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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.
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Our understanding of the evolution of growth rate and its temperature- and predator-induced plasticity can be advanced by identifying the underlying proximate mechanisms. Both behaviour (food intake) and digestive physiology (growth efficiency) may shape growth rate and its plasticity. Evaluating the contribution of these proximate mechanisms has been identified as an outstanding question in life-history theory (Angilletta et al. 2003). Higher growth rates mediated by a higher food intake generate food acquisition trade-offs and make animals more vulnerable to predation (e.g. Brodin & Johansson 2004; Stoks et al. 2005). Higher growth rates mediated by a higher growth efficiency will generate energy allocation trade-offs and result in reduced investments in other functions such as energy storage, immune function and antioxidant defence (e.g. Stoks et al. 2006; De Block & Stoks 2008). Unravelling the underlying proximate mechanisms of latitudinal patterns in growth rate (plasticity) would therefore inform about the selective factors (including costs) shaping growth rate evolution. Identifying whether and how growth efficiencies change across latitudes, temperatures and predation risk levels is also relevant to understand how these factors may shape food chain length: growth efficiencies determine the efficiency of energy transfer in the food chain, which may eventually shape the food chain length (Trussell, Ewanchuk & Matassa 2006, 2008).
With regard to latitudinal patterns in growth rate, mechanistic studies so far focused on the widespread pattern in fishes, amphibians and insects of a faster life history at higher latitudes to counteract the higher time constraints associated with the shorter thermally favourable growth seasons in the north (so-called countergradient variation, Conover & Schultz 1995; Blanckenhorn & Demont 2004; Conover, Duffy & Hice 2009). Many insect species, however, change voltinism (the number of generations per year) along a latitudinal gradient and have more generations, hence higher time constraints per generation, in low-latitude populations (Nylin & Svard 1991; Corbet, Suhling & Söndgerath 2006). This selects for a more rapid growth and development in low-latitude populations (Ragland & Kingsolver 2007; Nygren, Bergstrom & Nylin 2008; Shama et al. 2011; Stoks & De Block 2011), thereby reversing the generally documented latitudinal fast–slow life-history continuum. The often observed faster growth in high-latitude populations of fishes, amphibians and insects has been explained by both behavioural (increased food intake) and physiological (increased growth efficiency) components (e.g. fish: Present & Conover 1992; Billerbeck, Schultz & Conover 2000; amphibians: Lindgren & Laurila 2005). Whether or not the same proximate mechanisms generate the reversed latitudinal growth pattern and whether such voltinism-driven latitudinal patterns in time constraints modulate latitudinal patterns in predator-induced growth rate plasticity remain unexplored.
In this study, we document latitudinal variation in growth rate as well as in predator-induced and temperature-induced growth rate plasticity and study the underlying proximate mechanisms in the damselfly Ischnura elegans. Damselfly larvae are important intermediate predators in aquatic food chains (Johnson 1991; Jonsson et al. 2007). The study species spans a broad latitudinal gradient in Europe, its range extending from mid-Spain to mid-Sweden (Gosden, Stoks & Svensson 2011). The multivoltine (2–3 generations per year) French populations have higher growth rates than the univoltine (1 generation per year) Swedish populations (Shama et al. 2011; Stoks & De Block 2011). Larvae accelerate growth rate under predation risk in the one (Belgian) population where this was studied (Slos & Stoks 2006). The major aims of the current study are to identify the proximate mechanisms underlying the higher growth rates in the southern populations as well as the higher growth rates under predation risk and to test for latitudinal variation in the predator-induced growth accelerations. This will also allow evaluating whether the underlying proximate mechanisms of predator-induced plasticity differ between latitudes. Such patterns can be expected, if the associated costs of behavioural versus physiological mediation differ between latitudes.
Materials and methods
To study the latitudinal patterns in growth rate and predator-induced growth rate plasticity, as well as the underlying proximate mechanisms, larvae were reared from the egg stage in a common garden experiment. There was a randomized full factorial design with larvae of two populations from each of two latitudes (southern France and southern Sweden) exposed to all combinations of two predation risk levels (absent and present) and two temperatures (18 and 24 °C). Large aeshnid dragonfly larvae were used as predators. Mean air temperatures (period 1990–2008) during the growth period of the larvae (temperatures >8°C, Corbet 1999) recorded at weather stations within 30 km from the study sites were 17·5 °C in southern Sweden (Malmö) and 24·1 °C in southern France (Tour-du-Valat). The chosen temperatures generate clear thermal reaction norms for growth rate in I. elegans (Shama et al. 2011; Stoks & De Block 2011) and span the natural temperature regime of the populations of this species during a large part of the growing season. At lower temperatures, growth and development is very slow, and at higher temperatures, mortality increases considerably (R. Stoks, personal observations).
Eggs oviposited on wet filter paper were obtained from 15 to 20 field-collected females in two populations in southern France (St-Martin De Crau 43°37′N, 4°46′E and La Saline 43°29′N, 4°39′E) and two populations in southern Sweden (Genarp 55°36′N, 13°23′E and Vallby 55°38′N, 13°17′E). The distance between the northern and southern populations is about 1500 km. Filter paper with eggs was transported to the laboratory in Belgium and kept at 21 °C. Larvae were placed individually in 180-mL plastic cups filled to a height of 5 cm with aged tap water. Cups were randomly placed in one of the three water baths per rearing temperature. Larvae were randomly re-allocated to another water bath at the same temperature on a weekly basis.
Larvae reared with predation risk received both visual risk cues from conspecifics and chemical risk cues from predators and conspecifics. Cues from conspecifics impose predation threat because damselfly larvae are cannibalistic (De Block & Stoks 2004). To impose these cues, larvae were kept in transparent cups, and their daily food ration was dissolved in 500 μL predator medium. Predator medium was obtained by placing individual dragonfly larvae (size 3–4 cm) in containers (10 × 10 cm filled to a height of 3 cm with aged tap water) for 24 h. All dragonfly larvae were fed every other day with I. elegans larvae. Per 200 mL of this solution, one grinded I. elegans was added just before feeding the damselfly larvae. Larvae reared without predation risk were kept in the same cups that were taped, so that larvae were visually isolated, and their daily food ration was dissolved in aged tap water without predator cues. All larvae were fed ad libitum with Artemia nauplii 6 days a week.
During a 4-day digestion period, we quantified growth rate, food intake and growth efficiency in final instar larvae based on McPeek, Grace & Richardson (2001) and Stoks & McPeek (2003). After larvae had moulted into the final instar, they were placed individually in transparent 60-mL vials. Vials with larvae reared in the absence of predation risk were placed separately in white 180-mL cups, so that larvae could not see each other. Vials with larvae reared with predation risk were placed next to each other, so that larvae could see each other and impose predator stress. Additionally, larvae received chemical predator cues as described previously. To quantify growth rates, each larva was weighed at the start and at the end of this 4-day period to the nearest 0·01 mg using an electronic balance. Growth rate was calculated as [loge(final mass) − loge(initial mass)]/4 days. This measure of growth is independent of initial larval mass (McPeek, Grace & Richardson 2001). Each day, larvae received a known ration of Daphnia of similar size. The first day, 20 Daphnia were given, and the following days, rations were individually adjusted based on the number of uneaten Daphnia. We daily removed any uneaten Daphnia and calculated the number of Daphnia eaten. To convert numbers eaten into the total dry mass of Daphnia eaten during the digestion period, each day, the dry mass of one set of Daphnia was measured to the nearest 1 μg using a Cahn electrobalance after drying them at 60 °C for 48 h. We expressed food intake as total dry mass of Daphnia eaten per mg larva. Growth efficiency was calculated as [(gain in dry mass by the larva)/(total dry mass of food eaten)]. For this, wet masses of the larvae were converted to dry masses using the general conversion factor for coenagrionid damselfly larvae (i.e. dry mass = 0·1497 × wet mass) (McPeek, Grace & Richardson 2001). We have shown in a parallel experiment on I. elegans with identical treatment combinations (latitude, predation risk and temperature) that the relationship between wet mass and dry mass did not differ among treatment combinations (R. Stoks, I. Swillen & M. De Block, unpublished data). Sample sizes per combination of latitude, predation risk and temperature varied between 34 and 39 (total n = 292).
Effects of latitude, predation risk and temperature on the response variables were tested using separate general linear models in proc MIXED of SAS v9·2. (SAS Institute, Cary, NC, USA). In all models, population nested in latitude, and their interactions with the other independent variables were entered as random factors. These factors were not significant for any response variable, indicating consistent results within a given latitude. To meet assumptions of parametric statistical models, we log-transformed food intake. Because larvae with a higher initial mass showed a higher growth efficiency, initial mass was included as a covariate when analysing growth efficiency. Correct degrees of freedom were obtained using the Satterthwaite option.
Southern larvae grew considerably faster than northern larvae (Fig. 1, latitude, F1,284 = 15·18, P <0·0001). Growth rates were higher under predation risk and at the high temperature (predation risk, F1,284 = 5·70, P =0·018, temperature, F1,284 = 62·93, P <0·0001). Both the predator-induced and temperature-induced growth accelerations did not differ between latitudes (latitude × predation risk, F1,284 = 0·31, P =0·58; latitude × temperature, F1,284 = 0·40, P =0·53) nor was there a predation risk × temperature interaction (F1,284 = 0·04, P =0·85).
The proximate mechanisms underlying growth rates and their plasticity, food intake and growth efficiency are shown in Fig. 2. Southern larvae had a higher (mass-corrected) food intake, especially at the low temperature (Fig. 2a, latitude, F1,284 = 35·16, P <0·0001, latitude × temperature, F1,284 = 11·84, P =0·0007). Food intake was lower under predation risk and higher at the high temperature (predation risk, F1,284 = 6·30, P =0·013, temperature, F1,284 = 23·89, P <0·0001). The predator-induced decrease in food intake did not differ between latitudes (latitude × predation risk, F1,284 = 0·09, P =0·77), nor was there a predation risk × temperature interaction (F1,284 = 2·17, P =0·14).
Southern larvae had a higher growth efficiency than northern larvae (Fig. 2b, latitude, F1,16·1 = 6·20, P =0·024). Growth efficiency was higher under predation risk and at the high temperature (predation risk, F1,8·52 = 8·30, P =0·019, temperature, F1,9·11 = 21·15, P =0·0012). Both the predator-induced and temperature-induced increases in growth efficiency did not differ between latitudes (latitude × predation risk, F1,8·9 = 0·66, P =0·44; latitude × temperature, F1,9·18 = 0·060, P =0·82), nor was there a predation risk × temperature interaction (F1,8·58 = 0·88, P =0·37).
As predicted by the voltinism-driven time constraints (Abrams et al. 1996), and conforming with previous empirical work on I. elegans (Shama et al. 2011; Stoks & De Block 2011) and other insects that change voltinism along the latitudinal gradient (Ragland & Kingsolver 2007; Nygren, Bergstrom & Nylin 2008), larvae from multivoltine, southern populations had a higher growth rate than larvae from univoltine, northern populations. Both behaviour (increased food intake) and physiology (increased growth efficiency) contributed to the faster growth rate in the southern populations. Mechanistic studies on the more often reported faster growth in high-latitude populations of fish, amphibians and insects have consistently shown that this was associated with a higher growth efficiency, but not always with an increased food intake (reviewed in Conover, Duffy & Hice 2009). The willingness to increase food intake will depend on the associated consumptive costs of predation at that latitude. In line with this, mortality rates owing to predation in laboratory trials were lower in the southern populations compared with the northern populations of the study species (Stoks et al., in preparation).
The mechanisms underlying latitudinal patterns in growth efficiency are poorly understood. One potentially contributing physiological mechanism is variation in maintenance costs, as can be estimated by resting metabolic rates (Burton et al. 2011). Yet, this cannot be a general explanation, because negative, positive or no relationship has been documented between resting metabolic rate, growth rate and latitude (Lindgren & Laurila 2009 and references therein). In I. elegans, resting metabolic rate is higher in the faster-growing, southern larvae (Stoks et al., in preparation), so this cannot explain the higher growth efficiency at southern latitudes. Other, unexplored mechanisms that may contribute to the latitudinal differences in growth efficiency include differences in gut length and isozyme variation in enzymes involved in digestion and energy metabolism (Lindgren & Laurila 2005 and references therein).
There was considerable growth rate plasticity in response to both temperature and predation risk. As expected, growth rates were higher at the high temperature (Angilletta 2009). In line with previous studies, this was because of a combination of increased food intake and increased growth efficiency (e.g. Van Doorslaer & Stoks 2005; Karl & Fischer 2008). Larvae also accelerated growth rate under predation risk by dragonfly predators. This confirms a previous study on I. elegans (Slos & Stoks 2006), where larvae increased growth rate when reared with fish predators. Similar predator-induced growth accelerations have been documented in other odonate species (Mikolajewski et al. 2005; Wohlfahrt et al. 2007) and in vertebrates (tadpoles: reviewed in Benard 2004; fish: Johansson & Andersson 2009). It is unlikely that this faster growth rate is an artefact of nutritional enrichment caused by adding the chemical predator cues. Any increase in algal, bacterial and protozoan growth cannot explain the growth changes as damselfly larvae are not grazers and only feed on protozoans when small (Corbet 1999), while we observed the growth acceleration in final instar larvae (see also Mikolajewski et al. 2005). It is noteworthy that the temperature-induced and predator-induced growth increases were the same at both latitudes, despite strong differences in growth rates between northern and southern populations. This suggests that the growth rate difference among northern and southern populations will persist under different levels of predation risk and different temperatures. If true, then southern animals may outcompete the local, northern animals when moving northwards in response to climate change (see Lindgren & Laurila 2010 for the opposite pattern).
Two types of ultimate reasons may explain the observed predator-induced growth acceleration in the study species. First, a faster growth rate under predation risk has been predicted under some conditions by optimality models (e.g. Abrams & Rowe 1996; Higginson & Ruxton 2010). When non-predatory costs of growth exist, growth rate may increase under predation risk to reduce total time of exposure to risk (Abrams & Rowe 1996). Several of such non-predatory costs of rapid growth have been identified in damselflies, including reductions in energy storage and immune function (Stoks et al. 2006), and in antioxidant defence (De Block & Stoks 2008). Specifically for the study species, we observed faster-growing larvae to show a lower immune function (Stoks et al., in preparation) and a reduced cold resistance (Stoks & De Block 2011). Secondly, a predator-induced growth increase is to be expected (Urban 2007a) and observed under size-selective predation (Urban 2007b; Beckerman, Rodgers & Dennis 2010). Small fish that were abundant in many natural I. elegans ponds are gape-limited predators (Stoks & De Block 2000; Taylor, Trexler & Lotus 2001). Invertebrate predators, such as large dragonfly larvae, are not typical size-selective predators; that is, they are not gape-limited predators, and hence, larger prey are not protected by an absolute size refuge. Yet, larger damselfly larvae swim faster and therefore have a higher probability to survive attacks by small fish and large dragonfly larvae (Stoks & De Block 2000; Stoks & McPeek 2006; Strobbe et al. 2009). Note that both ultimate reasons are valid with regard to predation risk imposed by fish and by dragonfly predators. In line with this, Ischnura accelerate their growth rate in the presence of both predator types (fish: Slos & Stoks 2006; dragonfly larvae: this study).
The proximate mechanisms underlying the growth acceleration under predation risk are largely unknown. Based on the observation of reduced activity levels in the presence of predation risk, previous studies hypothesized that this growth acceleration was because of a reduced energy consumption under predation risk (Mikolajewski et al. 2005; Johansson & Andersson 2009). Reduced activity levels under predation risk are observed in many taxa, including the study species (Stoks et al., in preparation). Our results advance this topic in two ways. First, we explicitly showed an increased growth efficiency under predation risk. This may reflect the hypothesized reduced energy consumption associated with reduced activity levels. Also, reduced maintenance costs may play a role in increasing growth efficiency, as animals reared under chronic predation risk have been shown to have a lower resting metabolic rate (in tadpoles: Steiner & Van Buskirk 2009; in the study species: Stoks et al., in preparation). It is of note that the mechanisms supposed to underlie the increased growth efficiency under predation risk, reduced activity levels and reduced resting metabolic rate have also been observed in species that reduce growth rate under predation risk. This suggests that reductions in these variables are not enough for growth efficiency to increase under predation risk and that the strength of the reductions is important or that other unexplored mechanisms also play a role. For example, upregulation of costly stress proteins has been identified as a factor underlying growth reductions under predation risk (Slos & Stoks 2008), but not all species exhibit this costly upregulation (Slos, De Meester & Stoks 2009). Secondly, we could exclude that this predator-induced growth acceleration was partly attributed to an increased food intake, as food intake decreased under predation risk. Apparently, in contrast with the growth acceleration in southern populations and at the high temperatures, animals under predation risk are under stronger selection to balance the food acquisition trade-off in favour of reducing food intake to reduce the risk of being killed by the predator (Werner & Anholt 1993). Whatever the reason, this indicates that an increased growth efficiency can overrule a decreased food intake in shaping growth rate and that the response in growth efficiency is decisive in determining whether growth rates will increase or decrease under predation risk.
The emerging pattern that higher growth rates, associated with latitude, temperature or 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 are likely to be widespread in shaping growth rate patterns. This contrasts with the fact that most studies explaining growth rate patterns rely on consumptive costs of predation associated with the food acquisition trade-off (Werner & Anholt 1993). Our results imply that the associated physiological costs of rapid growth may play a major role both in shaping large-scale geographic variation in growth rates and also in shaping the extent and direction of growth rate plasticity. Secondly, it indicates that the efficiency of energy transfer in aquatic food chains, where damselfly larvae are important intermediate predators (Johnson 1991; Jonsson et al. 2007), 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. Previous studies, all focusing on prey animals that decrease growth rate and growth efficiency under predation risk, concluded that energy transfer would reduce under predation risk and therefore contribute to the shortening of food chains (Trussell, Ewanchuk & Matassa 2006, 2008; Hawlena & Schmitz 2010). Our results highlight that besides the resource identity (Trussell, Ewanchuk & Matassa 2008) also the prey identity may determine the influence of predation risk on ecosystem function.
We thank Philippe Chauvelon and Patrick Grillas (Tour-du-Valat) and the Swedish Meteorological Institute (SMHI) for providing temperature data and two anonymous reviewers for constructive feedback. MDB was supported as a postdoctoral fellow of the Research Foundation Flanders (FWO). This study was funded by an ESF Thermal adaptation travel grant to IS and research grants from FWO and the KU Leuven Research Fund to MDB and RS.