Introduction

Parasites and pathogens are pervasive and cause substantial fitness costs to their hosts. Therefore hosts have evolved effective immune systems. However, both the maintenance and the activation of an immune system are energetically expensive [1–3] and therefore trade-offs between immune defence and other life-history traits are expected [4]. The extent to which an organism should invest in its immune defence depends on the efficiency of the defence, the risk of being attacked and the magnitude of costs associated with mounting an immune response [5]. Individual’s nutritional resources and body condition can influence immune investment directly, for example by individuals with fewer resources being able to allocate less to immunity [6–8]. Subsequent strategic decisions related to other life-history traits [9] can further influence immunity, for example in the form of increased trade-offs between immunity and other expensive life history traits [2].

One key life-history trait in many organisms is dispersal, as it determines the potential spread of individuals and populations, and by means of gene flow it can also influence the rate of adaptation to changing conditions [10]. Similarly to immune defence, dispersal is an energetically expensive trait [11]. Studies testing the relationship between dispersal or flight and immune defence are relatively scarce. However, it was recently shown that in a damselfly, Calopteryx splendens, an activation of the immune system increases dispersal rate, suggesting that immune function may play an important role in the evolution of dispersal [12]. On the contrary, in bumblebees (Bombus terrestris; foraging activity) and in crickets (Gryllus texensis; tethered flight) energetic activities have been shown to reduce immune defence, indicating a life history trade-off between these two traits [13,14].

One possible mechanism for flight to impact immune defence is via its effect on metabolism. In the Glanville fritillary butterfly, for example, one-third of the variation in the distance moved in one hour is attributable to variation in flight metabolic rate [15]. However, the impacts of metabolic rate on immune defence are not, in general, consistent. For example, in a study of crickets [16] no correlation between metabolic rate and immune defence was found, whereas across seven Drosophila species immune defence was shown to be negatively associated with mass specific metabolic rate [17]. In the cabbage butterfly, individuals challenged with a nylon implant (mimicking parasitism) raised their standard metabolic rate nearly 8% compared to the control individuals [1].

Environmental conditions can have a great impact on individual performance. Food limitation during developmentally critical periods, for example, has been shown to have long lasting negative influences on numerous adult life history traits [9,18]. In the Glanville fritillary butterfly, we have previously shown that even short term food limitation during development can reduce adult lifespan and have other negative fitness consequences [19]. Similarly, the immune defence has been shown to be influenced by genetic [20] as well as environmental factors, such as nutrition [6,21]. Less is known about the condition-dependency of dispersal or flight metabolism, even though the importance of environmental factors in determining dispersal propensity is becoming increasingly recognised [6,22–24]. Finally, based on life history theory, trade-offs between energetically expensive traits may only be apparent under suboptimal environmental conditions [2,25].

Insects in general are excellent model organism for immuno-ecological studies because their immune defence system is far less complex than the vertebrate immune system, even though many components are homologous [26]. Insects defend themselves against foreign invaders such as parasites and pathogens by cellular encapsulation, antimicrobial peptides, prophenoloxidase cascade, and phagocytosis nodulation [27,28].

In the present study, our aim was to investigate the influence of short-term food limitation during development on four energetically expensive traits: encapsulation rate (immune defence), flight metabolic rate (dispersal ability), resting metabolic rate, and lifespan (proxy of adult fitness) using the Glanville fritillary (Melitaea cinxia) butterfly as a model organism. In addition, we examined the direct costs of induced immune response and flight on adult lifespan and whether those costs are greater under restrictive (i.e. food-limited) environmental conditions.

Materials & Methods

Results

Development time and pupal mass

The final, 7^th instar development time was shorter in males than in females (males: 9 ± 0.3 & 14 ± 0.3 days ± s.e. and females: 12 ± 0.3 & 16 ± 0.3 days ± s.e. for control and food restriction treatment, respectively; Table 1 & Figure 1). Males were also lighter than females as pupae (males: 137.3 ± 2.6 & 123.7 ± 2.6 mg ± s.e. and females: 170.3 ± 2.7 & 158.4 ± 2.7 mg ± s.e. for control and food restriction treatment, respectively; Table 1 & Figure 2). Experiencing three days of food restriction during the final instar prolonged the development time by four days on average (Figure 1). Individuals were not, however, able to fully compensate for the food limitation via increased development time, as they remained lighter as pupae than individuals that were fed ad libitum (Table 1 & Figure 2). Pupal development time was unaffected by sex and food restriction (Table 1). The effect of food restriction was not sex-specific (non-significant interaction; P = 0.56 P = 0.98, P = 0.68 & P = 0.35 for development time, weight increase in final instar, pupal mass & pupal time, respectively).

	Estimate	df	F	P	Effect direction
Larval development time
Sex		1, 202	96.1	< 0.0001	-*
Food restriction		1, 202	343.1	< 0.0001	+
Family	0.33 ± 19.1
Residual	2.9 ± 0.28
Pupal mass
Sex		1, 202	270.6	< 0.0001	-*
Food restriction		1, 202	40.0	< 0.0001	-
Family	62.1 ± 27.5
Residual	227.5 ± 22.6
Weight increase in final instar
Sex		1, 202	181.9	< 0.0001	-*
Food restriction		1, 202	27.0	< 0.0001	-
Family	32.2 ± 17.8
Residual	236.5 ± 23.4
Pupal time
Sex		1, 202	1.6	0.213
Food treatment		1, 202	0.03	0.861
Family	0.01 ± 0.01

Table 1. The effect of food restriction treatment and sex on larval and pupal life history traits and the effect of food restriction and encapsulation rate on adult lifespan.

*males had lower values

Family was included as a random factor. F statistics are given for fixed effects and Wald’s Z statistics for random effects.

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Figure 1. Development time (mean ± s.e.) of the final instar of larvae under control (no stripes) and food restricted (striped) feeding treatments in females (A) and in males (B).

https://doi.org/10.1371/journal.pone.0081289.g001

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Figure 2. Growth in mass during the final instar of larvae under control (white circle and dashed line) and food restricted (black circle and solid line) feeding treatments in females (A) and in males (B).

https://doi.org/10.1371/journal.pone.0081289.g002

Immunity at pupal stage

Encapsulation rate at pupal stage was not affected by pupal mass (P = 0.726). There was a trend suggesting that individuals that experienced food restriction as larvae had increased encapsulation rate (Table 2). A significant interaction between larval food treatment and increase in mass during the final instar (Table 2) indicated that individuals that grew more after the food restricted conditions had higher immunity compared with those that grew less (P = 0.03 for food restricted group only; Figure 3). Increase in mass during the final instar alone had no significant effect on pupal encapsulation rate (Table 2).

	Estimate	df	F	P	Effect direction
Pupal encapsulation rate
Food restriction		1, 112	3.4	0.069	+
Increase in mass (7th instar)		1, 112	0.8	0.397
Food restriction x Increase in mass (7th instar)		1, 112	3.9	0.050
Family	1.0 ± 0.0
Residual	223.7 ± 27.2
Adult encapsulation rate
Food restriction		1, 194	12.3	< 0.001	+
Forced flight		1, 194	4.3	0.040	+
Family	0.001 ± 0.0004
Random	0.01 ± 0.001
Lifespan
Food restriction		1, 193	3.0	0.087
Encapsulation rate		1, 193	2.2	0.139
Food restriction x Encapsulation rate		1, 193	4.2	0.042
Family	2.0 ± 0.0
Residual	53.4 ± 5.1

Table 2. Factors influencing pupal and adult encapsulation rate and adult lifespan.

Family was included as a random factor. F statistics are given for fixed effects and Wald’s Z statistics for random effects.

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Figure 3. Influence of growth during the final instar on pupal encapsulation rate separately for individuals under control (white circle and dashed line) and food restricted (black circle and solid line) feeding treatments.

https://doi.org/10.1371/journal.pone.0081289.g003

Metabolic rates

There was no difference in the mass-corrected resting metabolic rate (RMR) between the sexes (Table 3 & Figure 4a), whereas males had higher mass-corrected flight metabolic rate than females (both total and peak; Table 3 & Figure 4b and c). Individuals that had experienced food restriction during their development had lower RMR as well as total and peak flight metabolic rates than those fed ad libitum (Table 3 & Figure 4). The interaction between sex and larval treatment was not significant in any of the analyses (P = 0.24, P = 0.55 & P = 0.34 for RMR, flight total and peak, respectively).

	Estimate ± S.E	df	F	P	Effect direction
RMR
Sex		1, 128	0.0	0.965
Food restriction		1, 128	5.5	0.020	-
Ambient temperature		1, 128	4.4	0.04	+
Family	0.0 ± 0.0
Residual	0.04 ± 0.005
Total CO2 produced during flight
Sex		1,128	5.0	0.027	+*
Food restriction		1,128	8.3	0.005	-
Ambient temperature		1,128	3.8	0.053	+
Family	0.0005 ± 0.0003
Residual	0.002 ± 0.0003
Peak flight MR
Sex		1,128	5.7	0.019	+*
Food restriction		1,128	13.9	< 0.001	-
Ambient temperature		1,128	3.5	0.063	+
Family	0.02 ± 0.01
Residual	0.07 ± 0.01

Table 3. The effect of food restriction and sex on metabolic rates.

*males had higher values

Family was included as a random factor. F statistics are given for fixed effects and Wald’s Z statistics for random effects. All metabolic rates are mass-corrected residuals.

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Figure 4. Influence (mean ± s.e.) of food conditions during development (control = no stripes, food restriction = stripes) on resting metabolic rate (RMR; A), total CO₂ production during flight (B), and peak flight CO₂ production separately for females (white bars) and for males (grey bars).

https://doi.org/10.1371/journal.pone.0081289.g004

Immunity at adult stage

Individuals from the food restriction group had higher encapsulation rates at adult stage than those fed ad libitum (Table 2 & Figure 5a). There was no difference in encapsulation rate between the sexes (P = 0.27) nor did the sexes respond differently to the nutritional treatment (P = 0.65). There was no significant relationship between metabolic rates and encapsulation rate at adult stage (P = 0.50, P = 0.57, P = 0.79, respectively for RMR, total and peak flight metabolic rate). The encapsulation rate at adult stage was, however, affected by whether or not individuals were forced to fly prior the encapsulation assay. Individuals that flew had higher encapsulation rate compared with individuals that did not fly (Table 2 & Figure 5b). The interaction between food and flight treatment was non-significant (P = 0.28), and sex did not influence the impact of flight on immunity (P = 0.23).

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Figure 5. Influence of food conditions during development (A) and adult flight (B) on adult encapsulation rate (mean ± s.e.).

https://doi.org/10.1371/journal.pone.0081289.g005

Lifespan

The impact of encapsulation rate on adult lifespan was dependent on the food treatment during development (Table 2). In the control group, higher encapsulation rate correlated positively with lifespan, whereas in the nutritional restriction group there was no relationship between these traits (Figure 6). Lifespan was not affected by the food treatment or encapsulation rate alone (Table 2). In addition, lifespan was not influenced by sex (P = 0.20) or whether or not individuals were flown (P = 0.34). Considering only those individuals that were assayed for flight: RMR, total and peak metabolic rate did not significantly influence lifespan (P = 0.79, P = 0.13 and P = 0.06), the direction of the latter two was, however, that individuals with higher flight metabolic rates lived longer.

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Figure 6. Correlations between encapsulation rate at adult stage and adult lifespan separately for individuals under control (white circle and dashed line) and food restricted (black circle and solid line) larval feeding treatments.

Higher encapsulation rate correlated positively with lifespan in the control group (r = 0.27, P = 0.004), whereas in the nutritional restriction group there was no relationship between these traits (r = - 0.03, P = 0.73).

https://doi.org/10.1371/journal.pone.0081289.g006

Discussion

Even a relatively short term food restriction during the final stages of development can have great impact on the life history of an individual, especially if individual’s resource pool and body condition are low [9,18]. In the present study, restricted feeding during development resulted in reduced pupal mass, reduced mass-corrected resting metabolic rate as well as reduced flight metabolic rate (proxy of dispersal capacity). However, no direct influence of food restriction was observed on adult lifespan and the strength of individual encapsulation rate was increased. Additionally, even though the strength of immune defence was not directly related to resting or flight metabolic rates, we found, contrary to our prediction, that flight itself induced rather than suppressed immune response in the Glanville fritillary butterfly.

Experiencing three days of food restriction, even with food available between the restricted days, had a great impact on individual’s life history in the Glanville fritillary butterfly. Development time was increased on average by four days over those individuals fed ad libitum throughout their development. This increased development time was not enough to fully compensate for the food restriction as individuals still remained lighter as pupae compared with individuals that developed under standard conditions. This result is interesting in the light of a previous experiment with the same species, in which otherwise identical but one day shorter food restriction resulted in similar change in development time but no difference in pupal mass [19]. There may hence be a critical threshold for food shortage after which individuals will not try to compensate for the lack of food but instead initiate pupation, even with a cost of reduced pupal mass. As in our previous study, short-term food restriction did not result in a fitness cost in terms of lifespan. It is noteworthy, however, that in the previous study food restricted individuals had reduced fecundity [19], a trait that was not assessed in the present experiment. In many organisms, including insects, restricted food intake has a positive rather than negative impact on lifespan [39]. The underlying mechanisms responsible for this are still unknown. One hypothesis is that food restriction induces responses required to survive stress, such as decreases resting metabolic rate which in turn may decrease the rate of aging [40]. In support of this pattern, we found that developmental food restriction reduced mass-corrected resting metabolic rate (RMR). However, RMR did not directly impact adult lifespan.

Developmental food restriction had a negative impact also on the dispersal proxies, mass-corrected peak and total flight metabolic rate. This suggests that dispersal in the Glanville fritillary may be partially condition-dependent and that individuals that have experienced poor conditions during development have reduced dispersal ability. Importantly, this effect is not due to smaller body size, as the dispersal proxies were corrected for initial difference in pupal mass. Hence, food restriction is likely to influence some other aspect of individual condition (i.e. body composition) resulting in poor dispersal ability. Previous studies on the Glanville fritillary have shown dispersal tendency to be heritable [41,42] and linked with variation in a single gene, Pgi, encoding a glycolytic enzyme Phosphoglucose isomerase [43]. Our new results add to the growing evidence indicating the importance of both genetic [44] and environmental factors [45,46], and/or the interaction of the two [47,48] in determining dispersal ability.

Individuals may also use poor developmental conditions (food quality or quantity and density) as cues for deteriorating environment, and alter their body composition, morphology, or behaviour in a way that enhances their ability to escape from that poor environment [49]. We found no indication of dispersal-related predictive adaptive responses in the Glanville fritillary butterfly. Such responses are likely to depend on a number of species specific factors such as life history, dispersal propensity and the spatial and temporal population structure [50], as well as the strength of resource limitation.

We found an indication that food restricted larvae had increased encapsulation rate as pupae. This effect became more evident after the metamorphosis, when immunity was assessed at adult stage. This result is in the opposite direction to previous studies of some other insects showing that stressful conditions such as starvation reduce the strength of immune defence [51,52] but see 53. Studies in other Lepidoptera, on the other hand, have found that poor food quality [7], chemical defences in host plants [35], and heavy metal pollution [54] increase the strength of encapsulation response. It was recently also shown that in the butterfly Bicyclus anynana experiencing short cold thermal stress during the adult stage increased immune response (number of haemocytes) [55]. Thus, so far it seems that in Lepidoptera, stressful conditions, at least when relatively short term, in general increase immune response. Furthermore, we found that, consistent with previous studies on other taxa [16], the strength of encapsulation response was not associated with mass-corrected metabolic rate.

As both dispersal and immune defence are known to be costly [1,11], we expected to find a trade-off between encapsulation rate and the dispersal proxies: peak flight metabolic rate (capacity) and/or total flight metabolic rate (endurance), at least for individuals that experienced food restriction during their development. In insects, intense physical activity can result in immune suppression due to competition between lipid transport and immune function for the same protein [14,56]. Similarly, in migrating birds migratory individuals have been shown to have lower innate immunity than non-migratory ones [57]. In contradiction to our general prediction, we found no relationship between peak or total flight metabolic rates and immune response in either control of food deprived butterflies. Even more surprisingly, we found that when immune response was compared between individuals that were flown and those that were not, the former had higher encapsulation rate. Based on our data it is impossible to distinguish between increased immune response after flight as a general stress response (i.e. flight itself can be viewed stressful), or as a specific response to flight and/or even dispersal. The latter could be adaptive assuming that dispersing individuals in the wild may be faced with higher pathogen and/or parasite risk during the dispersal process itself or in the new habitat that they disperse to. Increased infection risk during dispersal or settlement could result from individuals being generally stressed or weak due to high energetic costs of dispersal. Alternatively, increased susceptibility to infection may arise from the fact that dispersers are exposed to a higher number of new habitats and maybe to new genotypes of pathogens to which they are not locally adapted. There is some evidence that dispersal interacts with the strength of immune defence. For example, using the damselfly Calopteryx virgo, it was found that activation of the immune system increased dispersal tendency [12]. Consistently, in the Monarch butterfly, Danaus plexippus, genes related to innate immunity are up-regulated in non-reproductive (juvenile-hormone deficient) migrants [58]. Importantly, the relationships between dispersal, immunity and condition may also vary across populations, as was shown in the mormon cricket, for which the influence of nutrition on immunity differed between two migratory populations [6]. Immune responses, in general, are likely to vary across local populations due to coevolutionary interactions such as differences in pathogen and parasitoid prevalence [59].

It is noteworthy that the immune response to flight was assessed almost immediately after flight and hence it is possible that the observed increased immune response is not long lasting. This idea is supported by the result that increased immune response, which presumably is energetically costly, did not have a negative effect on lifespan. Finally, we did not find a negative correlation between immune defence and flight metabolic rates, even on individuals with fewer resources (i.e. food deprived during development). It would be interesting in the future to assess the impact of adult nutrition [60], as the trade-offs may be evident only under unfavourable adult conditions [2] but see 61.

The effects of nutrition restriction and flight that we observed may be specific to the immune defence measured, and hence somewhat different results may have been obtained if we would have measured other responses than encapsulation. For example, it was recently shown in mice that selection for maximal metabolic rate supresses innate (cytokine production) but not adaptive immune function (antibody production) [62]. Similarly, in the present study immune response at adult stage was assessed on young and non-reproductive individuals, and hence future studies should also assess the possible trade-offs in older individuals at different reproductive stages when energy demands may be higher and trade-offs more evident. Finally, as our study was conducted in the laboratory, individuals were not exposed to the normal range of pathogens. The influence of nutritional restriction under more variable pathogen conditions could be very different [28].

In summary, we have shown that environmental conditions during development can have a great impact on adult life history, namely on metabolic rates, including a proxy for dispersal propensity, and encapsulation rate in the Glanville fritillary butterfly. Stressful events during both development (food restriction) and adult life (forced flight) induce an increased immune response, possibly allowing individuals to cope with later stressful events. Identifying the actual mechanisms that link both developmental conditions and flight to encapsulation rate, our measure of immune response, will remain a task for the future.

Acknowledgments

We would like to thank Saskya van Nouhuys and two anonymous referees for their useful comments. Suvi Ikonen, Alma Oksanen, and Linda Peltola are acknowledged for their help with the experiment.