Keep the ball rolling: sexual differences in conglobation behavior of a terrestrial isopod under different degrees of perceived predation pressure.

Background: Antipredator behaviors are theoretically subjected to a balance by which their display should be minimized when their benefits do not outweigh their costs. Such costs may be not only energetic, but also entail a reduction in the time available for other fitness-enhancing behaviors. However, these behaviors are only beneficial under predation risk. Therefore, antipredator behaviors are predicted to be maximized under strong predation risk. Moreover, predation pressure can differ among individuals according to traits such as sex or body size, if these traits increase vulnerability. Antipredator behaviors are expected to be maximized in individuals whose traits make them more conspicuous to predators. However, how sex, body size and antipredator behaviors interact is not always understood. Methods: In this work, I tested the interaction between sex, body size and antipredator behavior in the common pill woodlouse (Armadillidium vulgare), which conglobate (i.e., they roll up their bodies almost conforming a sphere that conceals their appendages) in response to predator attacks. Specifically, I tested whether latency to unroll after a standardized mechanical induction was greater in animals exposed to predator chemical cues (toad feces) than in conspecifics exposed to cues of non-predatory animals (rabbits) or no chemical cues whatsoever (distilled water), incorporating sex and body mass in the analyses. Results: In agreement with my prediction, latency to unroll was greater in individuals exposed to predator chemical cues. In other words, these animals engage in conglobation for longer under perceived predator vicinity. However, this result was only true for males. This sexual dimorphism in antipredator behavior could result from males being under greater predation risk than females, thus having evolved more refined antipredator strategies. Indeed, males of this species are known to actively search for females, which makes them more prone to superficial ground mobility, and likely to being detected by predators. Body size was unrelated to latency to unroll. As a whole, these results support the hypothesis that antipredator behavior is tuned to predator cues in a way consistent with a balance between costs and benefits, which might differ between the sexes.

Ammonium effects on oxidative stress, telomere length, and locomotion across life stages of an anuran from habitats with contrasting land-use histories.

Understanding the mechanistic implications behind wildlife responses to global changes is a central topic in eco-evolutionary research. In particular, anthropic pollution is known to impact wild populations across the globe, which may have even stronger consequences for species with complex life cycles. Among vertebrates, amphibians represent a paradigmatic example of metamorphosis, and their characteristics make them highly vulnerable to pollution. Here, we tested for differences in the redox status, telomere length, and locomotor performance across life stages of green frogs (Pelophylax perezi) from agrosystem and natural habitats, both constitutively and in response to an experimental ammonium exposure (10 mg/L). We found that larvae from the agrosystem constitutively showed an enhanced redox status (better antioxidant balance against H2O2, lower lipid peroxidation) but shorter telomeres as compared to larvae from the natural environment. The larval redox response to ammonium was, overall, similar in both habitats. In contrast, after metamorphosis, the redox status of individuals from the natural habitat seemed to cope better with ammonium exposure (denoted by lower lipid peroxidation), and differences between habitats in telomere length were no longer present. Intriguingly, while the swimming performance of larvae did not correlate with individual's physiology, metamorphs with lower glutathione reductase activity and longer telomeres had a better jumping performance. This may suggest that locomotor performance is both traded off with the production of reactive oxygen species and potentiated directly by longer telomeres or indirectly by the mechanisms that buffer their shortening. Overall, our study suggests that contrasting land-use histories can drive divergence in physiological pathways linked to individual health and lifespan. Since this pattern was life-stage dependent, divergent habitat conditions can have contrasting implications across the ontogenetic development of species with complex life cycles.

Long-term sex-dependent inflammatory response of adult frogs to ammonium exposure during the larval stage.

Among others, the global change involves a worldwide increase in cropland area, with the concomitant rise in nitrogenous fertilizer supplementation and species range alterations, including parasites and pathogens. As most animals rely on their immune systems against these infectious agents, studying the potential effects of nitrogenous compounds on animal immune response is vital to understand their susceptibility to infections under these altered circumstances. Being subjected to an alarming process of global declines, amphibians are the object of particular attention, given their sensitivity to these compounds, especially to ammonium. Moreover, whereas adults can actively avoid polluted patches, larvae are confined within their waterbodies, thus exposed to contaminants in it. In this work, we test whether chronic exposure to a sublethal dose of ammonium during the larval stage of Pelophylax perezi frogs, released from all contamination after metamorphosis, leads to impaired inflammatory response to phytohemagglutinin in adults. We also test whether such a response differs between agrosystem individuals as compared with conspecifics from natural habitats. We found negative carryover effects of chronic exposure of larvae to ammonium on adult inflammatory response, which could imply a greater susceptibility to pathogens and parasites. However, this damage is only true for males, which, according to the immunocompetence handicap hypothesis, could be a consequence of a testosterone-triggered impairment of male immune function. In disagreement with our prediction, however, we detected no differences in the inflammatory response of agrosystem frogs to phytohemagglutinin as compared with natural habitat conspecifics.

Carryover effects of chronic exposure to ammonium during the larval stage on post-metamorphic frogs.

Water contamination poses an important challenge to aquatic fauna, including well-documented effects on amphibian larvae. However, little is known about how contamination during the larval stages may affect post-metamorphic phases, or whether resistance may have evolved in some populations. In this work, we tested the hypothesis that chronic exposure to ammonium (a common contaminant in agroecosystems with confirmed effects on anuran tadpoles) during the larval stage of Pelophylax perezi frogs would affect growth and locomotor performance of metamorph, juvenile, subadult and adult stages. We also predicted that the effects of ammonium would be milder in offspring originated from parental agroecosystem frogs than those originating from forests. We compared tadpoles from both habitats either reared in untreated water or chronically exposed to ammonium. We found that exposure to ammonium during the larval stage inflicted effects on morphology (different measures of body size) and swimming speed after metamorphosis until adulthood. However, these effects were not always consistent through post-metamorphic stages and the effects differed as a function of treatment and habitat. In adults, body size and condition were greater in non-ammonium and ammonium exposed individuals, respectively. These differences were not detectable in metamorphs, for which only ammonium-exposed individuals from agroecosystem showed reduced body size in intermediate post-metamorphic stages. In turn, treatment reduced jumping distance only in agroecosystem adults, subadults and juveniles, which was opposite to the trend observed just after metamorphosis. These changes of patterns throughout the ontogeny of P. perezi could be due to processes such as compensatory growth, delayed energy costs derived from it, or early sexual differences that could be present even before they can be accounted for. In summary, this study suggests that exposure to ammonium during larval stages can result in diverse biological and long-term outcomes in later life stages.

Individual contributions to group chorus dynamics influence access to mating opportunities in wood frogs.

A limitation in bioacoustic studies has been the inability to differentiate individual sonic contributions from group-level dynamics. We present a novel application of acoustic camera technology to investigate how individual wood frogs' calls influence chorus properties, and how variation influences mating opportunities. We recorded mating calls and used playback trials to gauge preference for different chorus types in the laboratory. Males and females preferred chorus playbacks with low variance in dominant frequency. Females preferred choruses with low mean peak frequency. Field studies revealed more egg masses laid in ponds where males chorused with low variance in dominant frequency. We also noted a trend towards more egg masses laid in ponds where males called with low mean frequency. Nearest-neighbour distances influenced call timing (neighbours called in succession) and distances increased with variance in chorus frequency. Results highlight the potential fitness implications of individual-level contributions to a bioacoustic signal produced by groups.

The relationships between toad behaviour, antipredator defences, and spatial and sexual variation in predation pressure.

BACKGROUND: Animal behaviour is under strong selection. Selection on behaviour, however, might not act in isolation from other fitness-related traits. Since predators represent outstanding selective forces, animal behaviour could covary with antipredator defences, such that individuals better suited against predators could afford facing the costs of riskier behaviours. Moreover, not all individuals undergo equivalent degrees of predation pressure, which can vary across sexes or habitats. Individuals under lower predation pressure might also exhibit riskier behaviours. METHODS: In this work, I tested these hypotheses on natterjack toads (Epidalea calamita). Specifically, I gauged activity time, exploratory behaviour and boldness in standard laboratory conditions, and assessed whether they correlated with body size and antipredator strategies, namely sprint speed, parotoid gland area and parotoid gland colour contrast. Additionally, I compared these traits between sexes and individuals from an agrosystem and pine grove, since there is evidence that males and agrosystem individuals are subjected to greater predation pressure. RESULTS: Sprint speed as well as parotoid gland contrast and size appeared unrelated to the behavioural traits studied. In turn, body mass was negatively related to activity time, boldness and exploration. This trend is consistent with the fact that larger toads could be more detectable to their predators, which are mostly gape unconstrained and could easily consume them. As predicted, females exhibited riskier behaviours. Nonetheless, agrosystem toads did not differ from pine grove toads in the behavioural traits measured, despite being under stronger predation pressure.

Direct and indirect effects of chronic exposure to ammonium on anuran larvae survivorship, morphology, and swimming speed.

Several constituents of the current global change are usually deemed accountable for the worldwide declines of amphibian populations. Among these, water contamination poses a major threat, especially to larval stages, which are unable to escape a polluted water body. This problem is remarkable in agrosystems, one of the main sources of water pollution and whose area is forecasted to increase in the forthcoming decades. However, pollutants represent a selective pressure that may result in tolerance in affected areas. In this work, we tested whether chronic exposure to a sublethal concentration of ammonium (10 mg/L), one of the most frequent agrochemicals, affects differently hatching success, survivorship, morphology and swimming performance of Pelophylax perezi tadpoles from agrosystem and pine grove habitats. Ammonium diminished survivorship at the earliest stages after hatching. Thus, lower density was a by-product of exposure to ammonium. Higher density slowed down development, reduced snout-vent length, and had a sharper negative effect on body mass and tail length and depth of ammonium treated individuals with respect to the control. In turn, ammonium accelerated development and increased body mass, SVL, and tail length and depth. These effects did not depend on provenance habitat. However, only pine grove tadpoles' swimming speed was negatively affected by ammonium, which supports the hypothesis that agrosystem tadpoles are more tolerant to ammonium. Finally, corroborating previous findings, tadpoles with larger bodies and tails were faster swimmers, whereas proportionally more massive individuals were slower, and tail depth was unrelated to swimming speed.

Sex and habitat differences in size and coloration of an amphibian's poison glands match differential predator pressures.

Chemical defenses are frequently accompanied by salient color patterns actively avoided by predators, a phenomenon referred to as aposematism. However, the production of both chemical defenses and pigments is costly, and is thus expected to be reduced under mild predator pressure. In this work, I compared the size and coloration of parotoid glands (2 dorsal, external swollen structures that secrete toxins in toads) of male and female Epidalea calamita toads from agrosystems and from pine groves. I also quantified the predator attacks received by plasticine toad models, whose "parotoid glands" differed in size and color conspicuousness, exposed in each habitat. Predators avoided models with large and conspicuous parotoid glands, but models in agrosystems were more often attacked. Concerning actual toads, agrosystem and male individuals had larger parotoid glands, presumably implying greater production of chemical defenses than in pine grove and female conspecifics. These findings are aligned with previous research suggesting that both agrosystem toads and males in this system are subjected to a more intense predator pressure. Difference between parotoid gland and dorsum coloration was greater in agrosystem toads. A marked internal pattern could function as an aposematic signal, which could counteract increased predator pressure.

Telomere length mirrors age structure along a 2200-m altitudinal gradient in a Mediterranean lizard.

The timing of organisms' senescence is developmentally programmed but also shaped by the interaction between environmental inputs and life-history traits. In ectotherms, ageing dynamics are still poorly understood even though their body temperature, metabolism, or growth trajectory are very sensitive to environmental changes. Here, we investigated the role of life-history traits such as age, sex, body size, body condition, and tail autotomy (i.e self-amputation) in shaping telomere length in six populations of the Algerian sand lizard (Psammodromus algirus) distributed along an elevational gradient from 300 to 2500 m above the sea level. Additionally, we compiled the available information on reptiles' telomere length in a review table. Our cross-sectional study shows that older lizards have longer telomeres, which might be mostly linked to the selective disappearance of individuals with shorter telomeres or, alternatively, mediated by a higher expression of telomerase across their life. In fact, variation in telomere length across elevation was explained by age structure of lizards; thus, in contrast to our predictions, altitude had no effect on telomere length in this study system. Telomere length was unaffected by tail regeneration and was sex-independent, but positively correlated with body condition, which might be linked to high somatic investment. Hence, our results suggest that life-history traits such as age or body condition can be major drivers of telomere dynamics for this species, whereas environmental conditions apparently had scarce or no effects on lizard telomeres. Our findings emphasize the relevance of understanding species' life histories for fully disentangling the causes and consequences of differences in ageing in ectotherms.

Toads modulate flight strategy according to distance to refuge.

Among antipredator behaviours, escaping and hiding in a refuge are widespread in nature. Frequently, threatened prey flee towards a refuge nearby, if available. Therefore, refuge proximity may affect the fleeing strategy of a prey. In this work, I tested this hypothesis in Epidalea calamita, a cursorial toad that flees by means of intermittent runs. In a linear runway in standardized conditions, toads were recorded while conducting a short-distance (refuge at 70 cm), a medium-distance (refuge at 140 cm, divided in two 70-cm tracks), and a long-distance trial (refuge at 210 cm, divided in three 70-cm tracks), in a random sequence. Video analyses permitted to calculate sprint speed and run rates (number of runs per meter) in each track. Distance to refuge affected toad flight strategy. Toads started flights at a faster speed in the short-distance trials. In the medium- and the long-distance trials, toads accelerated after the first track, seemingly not motivated by refuge proximity. In these trials, run rate was greater in the first tracks. Altogether, these findings suggest that threatened toads respond firstly with slow, intermittent movements, and only shift to less intermittent, faster sprints if the threat persists. However, run rate was lower in the short-distance trial than in the first tracks of the other trials, suggesting straighter (and faster) flight toward the refuge when it is close. The effects of refuge proximity were greater in males, which (jointly with faster sprint speed) could reflect a greater conspicuousness of males to predator resulting in better escape strategies.

Simulated predation pressure in Pelobates cultripes tadpoles modulates morphology at the metamorphic stage.

Studies on the impacts of variation of biotic interactions at key life cycle stages are crucial to understand the interface between ecological and developmental processes. Predators exert a major impact on prey fitness. Although direct consumption entails the greatest effect, predators can affect prey by means of other mechanisms. For instance, injuries inflicted by failed predation attempts can jeopardize prey fitness, even beyond the short-term. In anuran tadpoles, failed predation typically results in partial tail loss, which is known to reduce swimming speed. However, the potential consequences of tadpole partial tail loss after metamorphosis remain understudied. Because tail materials could be important in conforming metamorph body, we assess the effects of tadpole partial tail loss on metamorph body size in Iberian spadefoot toads Pelobates cultripes. We clipped 55% tail length of pre-tail-resorption stage anesthetized tadpoles, and compared their body size as metamorphs with anesthetized and non-anesthetized non-tail-clipped controls. Also, we tested whether tail length correlated with metamorph body size of individuals of the control groups. Tail-clipped tadpoles produced smaller metamorphs than both controls (the bdy size of metamorphs from both controls was similar), which could incur costs in mid-term survival or time to first reproduction. This effect could be particularly important in areas with introduced predators, if autochthonous tadpoles lack defenses against them. Results suggest that materials resorbed from tadpole tail tissues might be reallocated into metamorph body, according to the negative effect of shorter tails in a correlational analysis, and clipped tails in an experimental test, on metamorph body size.

Integrating time progression in ecoimmunology studies: beyond immune response intensity.

Habitat alterations in agroecosystems may damage amphibian immune capacity. As agroecosystem extension is increasing worldwide, broader-context knowledge on the effects of agroecosystem stressful conditions on amphibian immune capacity is crucial for understanding and management of amphibian global declines. However, most studies on ecoimmunology assume synchronal among-group immune-response peaks, and focus on immune response after standardized time lapses, neglecting its progression. Herein, I compared inflamatory response short-term progression of agroecosystem and natural-habitat female and male natterjack toads Epidalea calamita, by measuring swelling response, once per hour, 6 h following an artificial immune challenge with innocuous antigen phytohemagglutinin. I also compared maximum magnitude of swelling response, irrespective of the moment when it was reached. Habitat differences arose only 3 h after challenge, when natural-habitat toads showed greater swelling response. Maximum magnitude of swelling response was similar in toads from both habitats. However, agroecosystem toads showed a delayed swelling response as compared with natural-habitat conspecifics, probably as a consequence of agroecosystem stressful conditions. Such a delay suggests a weaker immune capacity, and consequently impaired anti-pathogen performance. Regarding sex, swelling response magnitude did not differ between males and females. Female swelling response peaked earlier, but that of males was more sustained in time, which contradicts general across-taxa findings that males show impaired immune response. Interestingly, results suggest that measuring swelling response only after some standardized period following stimulation may be a simplistic approach and produce unrealistic results. Consequently, studies on ecoimmunology should implement swelling response progression in order to produce unbiased science.

Early swelling response to phytohemagglutinin is lower in older toads.

The effects of age on performance of life-history traits are diverse, but a common outcome is senescence, an irreversible deterioration of physical and physiological capabilities of older individuals. Immune response is potentially bound to senescence. However, little is known about immune response ageing in amphibians. In this work, we test the hypothesis that amphibian early immune response is reduced in older individuals. To this end, we captured adult natterjack toads (Epidalea calamita) and inoculated them with phytohemagglutinin, an innocuous protein that triggers a skin-swelling immune response whose magnitude is directly proportional to the ability of the individual to mount an immune response. We measured early swelling immune response (corresponding to an innate-response stage) hourly, for six hours, and we calculated the area under the curve (AUC) for each individual's time series, as a measure of immune response magnitude incorporating time. We estimated toad age by means of phalanx skeletochronology. Swelling and AUC decreased with age. Therefore, in accordance with our predictions, early immune response seems subject to senescence in these toads. Reduced ability to get over infections due to senescence of immune respose might be-together with a worse functioning of other organs and systems-among the causes of lower survival of older specimens.

Greater reproductive investment, but shorter lifespan, in agrosystem than in natural-habitat toads.

Global amphibian decline is due to several factors: habitat loss, anthropization, pollution, emerging diseases, and global warming. Amphibians, with complex life cycles, are particularly susceptible to habitat alterations, and their survival may be impaired in anthropized habitats. Increased mortality is a well-known consequence of anthropization. Life-history theory predicts higher reproductive investment when mortality is increased. In this work, we compared age, body size, and different indicators of reproductive investment, as well as prey availability, in natterjack toads (Epidalea calamita) from agrosystems and adjacent natural pine groves in Southwestern Spain. Mean age was lower in agrosystems than in pine groves, possibly as a consequence of increased mortality due to agrosystem environmental stressors. Remarkably, agrosystem toads were larger despite being younger, suggesting accelerated growth rate. Although we detected no differences in prey availability between habitats, artificial irrigation could shorten aestivation in agrosystems, thus increasing energy trade. Moreover, agrosystem toads exhibited increased indicators of reproductive investment. In the light of life-history theory, agrosystem toads might compensate for lesser reproductive events-due to shorter lives-with a higher reproductive investment in each attempt. Our results show that agrosystems may alter demography, which may have complex consequences on both individual fitness and population stability.

Elevational variation in body-temperature response to immune challenge in a lizard.

Immunocompetence benefits animal fitness by combating pathogens, but also entails some costs. One of its main components is fever, which in ectotherms involves two main types of costs: energy expenditure and predation risk. Whenever those costs of fever outweigh its benefits, ectotherms are expected not to develop fever, or even to show hypothermia, reducing costs of thermoregulation and diverting the energy saved to other components of the immune system. Environmental thermal quality, and therefore the thermoregulation cost/benefit balance, varies geographically. Hence, we hypothesize that, in alpine habitats, immune-challenged ectotherms should show no thermal response, given that (1) hypothermia would be very costly, as the temporal window for reproduction is extremely small, and (2) fever would have a prohibitive cost, as heat acquisition is limited in such habitat. However, in temperate habitats, immune-challenged ectotherms might show a febrile response, due to lower cost/benefit balance as a consequence of a more suitable thermal environment. We tested this hypothesis in Psammodromus algirus lizards from Sierra Nevada (SE Spain), by testing body temperature preferred by alpine and non-alpine lizards, before and after activating their immune system with a typical innocuous pyrogen. Surprisingly, non-alpine lizards responded to immune challenge by decreasing preferential body-temperature, presumably allowing them to save energy and reduce exposure to predators. On the contrary, as predicted, immune-challenged alpine lizards maintained their body-temperature preferences. These results match with increased costs of no thermoregulation with elevation, due to the reduced window of time for reproduction in alpine environment.

Thermoregulation in the lizard Psammodromus algirus along a 2200-m elevational gradient in Sierra Nevada (Spain).

Achieving optimal body temperature maximizes animal fitness. Since ambient temperature may limit ectotherm thermal performance, it can be constrained in too cold or hot environments. In this sense, elevational gradients encompass contrasting thermal environments. In thermally pauperized elevations, ectotherms may either show adaptations or suboptimal body temperatures. Also, reproductive condition may affect thermal needs. Herein, we examined different thermal ecology and physiology capabilities of the lizard Psammodromus algirus along a 2200-m elevational gradient. We measured field (T(b)) and laboratory-preferred (T(pref)) body temperatures of lizards with different reproductive conditions, as well as ambient (T(a)) and copper-model operative temperature (T(e)), which we used to determine thermal quality of the habitat (d(e)), accuracy (d(b)), and effectiveness of thermoregulation (de-db) indexes. We detected no Tb trend in elevation, while T(a) constrained T(b) only at high elevations. Moreover, while Ta decreased more than 7 °C with elevation, T(pref) dropped only 0.6 °C, although significantly. Notably, low-elevation lizards faced excess temperature (T(e) > T(pref)). Notably, de was best at middle elevations, followed by high elevations, and poorest at low elevations. Nonetheless, regarding microhabitat, high-elevation de was more suitable in sun-exposed microhabitats, which may increase exposition to predators, and at midday, which may limit daily activity. As for gender, d(b) and d(e)-d(b) were better in females than in males. In conclusion, P. algirus seems capable to face a wide thermal range, which probably contributes to its extensive corology and makes it adaptable to climate changes.

Thermal dependence of sprint performance in the lizard Psammodromus algirus along a 2200-meter elevational gradient: Cold-habitat lizards do not perform better at low temperatures.

Sprint speed has a capital relevance in most animals' fitness, mainly for fleeing from predators. Sprint performance is maximal within a certain range of body temperatures in ectotherms, whose thermal upkeep relies on exogenous thermal sources. Ectotherms can respond to diverse thermal environments either by shifting their thermal preferences or maintaining them through different adaptive mechanisms. Here, we tested whether maximum sprint speed of a lizard that shows conservative thermal ecology along a 2200-meter elevational gradient differs with body temperature in lizards from different elevations. Lizards ran faster at optimum than at suboptimum body temperature. Notably, high-elevation lizards were not faster than mid- and low-elevation lizards at suboptimum body temperature, despite their low-quality thermal environment. This result suggests that both preferred body temperature and thermal dependence of speed performance are co-adapted along the elevational gradient. High-elevation lizards display a number of thermoregulatory strategies that allow them to achieve high optimum body temperatures in a low thermal-quality habitat and thus maximize speed performance. As for reproductive condition, we did not find any effect of it on sprint speed, or any significant interaction with elevation or body temperature. However, strikingly, gravid females were significantly slower than males and non-gravid females at suboptimum temperature, but performed similarly well at optimal temperature.