Optimal husbandry of hatchling Eastern Indigo Snakes (Drymarchon couperi) during a captive head-start program.

Optimal husbandry techniques are desirable for any headstart program, but frequently are unknown for rare species. Here we describe key reproductive variables and determine optimal incubation temperature and diet diversity for Eastern Indigo Snakes (Drymarchon couperi) grown in laboratory settings. Optimal incubation temperature was estimated from two variables dependent on temperature, shell dimpling, a surrogate for death from fungal infection, and deviation of an egg from an ovoid shape, a surrogate for death from developmental anomalies. Based on these relationships and size at hatching we determined optimal incubation temperature to be 26°C. Additionally, we used incubation data to assess the effect of temperature on duration of incubation and size of hatchlings. We also examined hatchling diets necessary to achieve optimal growth over a 21-month period. These snakes exhibited a positive linear relationship between total mass eaten and growth rate, when individuals were fed less than 1711 g of prey, and displayed constant growth for individuals exceeding 1711 g of prey. Similarly, growth rate increased linearly with increasing diet diversity up to a moderately diverse diet, followed by constant growth for higher levels of diet diversity. Of the two components of diet diversity, diet evenness played a stronger role than diet richness in explaining variance in hatchling growth. These patterns document that our goal of satiating snakes was achieved for some individuals but not others and that diets in which total grams consumed over the first 21 months of life is distributed equivalently among at least three prey genera yielded the fastest growth rates for hatchling snakes.

Cryptosporidium serpentis Surveillance in Free-Ranging Snakes to Inform a Reintroduction Strategy for the Eastern Indigo Snake (Drymarchon couperi).

Understanding risk factors associated with reintroductions is important for making informed decisions within an adaptive framework. Biosecurity measures minimizing the risk of the introduction or spread of transmissible diseases are a priority when considering the release of captive-reared wildlife. Eastern indigo snake (EIS; Drymarchon couperi) reintroductions have been occurring in Alabama since 2010 and in Florida since 2017. During this effort the pathogen Cryptosporidium serpentis was detected, affecting several of the captive breeding snakes. Infected snakes were quarantined and removed from breeding efforts, which reduced snakes available for the reintroduction projects. To make informed management decisions about future reintroduction strategies, 155 free-ranging snakes were sampled at the two release sites and a third site in Georgia to evaluate the natural occurrence of C. serpentis. Additionally, 72 free-ranging EIS and other species incidentally encountered throughout the EIS range were tested opportunistically. All snakes sampled at the three focal sites tested negative, but one opportunistically tested EIS from South Florida tested positive. These results indicate that C. serpentis is present in the environment in at least one location, but at low levels. Our results suggest that, pending additional surveillance, C. serpentispositive snakes should not be included in reintroduction efforts, and that maintaining a high level of biosecurity is important in captive breeding programs.

Upper respiratory tract disease and associated diagnostic tests of mycoplasmosis in Alabama populations of Gopher tortoises, Gopherus polyphemus.

Upper respiratory tract disease (URTD) in North American tortoises (Gopherus) has been the focus of numerous laboratory and field investigations, yet the prevalence and importance of this disease remains unclear across many tortoise populations. Furthermore, much research has been focused on understanding diagnostic biomarkers of two known agents of URTD, Mycoplasma agassizii and Mycoplasma testudineum, yet the reliability and importance of these diagnostic biomarkers across populations is unclear. Gopher Tortoises (Gopherus polyphemus) have experienced significant declines and are currently protected range wide. Geographically, Alabama represents an important connection for Gopher Tortoise populations between the core and periphery of this species' distribution. Herein, we systematically sampled 197 Gopher Tortoises for URTD across seven sites in south-central and south-eastern Alabama. Plasma samples were assayed for antibodies to M. agassizii and M. testudineum; nasal lavage samples were assayed for the presence of viable pathogens as well as pathogen DNA. Lastly, animals were scored for the presence of external symptoms and nasal scarring consistent with URTD. External symptoms of URTD were present in G. polyphemus in all sites sampled in Alabama. There was no relationship between active symptoms of URTD and Mycoplasma antibodies, however the presence of URTD nasal scarring was positively related to M. agassizii antibodies (P = 0.032). For a single site that was sampled in three sequential years, seroprevalence to M. agassizii significantly varied among years (P < 0.0001). Mycoplasma agassizii DNA was isolated from four of the seven sites using quantitative PCR, yet none of the samples were culture positive for either of the pathogens. An analysis of disease status and condition indicated that there was a significant, positive relationship between the severity of URTD symptoms and relative body mass (P < 0.05). This study highlights the need for continued monitoring of disease in wild populations. Specifically, focus must be placed on identifying other likely pathogens and relevant biomarkers that may be important drivers of URTD in North American tortoises. Special consideration should be given to environmental contexts that may render wild populations more susceptible to disease.

Patterns of head shape and scutellation in Drymarchon couperi (Squamata: Colubridae) reveal a single species.

Krysko et al. (2016a) used analyses of DNA sequence data to reveal two genetic lineages of Drymarchon couperi. The Atlantic lineage contained specimens from southeastern Georgia and eastern peninsular Florida, and the Gulf Coast lineage contained specimens from western and southern peninsular Florida as well as western Florida, southern Alabama, and southern Mississippi. In a second paper Krysko et al. (2016b) analyzed morphological variation of the two lineages, which allowed them to restrict D. couperi to the Atlantic lineage and to describe the Gulf Coast lineage as a new species, Drymarchon kolpobasileus. This taxonomic discovery was remarkable for such a large, wide-ranging species and was notable for its impact on conservation. Because of population declines, particularly in western Florida, southern Alabama, and southern Mississippi, D. couperi (sensu lato) was listed as Threatened under the Endangered Species Act (United States Fish and Wildlife Service 1978, 2008) and repatriation of the species to areas where it had been extirpated was listed as a priority conservation goal (United States Fish and Wildlife Service 1982, 2008). Such repatriation efforts were attempted in Alabama, Florida, Georgia, and South Carolina, starting in 1977 (Speake et al. 1987), but failed to create viable populations, likely because too few snakes were released at too many sites (Guyer et al. 2019; Folt et al. 2019a). A second attempt at repatriation was started in 2010 and concentrated on release of snakes at a single site in Alabama (Stiles et al. 2013). However, Krysko et al. (2016a) criticized this repatriation effort because it appeared to involve release of D. couperi (sensu stricto) into the geographic region occupied by D. kolpobasileus (as diagnosed in Krysko et al. 2016b).

Environmental DNA (eDNA) Detection Probability Is Influenced by Seasonal Activity of Organisms.

Environmental DNA (eDNA) holds great promise for conservation applications like the monitoring of invasive or imperiled species, yet this emerging technique requires ongoing testing in order to determine the contexts over which it is effective. For example, little research to date has evaluated how seasonality of organism behavior or activity may influence detection probability of eDNA. We applied eDNA to survey for two highly imperiled species endemic to the upper Black Warrior River basin in Alabama, US: the Black Warrior Waterdog (Necturus alabamensis) and the Flattened Musk Turtle (Sternotherus depressus). Importantly, these species have contrasting patterns of seasonal activity, with N. alabamensis more active in the cool season (October-April) and S. depressus more active in the warm season (May-September). We surveyed sites historically occupied by these species across cool and warm seasons over two years with replicated eDNA water samples, which were analyzed in the laboratory using species-specific quantitative PCR (qPCR) assays. We then used occupancy estimation with detection probability modeling to evaluate both the effects of landscape attributes on organism presence and season of sampling on detection probability of eDNA. Importantly, we found that season strongly affected eDNA detection probability for both species, with N. alabamensis having higher eDNA detection probabilities during the cool season and S. depressus have higher eDNA detection probabilities during the warm season. These results illustrate the influence of organismal behavior or activity on eDNA detection in the environment and identify an important role for basic natural history in designing eDNA monitoring programs.