Voltinism-associated differences in winter survival across latitudes: integrating growth, physiology, and food intake.

Species that span large latitudinal gradients face strong differences in voltinism and in winter conditions within their range. Latitudinal gradients in winter survival and especially their underlying mechanisms and association with voltinism patterns are poorly studied. We tested in the damselfly Enallagma cyathigerum whether high-latitude populations were better in dealing with the longer winters compared to central- and low-latitude populations and whether this was associated with changes in voltinism. We thereby evaluated whether higher initial levels and/or lower reductions during winter of energy storage (measured as fat content) and investment in immune function [measured as the activity of phenoloxidase (PO)], and/or stronger compensatory responses in food intake contributed to the higher winter survival in high-latitude populations. To this end, we simulated a long high-latitude winter at 4 °C under manipulated food conditions. Across food levels, winter survival was highest in Swedish larvae, intermediate in Belgian larvae, and lowest in Spanish larvae, indicating latitude-specific thermal adaptation that could be partly linked to differences in voltinism. The semi-voltine Swedish larvae were growing slower before winter and as a result accumulated the highest fat content and PO activity when the winter started compared to the univoltine, faster growing Belgian, and Spanish larvae. Fat content and PO activity declined during the winter, yet equally across latitudes, and were not buffered by compensatory food intake. Our data identified possible underlying physiological mechanisms of winter survival and support the hypothesis that widespread latitude-associated voltinism shifts may be a selective factor contributing to latitudinal shifts in winter survival.

The interplay of adult and larval time constraints shapes species differences in larval life history.

In animals with a complex life cycle, larval life-history plasticity is likely shaped by the interplay of selective factors in both larval and adult stages. A wide interspecific variation in responses to larval time constraints imposed by seasonality has been documented. Few studies have addressed differences among closely related species in the evolutionary trajectories of age and size at metamorphosis and their link with larval growth rate under time constraints. None have considered how species-specific length of the reproductive season affects larval developmental responses to time constraints. We tested in four Coenagrion damselfly species whether species with a longer reproductive season, facing a smaller threat of missing out on reproduction, react less to larval time constraints and pre-winter food shortage by accelerating development rate and growth rate, and therefore pay less physiological costs. All species increased development and growth rates under larval time constraints. The magnitude of this increase negatively correlated across species with the length of the reproductive season. Under larval time constraints, only the species exhibiting the longest reproductive season suffered a delayed emergence and a reduced investment in energy storage, yet also showed an increased immune function. Under a longer reproductive season, evolution may favor compensation for larval constraints after metamorphosis. Growth rate was accelerated after pre-winter food shortage to the same extent across species; effects on age and mass at emergence also did not differ among species. Time constraints associated with the length of the reproductive season may predictably contribute to species differences in their response to time constraints imposed in the larval stage. Our study adds empirical proof that the interplay of selective factors in the larval and adult stages may determine life-history plasticity with regard to larval time constraints.

Local genetic adaptation generates latitude-specific effects of warming on predator-prey interactions.

Temperature effects on predator-prey interactions are fundamental to better understand the effects of global warming. Previous studies never considered local adaptation of both predators and prey at different latitudes, and ignored the novel population combinations of the same predator-prey species system that may arise because of northward dispersal. We set up a common garden warming experiment to study predator-prey interactions between Ischnura elegans damselfly predators and Daphnia magna zooplankton prey from three source latitudes spanning >1500 km. Damselfly foraging rates showed thermal plasticity and strong latitudinal differences consistent with adaptation to local time constraints. Relative survival was higher at 24 °C than at 20 °C in southern Daphnia and higher at 20 °C than at 24 °C, in northern Daphnia indicating local thermal adaptation of the Daphnia prey. Yet, this thermal advantage disappeared when they were confronted with the damselfly predators of the same latitude, reflecting also a signal of local thermal adaptation in the damselfly predators. Our results further suggest the invasion success of northward moving predators as well as prey to be latitude-specific. We advocate the novel common garden experimental approach using predators and prey obtained from natural temperature gradients spanning the predicted temperature increase in the northern populations as a powerful approach to gain mechanistic insights into how community modules will be affected by global warming. It can be used as a space-for-time substitution to inform how predator-prey interaction may gradually evolve to long-term warming.

Latitudinally structured variation in the temperature dependence of damselfly growth rates.

The Metabolic Theory of Ecology predicts that the slope of the rate-temperature relationship, E, remains consistent across traits and organisms, acting as a major determinant of large-scale ecological patterns. Although E has recently been shown to vary systematically, we have a poor understanding of its ecological significance. To address this question, we conducted a common-garden experiment involving six damselfly species differing in distribution, estimating E at the level of full-sib families. Each species was sampled throughout its latitudinal range, allowing us to characterise variation in E along a latitudinal gradient spanning 3600 km. We show that E differs among populations and increases with latitude. E was right-skewness across species, but this was largely an artefact of the latitudinal trend. Increased seasonality towards higher latitude may contribute to the latitudinal trend in E. We conclude that E should be seen as a trait involved in local adaptation.

Generalists and specialists along a latitudinal transect: patterns of thermal adaptation in six species of damselflies.

Tropical organisms colonizing temperate environments face reduced average temperatures and dramatic thermal fluctuations. Theoretical models postulate that thermal specialization should be favored either when little environmental variation is experienced within generations or when among-generation variation is small relative to within-generation variation. To test these predictions, we studied six temperate species of damselflies differing in latitudinal distribution. We developed a computer model simulating how organisms experience environmental variation (accounting for diapause and voltinism) and performed a laboratory experiment assaying thermal sensitivities of growth rates. The computer model showed opposing latitudinal trends in among- and within-generation thermal variability: within-generation thermal variability decreased toward higher latitudes, whereas relative levels of among-generation thermal variability peaked at midlatitudes (where a shift in voltinism occurred). The growth experiment showed that low-latitude species were more thermally generalized than mid- and high-latitude species, supporting the prediction that generalists are favored under high levels of within-generation variation. Northern species had steeper, near-exponential reaction norms suggestive of thermal specialization. However, they had strikingly high thermal optima and grew very slowly over most of the thermal range they are expected to experience in the field. This observation is at present difficult to explain. These results highlight the importance of considering interactions between life history and environmental variation when deriving expectations of thermal adaptation.

Behaviour and physiology shape the growth accelerations associated with predation risk, high temperatures and southern latitudes in Ischnura damselfly larvae.

1. To better predict effects of climate change and predation risk on prey animals and ecosystems, we need studies documenting not only latitudinal patterns in growth rate but also growth plasticity to temperature and predation risk and the underlying proximate mechanisms: behaviour (food intake) and digestive physiology (growth efficiency). The mechanistic underpinnings of predator-induced growth increases remain especially poorly understood. 2. We reared larvae from replicated northern and southern populations of the damselfly Ischnura elegans in a common garden experiment manipulating temperature and predation risk and quantified growth rate, food intake and growth efficiency. 3. The predator-induced and temperature-induced growth accelerations were the same at both latitudes, despite considerably faster growth rates in the southern populations. While the higher growth rates in the southern populations and the high rearing temperature were driven by both an increased food intake and a higher growth efficiency, the higher growth rates under predation risk were completely driven by a higher growth efficiency, despite a lowered food intake. 4. The emerging pattern that higher growth rates associated with latitude, temperature and predation risk were all (partly or completely) mediated by a higher growth efficiency has two major implications. First, it indicates that energy allocation trade-offs and the associated physiological costs play a major role both in shaping large-scale geographic variation in growth rates and in shaping the extent and direction of growth rate plasticity. Secondly, it suggests that the efficiency of energy transfer in aquatic food chains, where damselfly larvae are important intermediate predators, will be higher in southern populations, at higher temperatures and under predation risk. This may eventually contribute to the lengthening of food chains under these conditions and highlights that the prey identity may determine the influence of predation risk on food chain length.

Latitudinal and voltinism compensation shape thermal reaction norms for growth rate.

Latitudinal variation in thermal reaction norms of key fitness traits may inform about the response of populations to climate warming, yet their adaptive nature and evolutionary potential are poorly known. We assessed the contribution of quantitative genetic, neutral genetic and environmental effects to thermal reaction norms of growth rate for populations of the damselfly Ischnura elegans. Among populations, reaction norms differed primarily in elevation, suggesting that time constraints associated with shorter growth seasons in univoltine, high-latitude as well as multivoltine, low-latitude populations selected for faster growth rates. Phenotypic divergence among populations is consistent with selection rather than drift as Q(ST) was greater than F(ST) in all cases. Q(ST) estimates increased with experimental temperature and were influenced by genotype by environment interactions. Substantial additive genetic variation for growth rate in all populations suggests that evolution of trait means in different environments is not constrained. Heritability of growth rates was higher at high temperature, driven by increased genetic rather than environmental variance. While environment-specific nonadditive effects also may contribute to heritability differences among temperatures, maternal effects did not play a significant role (where these could be accounted for). Genotype by environment interactions strongly influenced the adaptive potential of populations, and our results suggest the potential for microevolution of thermal reaction norms in each of the studied populations. In summary, the observed latitudinal pattern in growth rates is adaptive and results from a combination of latitudinal and voltinism compensation. Combined with the evolutionary potential of thermal reaction norms, this may affect populations' ability to respond to future climate warming.

Rapid growth reduces cold resistance: evidence from latitudinal variation in growth rate, cold resistance and stress proteins.

BACKGROUND: Physiological costs of rapid growth may contribute to the observation that organisms typically grow at submaximal rates. Although, it has been hypothesized that faster growing individuals would do worse in dealing with suboptimal temperatures, this type of cost has never been explored empirically. Furthermore, the mechanistic basis of the physiological costs of rapid growth is largely unexplored. METHODOLOGY/PRINCIPAL FINDING: Larvae of the damselfly Ischnura elegans from two univoltine northern and two multivoltine southern populations were reared at three temperatures and after emergence given a cold shock. Cold resistance, measured by chill coma recovery times in the adult stage, was lower in the southern populations. The faster larval growth rates in the southern populations contributed to this latitudinal pattern in cold resistance. In accordance with their assumed role in cold resistance, Hsp70 levels were lower in the southern populations, and faster growing larvae had lower Hsp70 levels. Yet, individual variation in Hsp70 levels did not explain variation in cold resistance. CONCLUSIONS/SIGNIFICANCE: WE PROVIDE EVIDENCE FOR A NOVEL COST OF RAPID GROWTH: reduced cold resistance. Our results indicate that the reduced cold resistance in southern populations of animals that change voltinism along the latitudinal gradient may not entirely be explained by thermal selection per se but also by the costs of time constraint-induced higher growth rates. This also illustrates that stressors imposed in the larval stage may carry over and shape fitness in the adult stage and highlights the importance of physiological costs in the evolution of life-histories at macro-scales.

Predator-driven trait diversification in a dragonfly genus: covariation in behavioral and morphological antipredator defense.

Proof for predation as an agent shaping evolutionary trait diversification is accumulating, however, our understanding how multiple antipredator traits covary due to phenotypic differentiation is still scarce. Species of the dragonfly genus Leucorrhinia underwent shifts from lakes with fish as top predators to fishless lakes with large dragonfly predators. This move to fishless lakes was accompanied by a partial loss and reduction of larval spines. Here, we show that Leucorrhinia also reduced burst swimming speed and its associated energy fuelling machinery, arginine kinase activity, when invading fishless lakes. This results in patterns of positive phylogenetic trait covariation between behavioral and morphological antipredator defense (trait cospecialization) and between behavioral antipredator defense and physiological machinery (trait codependence). Across species patterns of trait covariation between spine status, burst swimming speed and arginine kinase activity also matched findings within the phenotypically plastic L. dubia. Our results highlight the importance of predation as a factor affecting patterns of multiple trait covariation during phenotypic diversification.

Autotomy reduces immune function and antioxidant defence.

Costs of autotomy, an antipredator defence, are typically explained by impaired mobility; yet physiologically mediated costs may also play a role. Given the resemblance to wounding, a decreased immune function and an associated reduction in antioxidant defence is expected after autotomy. In line with this, after lamellae autotomy, larvae of the damselfly Lestes viridis showed lower levels of innate immunity (i.e. phenoloxidase, PO) and antioxidant defence (superoxide dismutase, SOD). Levels of catalase (CAT) remained, however, unaffected. In line with its cytotoxicity, PO covaried positively with CAT, yet negatively with SOD. We identified a novel cost of autotomy in terms of a reduced innate immunity, which may provide an alternative explanation for the often observed costs of autotomy and which may generate indirect interactions between predators and parasites.

Compensatory growth and oxidative stress in a damselfly.

Physiological costs of compensatory growth are poorly understood, yet may be the key components in explaining why growth rates are typically submaximal. Here we tested the hypothesized direct costs of compensatory growth in terms of oxidative stress. We assessed oxidative stress in a study where we generated compensatory growth in body mass by exposing larvae of the damselfly Lestes viridis to a transient starvation period followed by ad libitum food. Compensatory growth in the larval stage was associated with higher oxidative stress (as measured by induction of superoxide dismutase and catalase) in the adult stage. Our results challenge two traditional views of life-history theory. First, they indicate that age and mass at metamorphosis not necessarily completely translate larval stress into adult fitness and that the observed physiological cost may explain hidden carry-over effects. Second, they support the notion that costs of compensatory growth may be associated with free-radical-mediated trade-offs and not necessarily with resource-mediated trade-offs.

Correcting the short-term effect of food deprivation in a damselfly: mechanisms and costs.

1. Mass at emergence is a life-history trait strongly linked to adult fitness. Therefore, when faced with transient food shortage in the larval stage, mass-correcting mechanisms are common. 2. These correcting mechanisms may carry costs with them. On one hand, these costs may be overestimated because they can be confounded with the direct effects of the transient food shortage itself. On the other hand, costs may be underestimated by ignoring physiological costs. Another largely neglected topic is that correcting mechanisms and costs may critically depend upon other stressors that often co-occur. 3. Here, we identify the mass-correcting mechanisms and their associated costs at emergence in the damselfly Coenagrion puella, after being stressed by a transient period of starvation and a subsequent exposure to pesticide stress during the larval stage. We introduce path analysis to disentangle direct costs of starvation and the mass-correcting mechanisms in terms of immune response. 4. As predicted, we found no differences in mass at emergence. Starvation directly resulted in a costly delayed emergence and a decreased immune response at emergence. Mass-correcting mechanisms included a prolonged post-starvation period, reduced mass loss at emergence and compensatory growth, although the latter only in females under pesticide stress. 5. The mass-correcting mechanisms were associated with beneficial effects on investment in immune response, but only in the absence of pesticide stress. Under pesticide stress, these beneficial effects were mostly undone or overruled, resulting in negative effects of the mass-correcting mechanisms in terms of immune response. 6. Our results stress the importance of and introduce a statistical way of disentangling direct costs of starvation and the mass-correcting mechanisms themselves, and the importance of including physiological endpoints in this kind of studies.

Life-history evolution when Lestes damselflies invaded vernal ponds.

We know little about the macroevolution of life-history traits along environmental gradients, especially with regard to the directionality compared to the ancestral states and the associated costs to other functions. Here we examine how age and size at maturity evolved when Lestes damselflies shifted from their ancestral temporary pond habitat (i.e., ponds that may dry once every decade or so) to extremely ephemeral vernal ponds (ponds that routinely dry completely each year). Larvae of three species were reared from eggs until emergence under different levels of photoperiod and transient starvation stress. Compared to the two temporary-pond Lestes, the phylogenetically derived vernal-pond Lestes dryas developed more rapidly across photoperiod treatments until the final instar, and only expressed plasticity in development time in the final instar under photoperiod levels that simulated a later hatching date. The documented change in development rate can be considered adaptive and underlies the success of the derived species in vernal ponds. Results suggest associated costs of faster development are lower mass at maturity and lower immune function after transient starvation stress. These costs may not only have impeded further evolution of the routine development rate to what is physiologically maximal, but also maintained some degree of plasticity to time constraints when the habitat shift occurred.

Physiological costs of compensatory growth in a damselfly.

Little is known about physiological costs of rapid growth. We successfully generated compensatory growth to time stress and transient food stress in the damselfly Lestes viridis and studied the physiological correlates of the resulting reduced ability to cope with starvation. We found evidence for both mechanisms proposed to underlie the physiological trade-off: compensatory growth was associated with (1) a higher metabolic rate, as indicated by a higher oxygen consumption and a faster depletion of energy storage molecules (glycogen and triglycerides), and (2) a smaller investment in energy storage. The former may also explain why storage molecules after emergence were negatively affected by time stress and food stress, despite the successful compensation before emergence. These deferred physiological costs of rapid growth have the potential to couple larval stresses to adult fitness irrespective of age and size at emergence, and they may partly explain why many animals do not show their maximum achievable growth rate.

Time constraints mediate predator-induced plasticity in immune function, condition, and life history.

The simultaneous presence of predators and a limited time for development imposes a conflict: accelerating growth under time constraints comes at the cost of higher predation risk mediated by increased foraging. The few studies that have addressed this tradeoff have dealt only with life history traits such as age and size at maturity. Physiological traits have largely been ignored in studies assessing the impact of environmental stressors, and it is largely unknown whether they respond independently of life history traits. Here, we studied the simultaneous effects of time constraints, i.e., as imposed by seasonality, and predation risk on immune defense, energy storage, and life history in lestid damselflies. As predicted by theory, larvae accelerated growth and development under time constraints while the opposite occurred under predation risk. The activity of phenoloxidase, an important component of insect immunity, and investment in fat storage were reduced both under time constraints and in the presence of predators. These reductions were smaller when time constraints and predation risk were combined. This indicates that predators can induce sublethal costs linked to both life history and physiology in their prey, and that time constraints can independently reduce the impact of predator-induced changes in life history and physiology.

Spatiotemporal allozyme variation in the damselfly, Lestes viridis (Odonata: Zygoptera): gene flow among permanent and temporary ponds.

Several insect species seem to persist not only in permanent but also in temporary ponds where they face particularly harsh conditions and frequent extinctions. Under such conditions, gene flow may prevent local adaptation to temporary ponds and may promote phenotypic plasticity, or maintain apparent population persistence. The few empirical studies on insects suggest the latter mechanism, but no studies so far quantified gene flow including both pond types. We investigated the effects of pond type and temporal variation on population genetic differentiation and gene flow in the damselfly Lestes viridis in northern Belgium. We report a survey of two allozyme loci (Gpi, Pgm) with polyacrylamide gel electrophoresis in 14 populations from permanent and temporary ponds, and compared these results with similar data from the same permanent populations one year before. The data suggested that neither pond-drying regime, nor temporal variation have a substantial effect on population genetic structuring and did not provide evidence for stable population differentiation in L. viridis in northern Belgium. Gene flow estimates were high within permanent and temporary ponds, and between pond types. Our data are consistent with a source-sink metapopulation system where temporary ponds act as sinks in dry years, and are quickly recolonized after local population extinction. This may create a pattern of apparent population persistence of this species in permanent and temporary ponds without clear local adaptation.

Life-history variation in relation to time constraints in a damselfly.

Although variation within populations in plasticity to time constraints is expected with regard to hatching date, empirical studies are largely lacking. We studied life-history responses to time constraints manipulated by photoperiod and associated with hatching date in larvae of the damselfly Lestes viridis for two populations with a different hydroperiod. In a common garden experiment, early- and late-hatched larvae from both populations were reared at two photoperiods mimicking the start and the end of the egg-hatching season. In a reciprocal transplant experiment, early- and late-hatched larvae from both populations were reared in both ponds. In all these experiments, larvae were reared from egg hatching until adult emergence. Within both populations, larvae reared at the photoperiod indicating a late time point in the growing season, reduced development time to compensate for their perceived shorter development period. Growth rate, however, did not respond to photoperiod, resulting in a lower mass at emergence. As expected, both in the laboratory and in the field, larvae from eggs that hatched later in the season generally had a shorter development time and a faster growth rate, resulting in a higher mass at emergence compared to early-hatched larvae. This may explain the intriguing seasonal increase in mass at emergence in this species, and affect the predictions of optimality models. None of these life-history responses differed between the two populations, despite clear differences in time constraints linked to hydroperiod, suggesting the robustness of the observed patterns. Given the ubiquity of asynchronous hatching in nature, and the adaptive value of the observed differences between early- and late-hatched larvae, we expect the effects of hatching date on life-history plasticity to be widespread.

Lethal and sublethal costs of autotomy and predator presence in damselfly larvae.

We studied the costs of lamellae autotomy with respect to growth and survival of Lestes sponsa damselfly larvae in field experiments. We manipulated predation risk by Aeshna cyanea dragonfly larvae and lamellae status of L. sponsa larvae in field enclosures and compared differences in numbers, size and mass of survivors among treatments. In the absence of a free-ranging A. cyanea larva, about 29% of the L. sponsa larvae died. This was probably due to cannibalism. The presence of a free-ranging A. cyanea reduced larval survival by 68% compared to treatments in which it was absent or not permitted to forage on L. sponsa damselflies. Across all predator treatments, lamellae autotomy reduced survival by about 20%. The mean head width and mass of survivors was lower in the enclosures with a free-ranging A. cyanea compared to the other two predator treatments. This suggested that larvae grew less in the presence of a free-ranging predator, indicating that increased antipredator behaviours were more important in shaping growth responses than reduced population density. Mass, but not head width, of survivors was also reduced after autotomy. The fitness consequences of these effects for the adults may be pronounced. In general, these field data strongly suggest that lamellae autotomy affects population regulation of damselflies.