. 207: 993-1004 Siva-Jothy M.T. and Thompson J.J.W. 2002. Short-term nutrient deprivation affects immune function. Physiol. Entomol. 27: 206-212 So¨derha¨ll K. and Cerenius L. 1998. Role of the prophenoloxidase activating system in invertebrate immunity. Curr. Opin. Immunol. 10: 23-28 Sorvari J. and Hakkarainen H. 2009. Forest clear-cutting causes small workers in the polydomous wood ant Formica aquilonia. Ann Zool Fenn 46: 431-438 Sorvari J., Haatanen M.-K. and Vesterlund S.-R. 2011. Combined effects of overwintering temperature and habitat degradation on the survival of boreal wood ant. J. Insect Cons. 15: 727-731 Stelzer R.J., Carlton M., Chittka L. and Ings T.C. 2010. Winter active bumblebees (Bombus terrestris) achieve high foraging rates in urban Britain. PLoS One 5: e9559 Sugumaran M. and Kanost M.R. 1993. Regulation of insect hemolymph phenoloxidases. In: Parasites and Pathogens in Insects. Volume 1: Parasites (Beckage N.E., Thompson S.N. and Federici B.A., Eds) Academic Press, San Diego. pp 317-342 Sundstro¨m L. 1995. Dispersal polymorphism and physiological condition of males and females in the ant Formica truncorum. Behav. Ecol. 6: 132-139 Tauber M.J., Tauber C.A. and Masaki S. 1986. Seasonal Adaptations of Insects. Oxford University Press, Oxford Thompson A.C. and Davis F.M. 1981. The effect of temperature on the rate of metabolism of lipids and glycogen in diapausing southwestern corn borer, Diatraea grandiosella. Comp. Biochem. Physiol. 70A: 555-558 Tucker T.M. and Stevens L. 2003. Geographical variation and sexual dimorphism of phenoloxidase levels in Japanese beetles (Popillia japonica). Proc. R. Soc. B 270: 245-247 Waters J.S. and Harrison J.F. 2012. Insect metabolic rates. In: Metabolic Ecology: A Scaling Approach (Sibly R.M., Brown J.H. and Kodrich-Brown A., Eds) 1st edition. John Wiley & Sons. Ltd. pp 198-217 Whitehorn P.R., Tinsley M.C., Brown M.J.F., Darvill B. and Goulson D. 2011. Genetic diversity, parasite prevalence and immunity in wild bumblebees. Proc. R. Soc. B 278: 1195-1202 Williams C.M., Hellmann J. and Sinclair B.J. 2012. Lepidopteran species differ in susceptibility to winter warming. Climate Res. 53: 119-130 Williams J.B., Shorthouse J.D. and Lee R.E. 2003. Deleterious effects of mild simulated overwintering temperatures on survival and potential fecundity of rose-galling Diplolepis wasps. J. Exp. Zool. A 298: 23-31 Zhuo Z., Li X.-K., Jin L.-B. and Ren B.-Z. 2011. Temperature effect on phenoloxidase activity in different stage of Locusta migratoria manilensis Meyen. J. Jilin Agric. Univ. 33: 613-616