Fear is the mother of invention: anuran embryos exposed to predator cues alter life-history traits, post-hatching behaviour and neuronal activity patterns.

Neurophysiological modifications associated to phenotypic plasticity in response to predators are largely unexplored, and there is a gap of knowledge on how the information encoded in predator cues is processed by prey sensory systems. To explore these issues, we exposed Rana dalmatina embryos to dragonfly chemical cues (kairomones) up to hatching. At different times after hatching (up to 40 days), we recorded morphology and anti-predator behaviour of tadpoles from control and kairomone-treated embryo groups as well as their neural olfactory responses, by recording the activity of their mitral neurons before and after exposure to a kairomone solution. Treated embryos hatched later and hatchlings were smaller than control siblings. In addition, the tadpoles from the treated group showed a stronger anti-predator response than controls at 10 days (but not at 30 days) post-hatching, though the intensity of the contextual response to the kairomone stimulus did not differ between the two groups. Baseline neuronal activity at 30 days post-hatching, as assessed by the frequency of spontaneous excitatory postsynaptic events and by the firing rate of mitral cells, was higher among tadpoles from the treated versus the control embryo groups. At the same time, neuronal activity showed a stronger increase among tadpoles from the treated versus the control group after a local kairomone perfusion. Hence, a different contextual plasticity between treatments at the neuronal level was not mirrored by the anti-predator behavioural response. In conclusion, our experiments demonstrate ontogenetic plasticity in tadpole neuronal activity after embryonic exposure to predator cues, corroborating the evidence that early-life experience contributes to shaping the phenotype at later life stages.

Sperm traits negatively covary with size and asymmetry of a secondary sexual trait in a freshwater crayfish.

In species where females mate promiscuously, the reproductive success of males depends both on their ability to acquire mates (pre-copulatory sexual selection) and ability of their ejaculates to outcompete those of other males (post-copulatory sexual selection). Sperm competition theory predicts a negative relationship between investment in body traits favouring mate acquisition (secondary sexual characters, SSCs) and investment in ejaculate size or quality, due to the inherent costs of sperm production. In contrast, the phenotype-linked fertility hypothesis posits that male fertilizing efficiency is reliably reflected by the phenotypic expression of male SSCs, allowing females to obtain direct benefits by selecting more ornamented males as copulation partners. In this study, we investigated the relationships between male SSCs and size and quality (viability and longevity) of ejaculates allocated to females in mating trials of the freshwater crayfish Austropotamobius italicus. We showed that the relative size of male weapons, the chelae, was negatively related to ejaculate size, and that chelae asymmetry, resulting from regeneration of lost chelipeds, negatively covaried with sperm longevity. Moreover, males allocated more viable sperm to mates from their own rather than different stream of origin. Our findings thus suggest that, according to sperm competition theory, pre-copulatory sexual selection for large weapons used in male fighting may counteract post-copulatory sperm competition in this crayfish species, and that investment in cheliped regeneration may impair ejaculate quality.

Sex-, morph- and size-specific susceptibility to stress measured by haematological variables in captive common wall lizard Podarcis muralis.

In polymorphic species of animals, colour morphs may show alternative physiological properties, and hence evolve or be maintained as an indirect response to selection exerted on these physiological attributes. In this study, we investigated if different colour morphs (white, red and yellow) of the polymorphic common wall lizard differed in their physiological responses to a long-term stress by determining variation between capture and release in leukocytes profiles, haemoparasite loads and body condition of male and females maintained in captivity throughout the breeding season. We found that most blood parameters of lizards varied significantly following captivity, and this variation was sex-, morph- and size-dependent. In particular, the heterophil:lymphocyte ratio (H:L), a sensitive measure of immunodepression and long-term stress, varied significantly among yellow females, larger individuals significantly increasing and smaller individuals decreasing their H:L ratio after captivity. This trend was reversed in red females, where smaller individuals presented raised H:L index at release. Our study indicated that response to long-term stressful conditions, such as those induced by captivity, differed among common wall lizard colour morphs, implying a sex-, size-(i.e. age) and morph-specific sensitivity to stress, and hence a different physiological profile of colour morphs, which may contribute to the maintenance of colour polymorphism in this species.

Global changes and animal phenotypic responses: melanin-based plumage redness of scops owls increased with temperature and rainfall during the last century.

The ecological effects of global climate changes include shifts of species' distribution and changes in migration strategies and phenotype. Colour polymorphism, which can be envisaged as a species' evolutionary response to alternating conditions or to a wide range of habitats, may be affected by climate changes as well. The scops owl (Otus scops) shows two main colour morphs, dark- and pale-reddish, as well as intermediate morphs. We investigated temporal trends in an index of plumage colour of Italian scops owls from museum collections (1870-2007). We found a significant increase in plumage redness over the last century, which was correlated with an increase in temperature and rainfall of the years before specimen collection. However, the temporal increase in plumage redness persisted after controlling for climatic variables, suggesting that other environmental factors could be involved. Our study indicates that ongoing climate changes might have either shifted the selective balance between colour morphs, or differentially affected migration and movement patterns of colour morphs.

Male-male combats in a polymorphic lizard: residency and size, but not color, affect fighting rules and contest outcome.

Theoretical models predict that the outcome of dyadic agonistic encounters between males is influenced by resource-holding potential, resource value, and intrinsic aggressiveness of contestants. Moreover, in territorial disputes residents enjoy a further obvious competitive advantage from the residency itself, owing to the intimate familiarity with their territory. Costs of physical combats are, however, dramatically high in many instances. Thus, signals reliably reflecting fighting ability of the opponents could easily evolve in order to reduce these costs. For example, variation in color morph in polymorphic species has been associated with dominance in several case studies. In this study, we staged asymmetric resident-intruder encounters in males of the common wall lizard Podarcis muralis, a species showing three discrete morphs (white, yellow, and red) to investigate the effects of asymmetries in color morph, residency, and size between contestants on the outcome of territorial contests. We collected aggression data by presenting each resident male with three intruders of different color morph, in three consecutive tests conducted in different days, and videotaping their interactions. The results showed that simple rules such as residency and body size differences could determine the outcome of agonistic interactions: residents were more aggressive than intruders, and larger males were competitively superior to smaller males. However, we did not find any effect of color on male aggression or fighting success, suggesting that color polymorphism in this species is not a signal of status or fighting ability in intermale conflicts.

Female freshwater crayfish adjust egg and clutch size in relation to multiple male traits.

Females may invest more in reproduction if they acquire mates of high phenotypic quality, because offspring sired by preferred partners may be fitter than offspring sired by non-preferred ones. In this study, we tested the differential maternal allocation hypothesis in the freshwater crayfish, Austropotamobius italicus, by means of a pairing experiment aimed at evaluating the effects of specific male traits (body size, chelae size and chelae asymmetry) on female primary reproductive effort. Our results showed that females laid larger but fewer eggs for relatively small-sized, large-clawed males, and smaller but more numerous eggs for relatively large-sized, small-clawed males. Chelae asymmetry had no effects on female reproductive investment. While the ultimate consequences of this pattern of female allocation remain unclear, females were nevertheless able to adjust their primary reproductive effort in relation to mate characteristics in a species where inter-male competition and sexual coercion may mask or obscure their sexual preferences. In addition, our results suggest that female allocation may differentially affect male characters, thus promoting a trade-off between the expression of different male traits.

Sexual dimorphism in digit length ratios in two lizard species.

Sexual dimorphism in digit length ratios has been reported for humans, a few other mammals, and two bird species. This dimorphism is thought to arise via an interaction between the prenatal exposure of the embryo to sex hormones and the Hox genes, which are highly conserved among vertebrates and control the development of both the appendices, including fingers and toes, and the urogenital system. In this study, we report on sexual dimorphism in 2D:3D, 2D:4D, and 3D:4D contralateral ratios of the forelimbs in two species of oviparous lizards, the common wall lizard (Podarcis muralis) and the tree skink (Mabuya planifrons), as measured on museum specimens. We found that male P. muralis had a larger 2D:4D ratio on both sides and larger 2D:3D ratio on the left side than females, whereas in M. planifrons, males had lower 2D:3D ratios than females on the left side. The two species show opposite patterns of sexual dimorphism in body size, males being larger than females in P. muralis, and the reverse in M. planifrons, suggesting that interspecific variation of sex differences in digit ratios could be associated with sex-specific growth trajectories. There was a limited evidence for directional asymmetry in digit ratios. Therefore, our findings provide the first evidence that digit ratios are sexually dimorphic in any reptile species and are consistent with the idea that the genetic link between limb development and the urogenital system had been established with the evolution of the earliest terrestrial tetrapods. Importantly, many lizard species with genetic sex determination, including the ones we studied, are oviparous and may represent valuable animal models for experimental tests of the association between prenatal exposure to androgens or estrogens and digit ratios.

Effects of elevated egg corticosterone levels on behavior, growth, and immunity of yellow-legged gull (Larus michahellis) chicks.

Eggs of vertebrates contain steroid hormones of maternal origin that may influence offspring performance. Recently, it has been shown that glucocorticoids, which are the main hormones mediating the stress response in vertebrates, are transmitted from the mother to the egg in birds. In addition, mothers with experimentally elevated corticosterone levels lay eggs with larger concentrations of the hormone, which produce slow growing offspring with high activity of the hypothalamo-adrenal axis under acute stress. However, the effects and function of transfer of maternal corticosterone to the eggs are largely unknown. In the present study, we injected corticosterone in freshly laid eggs of yellow-legged gulls (Larus michahellis), thus increasing the concentration of the hormone within its natural range of variation, and analyzed the effect of manipulation on behavioral, morphological, and immune traits of the offspring in the wild. Eggs injected with corticosterone had similar hatching success to controls, but hatched later. Mass loss during incubation was greater for corticosterone-treated eggs, except for the last laid ones. Corticosterone injection reduced rate and loudness of late embryonic vocalizations and the intensity of chick begging display. Tonic immobility response, reflecting innate fearfulness, was unaffected by hormone treatment. Elevated egg corticosterone concentrations depressed T-cell-mediated immunity but had no detectable effects on humoral immune response to a novel antigen, viability at day 10, or growth. Present results suggest that egg corticosterone can affect the behavior and immunity of offspring in birds and disclose a mechanism mediating early maternal effects whereby stress experienced by females may negatively translate to offspring phenotypic quality.

Larynx morphology and sound production in three species of Testudinidae.

Although the ability to vocalize is widespread among tortoises, the mechanisms of sound production in chelonians remain undescribed. In this study, we analyze the morphology and histology of the larynx of three species of Testudinidae (Testudo hermanni, T. graeca, and T. marginata) in order to ascertain the presence of vibrating acoustic structure, and based on our findings we propose a general model for phonation in tortoises. The structure of the larynx of the three tortoises analyzed is simple: three cartilages (the cricoid and two arytenoids) form the skeleton of the larynx, while two pairs of muscles (the dilators and constrictors) control the widening and closing of the glottis. The larynx is supported in the oral cavity by the hyoid cartilage, which in tortoises assumes the same functions of the thyroid cartilage of mammals. Two bands of elastic fibers are inserted in the lateral walls of the larynx just upstream of the glottis, and can be stretched away from the hyoid by the movements of the arytenoids. Their position and structure suggest that these bands are capable of vibrating during exhalation, and therefore may be considered vocal cords. The cricoid of T. marginata and T. graeca hold two diverticula, not previously reported, which might function as a low-frequency resonating chamber, improving the harmonic structure of tortoise calls. The structure of the larynx is compared with that of other vertebrates and the relationships between morphology and phonation are discussed. This is the first detailed description of anatomical structures possibly devoted to vocalization in chelonians.