Aggressive but not reproductive boldness in male green anole lizards correlates with baseline vasopressin activity.

Across species, individuals within a population differ in their level of boldness in social encounters with conspecifics. This boldness phenotype is often stable across both time and social context (e.g., reproductive versus agonistic encounters). Various neural and hormonal mechanisms have been suggested as underlying these stable phenotypic differences, which are often also described as syndromes, personalities, and coping styles. Most studies examining the neuroendocrine mechanisms associated with boldness examine subjects after they have engaged in a social interaction, whereas baseline neural activity that may predispose behavioral variation is understudied. The present study tests the hypotheses that physical characteristics, steroid hormone levels, and baseline variation in Ile3-vasopressin (VP, a.k.a., Arg8-vasotocin) signaling predispose boldness during social encounters. Boldness in agonistic and reproductive contexts was extensively quantified in male green anole lizards (Anolis carolinensis), an established research organism for social behavior research that provides a crucial comparison group to investigations of birds and mammals. We found high stability of boldness across time, and between agonistic and reproductive contexts. Next, immunofluorescence was used to colocalize VP neurons with phosphorylated ribosomal protein S6 (pS6), a proxy marker of neural activity. Vasopressin-pS6 colocalization within the paraventricular and supraoptic nuclei of the hypothalamus was inversely correlated with boldness of aggressive behaviors, but not of reproductive behaviors. Our findings suggest that baseline vasopressin release, rather than solely context-dependent release, plays a role in predisposing individuals toward stable levels of displayed aggression toward conspecifics by inhibiting behavioral output in these contexts.

Sperm Cryopreservation as a Tool for Amphibian Conservation: Production of F2 Generation Offspring from Cryo-Produced F1 Progeny.

Sperm cryopreservation and biobanking are emerging as tools for supporting genetic management of small and threatened populations in amphibian conservation programs. However, there is little to no evidence demonstrating reproductive maturity and viability of offspring generated with cryopreserved sperm, potentially limiting widespread integration of these technologies. The purpose of this report is to demonstrate that amphibian sperm can be cryopreserved and thawed to successfully produce individuals of an F1 generation that can reach adulthood and reproductive maturity, to generating viable gametes and an F2 generation. Species-specific exogenous hormones were administered to both F0 and F1 adults to stimulate spermiation and oviposition in the eastern tiger salamander (Ambystoma tigrinum), dusky gopher frog (Lithobates sevosa), and Puerto Rican crested toad (Peltophryne lemur). Sperm cells collected non-lethally from F0 adults were cryopreserved, thawed, and used for in vitro fertilization (IVF) to produce F1 offspring. Individuals of the F1 generation are shown to reach adulthood, express viable gametes, and produce offspring through facilitated breeding, or IVF. The production of amphibian F2 generations shown here demonstrates that amphibian sperm collected non-lethally can be banked and used to generate reproductively viable animals of subsequent generations, thus maintaining valuable genetic linages and diversity in threatened amphibian species. The incredible value that cryopreservation of sperm has for long-term genetic management aids in the sustainability of both in situ and ex situ conservation efforts for this taxon.

Social boldness correlates with brain gene expression in male green anoles.

Within populations, some individuals tend to exhibit a bold or shy social behavior phenotype relative to the mean. The neural underpinnings of these differing phenotypes - also described as syndromes, personalities, and coping styles - is an area of ongoing investigation. Although a social decision-making network has been described across vertebrate taxa, most studies examining activity within this network do so in relation to exhibited differences in behavioral expression. Our study instead focuses on constitutive gene expression in bold and shy individuals by isolating baseline gene expression profiles that influence social boldness predisposition, rather than those reflecting the results of social interaction and behavioral execution. We performed this study on male green anole lizards (Anolis carolinensis), an established model organism for behavioral research, which provides a crucial comparison group to investigations of birds and mammals. After identifying subjects as bold or shy through repeated reproductive and agonistic behavior testing, we used RNA sequencing to compare gene expression profiles between these groups within various forebrain, midbrain, and hindbrain regions. The ventromedial hypothalamus had the largest group differences in gene expression, with bold males having increased expression of neuroendocrine and neurotransmitter receptor and calcium channel genes compared to shy males. Conversely, shy males express more integrin alpha-10 in the majority of examined regions. There were no significant group differences in physiology or hormone levels. Our results highlight the ventromedial hypothalamus as an important center of behavioral differences across individuals and provide novel candidates for investigations into the regulation of individual variation in social behavior phenotype.