Sperm cryopreservation in an Australian skink (Eulamprus quoyii).

Assisted reproductive technologies for population and genetic management for threatened herpetofauna have grown substantially in the past decade. Here we describe experiments to optimise sperm cryopreservation in a model squamate, the eastern water skink Eulamprus quoyii . Small, concentrated volumes of highly motile spermatozoa were reliably collected from adult male E. quoyii by non-lethal ventral massage. Samples were used to: (1) test whether protein-rich diluents, namely Beltsville poultry semen extender (BPSE) and TES and Tris (TEST) yolk buffer (TYB), improve post-thaw quality metrics compared with Dulbecco's phosphate-buffered saline (DPBS); and (2) compare the efficacy of these diluents in combination with either 1.35M glycerol or 1.35M dimethyl sulfoxide (DMSO) at two freezing rates, fast (approximately -20°C min-1 ) versus slow (-6°C min-1 ). Glycerol and DMSO performed equally well in preserving spermatozoa under slow freezing rates. Under these conditions, the use of the complex diluents BPSE and TYB significantly improved post-thaw total motility compared with DPBS. Complex interactions occurred between cryodiluent type, cryoprotectant and freezing rate when testing fast versus slow freezing rates among treatment groups. Under slow freezing rates, DMSO was better at preserving membrane integrity and motility, regardless of diluent type, but successful fast freezing required complex diluents to support motility and membrane integrity, which has implications for implementation in a field setting.

Shrinking before our isles: the rapid expression of insular dwarfism in two invasive populations of guttural toad (Sclerophrys gutturalis).

Island ecosystems have traditionally been hailed as natural laboratories for examining phenotypic change, including dramatic shifts in body size. Similarly, biological invasions can drive rapid localized adaptations within modern timeframes. Here, we compare the morphology of two invasive guttural toad (Sclerophrys gutturalis) populations in Mauritius and Réunion with their source population from South Africa. We found that female toads on both islands were significantly smaller than mainland counterparts (33.9% and 25.9% reduction, respectively), as were males in Mauritius (22.4%). We also discovered a significant reduction in the relative hindlimb length of both sexes, on both islands, compared with mainland toads (ranging from 3.4 to 9.0%). If our findings are a result of natural selection, then this would suggest that the dramatic reshaping of an amphibian's morphology-leading to insular dwarfism-can result in less than 100 years; however, further research is required to elucidate the mechanism driving this change (e.g. heritable adaptation, phenotypic plasticity, or an interaction between them).

Conservation implications of physiological carry-over effects in bats recovering from white-nose syndrome.

Although it is well documented that infectious diseases can pose threats to biodiversity, the potential long-term consequences of pathogen exposure on individual fitness and its effects on population viability have rarely been studied. We tested the hypothesis that pathogen exposure causes physiological carry-over effects with a pathogen that is uniquely suited to this question because the infection period is specific and time limited. The fungus Pseudogymnoascus destructans causes white-nose syndrome (WNS) in hibernating bats, which either die due to the infection while hibernating or recover following emergence from hibernation. The fungus infects all exposed individuals in an overwintering site simultaneously, and bats that survive infection during hibernation clear the pathogen within a few weeks following emergence. We quantified chronic stress during the active season, when bats are not infected, by measuring cortisol in bat claws. Free-ranging Myotis lucifugus who survived previous exposure to P. destructans had significantly higher levels of claw cortisol than naïve individuals. Thus, cryptic physiological carry-over effects of pathogen exposure may persist in asymptomatic, recovered individuals. If these effects result in reduced survival or reproductive success, they could also affect population viability and even act as a third stream in the extinction vortex. For example, significant increases in chronic stress, such as those indicated here, are correlated with reduced reproductive success in a number of species. Future research should directly explore the link between pathogen exposure and the viability of apparently recovered populations to improve understanding of the true impacts of infectious diseases on threatened populations.

No evidence for innate differences in tadpole behavior between natural, urbanized, and invasive populations.

ABSTRACT: Animals are increasingly challenged to respond to novel or rapidly changing habitats due to urbanization and/or displacement outside their native range by humans. Behavioral differences, such as increased boldness (i.e., propensity for risk-taking), are often observed in animals persisting in novel environments; however, in many cases, it is unclear how these differences arise (e.g., through developmental plasticity or evolution) or when they arise (i.e., at what age or developmental stage). In the Guttural Toad (Sclerophrys gutturalis), adult urban toads from both native and invasive ranges are bolder than conspecifics in natural habitats. Here, we reared Guttural Toad tadpoles in a common garden experiment, and tested for innate differences in boldness across their development and between individuals whose parents and lineage came from rural-native, urban-native, and urban-invasive localities (i.e., origin populations). Tadpoles did not differ in their boldness or in how their boldness changed over ontogeny based on their origin populations. In general, tadpoles typically became less bold as they aged, irrespective of origin population. Our findings indicate that differences in boldness in free-living adult Guttural Toads are not innate in the tadpole stage and we discuss three possible mechanisms driving phenotypic divergence in adult boldness for the focus of future research: habitat-dependent developmental effects on tadpole behavior, decoupled evolution between the tadpole and adult stage, and/or behavioral flexibility, learning, or acclimatization during the adult stage. SIGNIFICANCE STATEMENT: To determine if animals can persist in urban areas or become invasive outside their native ranges, it is important to understand how they adapt to life in the city. Our study investigates if differences in boldness that have been found in adult Guttural Toads (Sclerophrys gutturalis) represent heritable differences that can also be found in early life stages by rearing tadpoles from eggs in a common garden experiment. We did not find any differences in boldness among tadpoles from rural-native, urban-native, and urban-invasive origin populations. Our findings suggest that differences in boldness are not innate and/or that boldness is a behavioral trait that is decoupled between the tadpole and the adult stage.

Island Hopping through Urban Filters: Anthropogenic Habitats and Colonized Landscapes Alter Morphological and Performance Traits of an Invasive Amphibian.

A prominent feature of the modern era is the increasing spread of invasive species, particularly within island and urban ecosystems, and these occurrences provide valuable natural experiments by which evolutionary and invasion hypotheses can be tested. In this study, we used the invasion route of guttural toads (Sclerophrys gutturalis) from natural-native and urban-native populations (Durban, South Africa) to their urban-invasive and natural-invasive populations (Mauritius and Réunion) to determine whether phenotypic changes that arose once the toads became urbanized in their native range have increased their invasive potential before they were transported (i.e., prior adaptation) or whether the observed changes are unique to the invasive populations. This urban/natural by native/invasive gradient allowed us to examine differences in guttural toad morphology (i.e., body size, hindlimb, and hindfoot length) and performance capacity (i.e., escape speed, endurance, and climbing ability) along their invasion route. Our findings indicate that invasive island populations have reduced body sizes, shorter limbs in relation to snout-vent length, decreased escape speeds, and decreased endurance capacities that are distinct from the native mainland populations (i.e., invasion-derived change). Thus, these characteristics did not likely arise directly from a pre-transport anthropogenic "filter" (i.e., urban-derived change). Climbing ability, however, did appear to originate within the urban-native range and was maintained within the invasive populations, thereby suggesting it may have been a prior adaptation that provided this species with an advantage during its establishment in urban areas and spread into natural forests. We discuss how this shift in climbing performance may be ecologically related to the success of urban and invasive guttural toad populations, as well as how it may have impacted other island-derived morphological and performance phenotypes.

Road avoidance and its energetic consequences for reptiles.

Roads are one of the most widespread human-caused habitat modifications that can increase wildlife mortality rates and alter behavior. Roads can act as barriers with variable permeability to movement and can increase distances wildlife travel to access habitats. Movement is energetically costly, and avoidance of roads could therefore impact an animal's energy budget. We tested whether reptiles avoid roads or road crossings and explored whether the energetic consequences of road avoidance decreased individual fitness. Using telemetry data from Blanding's turtles (Emydoidea blandingii; 11,658 locations of 286 turtles from 15 sites) and eastern massasaugas (Sistrurus catenatus; 1,868 locations of 49 snakes from 3 sites), we compared frequency of observed road crossings and use of road-adjacent habitat by reptiles to expected frequencies based on simulated correlated random walks. Turtles and snakes did not avoid habitats near roads, but both species avoided road crossings. Compared with simulations, turtles made fewer crossings of paved roads with low speed limits and more crossings of paved roads with high speed limits. Snakes made fewer crossings of all road types than expected based on simulated paths. Turtles traveled longer daily distances when their home range contained roads, but the predicted energetic cost was negligible: substantially less than the cost of producing one egg. Snakes with roads in their home range did not travel further per day than snakes without roads in their home range. We found that turtles and snakes avoided crossing roads, but road avoidance is unlikely to impact fitness through energetic expenditures. Therefore, mortality from vehicle strikes remains the most significant impact of roads on reptile populations.

Comparability and repeatability of three commonly used methods for measuring endurance capacity.

Measures of endurance (time to exhaustion) have been used to address a wide range of questions in ecomorphological and physiological research, as well as being used as a proxy for survival and fitness. Swimming, stationary (circular) track running, and treadmill running are all commonly used methods for measuring endurance. Despite the use of these methods across a broad range of taxa, how comparable these methods are to one another, and whether they are biologically relevant, is rarely examined. We used Australian water dragons (Intellagama lesueurii), a species that is morphologically adept at climbing, swimming, and running, to compare these three methods of endurance and examined if there is repeatability within and between trial methods. We found that time to exhaustion was not highly repeatable within a method, suggesting that single measures or a mean time to exhaustion across trials are not appropriate. Furthermore, we compared mean maximal endurance times among the three methods, and found that the two running methods (i.e., stationary track and treadmill) were similar, but swimming was distinctly different, resulting in lower mean maximal endurance times. Finally, an individual's endurance rank was not repeatable across methods, suggesting that the three endurance trial methods are not providing similar information about an individual's performance capacity. Overall, these results highlight the need to carefully match a measure of performance capacity with the study species and the research questions being asked so that the methods being used are behaviorally, ecologically, and physiologically relevant.

Mitigating reptile road mortality: fence failures compromise ecopassage effectiveness.

Roadways pose serious threats to animal populations. The installation of roadway mitigation measures is becoming increasingly common, yet studies that rigorously evaluate the effectiveness of these conservation tools remain rare. A highway expansion project in Ontario, Canada included exclusion fencing and ecopassages as mitigation measures designed to offset detrimental effects to one of the most imperial groups of vertebrates, reptiles. Taking a multispecies approach, we used a Before-After-Control-Impact study design to compare reptile abundance on the highway before and after mitigation at an Impact site and a Control site from 1 May to 31 August in 2012 and 2013. During this time, radio telemetry, wildlife cameras, and an automated PIT-tag reading system were used to monitor reptile movements and use of ecopassages. Additionally, a willingness to utilize experiment was conducted to quantify turtle behavioral responses to ecopassages. We found no difference in abundance of turtles on the road between the un-mitigated and mitigated highways, and an increase in the percentage of both snakes and turtles detected dead on the road post-mitigation, suggesting that the fencing was not effective. Although ecopassages were used by reptiles, the number of crossings through ecopassages was lower than road-surface crossings. Furthermore, turtle willingness to use ecopassages was lower than that reported in previous arena studies, suggesting that effectiveness of ecopassages may be compromised when alternative crossing options are available (e.g., through holes in exclusion structures). Our rigorous evaluation of reptile roadway mitigation demonstrated that when exclusion structures fail, the effectiveness of population connectivity structures is compromised. Our project emphasizes the need to design mitigation measures with the biology and behavior of the target species in mind, to implement mitigation designs in a rigorous fashion, and quantitatively evaluate road mitigation to ensure allow for adaptive management and optimization of these increasingly important conservation tools.

A novel technique to measure chronic levels of corticosterone in turtles living around a major roadway.

Conservation biology integrates multiple disciplines to expand the ability to identify threats to populations and develop mitigation for these threats. Road ecology is a branch of conservation biology that examines interactions between wildlife and roadways. Although the direct threats of road mortality and habitat fragmentation posed by roads have received much attention, a clear understanding of the indirect physiological effects of roads on wildlife is lacking. Chronic physiological stress can lower immune function, affect reproductive rates and reduce life expectancy; thus, it has the potential to induce long-lasting effects on populations. Reptiles are globally in decline, and roads are known to have negative effects on reptile populations; however, it is unknown whether individual responses to roads and traffic result in chronic stress that creates an additional threat to population viability. We successfully extracted reliable measures of corticosterone (CORT), a known, commonly used biomarker for physiological stress, from claw trimmings from painted turtles (Chrysemys picta) captured at three study sites (road-impacted site, control site and validation site). Corticosterone levels in claws were evaluated as a measure of chronic stress in turtles because CORT is deposited during growth of the claw and could provide an opportunity to examine past long-term stress levels. While male turtles had higher CORT levels on average than females, there was no difference in the level of CORT between the road-impacted and control site, nor was there a relationship between CORT and turtle body condition. In validating a novel approach for non-invasive measurement of long-term CORT levels in a keratinized tissue in wild reptiles, our study provides a new avenue for research in the field of stress physiology.