Snake Fungal Disease in Free-Ranging Northern Pine Snakes (Pituophis melanoleucus melanoleucus) in New Jersey: Lesions, Severity of Sores and Investigator's Perceptions.

Ophidiomyces ophidiicola, the fungus causing snake fungal disease (SFD), has been identified in northern pine snakes (Pituophis melanoleucus) in New Jersey. In this paper, we (1) review the positivity rate of SFD on different locations on snakes' bodies, (2) determine the relationship between the sores and quantitative polymerase chain reaction (qPCR) positivity rates, and (3) explore the relationship between the investigators' clinical evaluation of the severity of sores, their evaluation of the likelihood of the sores being positive, and the qPCR positivity of SFD for the sores. Swabbing the sores was more effective at determining whether the snakes tested positive for O. ophidiicola than ventrum swabbing alone. The perception of the severity of the sores did not relate to qPCR positivity for O. ophidiicola. We suggest that the assessment of the rate of SFD among snakes in the wild needs to include the sampling of snakes with no clinical signs, as well as those with sores, and the swabbing of all the sores collectively. Clear terminology for sores, the identification of clinical signs of SFD, and distinguishing the rates of O. ophidiicola by PCR testing should be adopted. Overall, the pine snakes exhibited a higher rate of sores and positivity of O. ophidiicola swabs by PCR testing compared to the other snakes.

Prevalence of Ophidiomyces ophidiicola and epizootiology of snake fungal disease in free-ranging Northern Pine Snakes (Pituophis melanoleucus melanoleucus) in New Jersey.

Snake fungal disease, caused by Ophidiomyces ophidiicola, is recognized as a potential concern for North American snakes. We tested skin swabs from Northern Pine Snakes (Pituophis melanoleucus melanoleucus) in the New Jersey pinelands for the presence of O. ophidiicola before emergence from hibernation. We used qPCR to test the collected swabs for the presence of O. ophidiicola, then determined pathogen prevalence as a function of sampling year, sampling location (skin lesion, healthy ventral skin, healthy head skin) sex, and age. There were no temporal trends in O. ophidiicola detection percentages on snakes, which varied from 58 to 83% in different years. Ophidiomyces ophidiicola detection on snakes was highest in swabs of skin lesions (71%) and lowest in head swabs (29%). Males had higher prevalence than females (82% versus 62%). The fungus was not detected in hatchling snakes (age 0) in the fall, but 75% of juveniles tested positive at the end of hibernation (age 1 year). We also screened hibernacula soil samples for the presence of O. ophidiicola. Where snakes hibernated, 69% of soil samples were positive for O. ophidiicola, and 85% of snakes lying on positive soil samples also tested positive for the pathogen. Although a high proportion of snakes (73%) tested positive for O. ophidiicola during our 4-year study, the snakes appeared healthy except for small skin lesions. We conclude that O. ophidiicola prevalence is high on hibernating Northern Pine Snakes and in the hibernacula soil, with a strong association between snakes and positive adjacent soil. This is the first demonstration that snakes likely become infected during hibernation.

Combining surface and soil environmental DNA with artificial cover objects to improve terrestrial reptile survey detection.

Reptiles are increasingly of conservation concern due to their susceptibility to habitat loss, emerging disease, and harvest in the wildlife trade. However, reptile populations are often difficult to monitor given the frequency of crypsis in their life history. This difficulty has left uncertain the conservation status of many species and the efficacy of conservation actions unknown. Environmental DNA (eDNA) surveys consistently elevate the detection rate of species they are designed to monitor, and while their use is promising for terrestrial reptile conservation, successes in developing such surveys have been sparse. We tested the degree to which inclusion of surface and soil eDNA sampling into conventional artificial-cover methods elevates the detection probability of a small, cryptic terrestrial lizard, Scincella lateralis. The eDNA sampling of cover object surfaces with paint rollers elevated per sample detection probabilities for this species 4-16 times compared with visual surveys alone. We readily detected S. lateralis eDNA under cover objects up to 2 weeks after the last visual detection, and at some cover objects where no S. lateralis were visually observed in prior months. With sufficient sampling intensity, eDNA testing of soil under cover objects produced comparable per sample detection probabilities as roller surface methods. Our results suggest that combining eDNA and cover object methods can considerably increase the detection power of reptile monitoring programs, allowing more accurate estimates of population size, detection of temporal and spatial changes in habitat use, and tracking success of restoration efforts. Further research into the deposition and decay rates of reptile eDNA under cover objects, as well as tailored protocols for different species and habitats, is needed to bring the technique into widespread use.

El interés por la conservación de los reptiles es cada vez mayor debido a su susceptibilidad ante la pérdida del hábitat, enfermedades emergentes y la captura para el mercado de fauna. Sin embargo, las poblaciones de reptiles son difíciles de monitorear por lo frecuente que es la cripsis en sus historias de vida. Esta dificultad deja incierto el estado de conservación de muchas especies y desconocida la eficacia de las acciones de conservación. Los censos de ADN ambiental (DNAa) elevan sistemáticamente la tasa de detección de las especies que monitorean, y aunque su uso es prometedor para la conservación de los reptiles terrestres, han sido escasos los éxitos en el desarrollo de dichos censos. Analizamos el grado al que la inclusión del muestreo de DNAa superficial y del suelo a los métodos convencionales de cobertura artificial eleva la probabilidad de detección de una pequeña lagartija terrestre críptica: Scincella lateralis. El muestreo de DNAa de las superficies con cobertura de objetos con rodillos de pintura elevó las probabilidades de detección por muestra para esta especie 4-16 veces más que los censos visuales. Detectamos fácilmente el DNAa de S. lateralis bajo los objetos de cubierta hasta dos semanas después de la última detección visual y en algunos objetos de cubierta en donde no se había observado en los meses previos a S. lateralis. Con suficiente intensidad de muestreo, el análisis de DNAa del suelo bajo objetos de cubierta produjo probabilidades de detección por muestra comparables como métodos de rodillo superficial. Nuestros resultados sugieren que la combinación del DNAa y los métodos de objetos de cobertura puede incrementar considerablemente el poder de detección de los programas de monitoreo de reptiles, lo que permite estimaciones más precisas del tamaño poblacional, detección de los cambios espaciales y temporales en el uso de hábitat y el éxito de rastreo de los esfuerzos de restauración. Además, se necesita la investigación sobre las tasas de depósito y descomposición del DNAa de reptiles bajo objetos de cubierta, así como los protocolos hechos para diferentes especies y hábitats, para que la técnica entre al uso difundido.

Soil Reservoir Dynamics of Ophidiomyces ophidiicola, the Causative Agent of Snake Fungal Disease.

Wildlife diseases pose an ever-growing threat to global biodiversity. Understanding how wildlife pathogens are distributed in the environment and the ability of pathogens to form environmental reservoirs is critical to understanding and predicting disease dynamics within host populations. Snake fungal disease (SFD) is an emerging conservation threat to North American snake populations. The causative agent, Ophidiomyces ophidiicola (Oo), is detectable in environmentally derived soils. However, little is known about the distribution of Oo in the environment and the persistence and growth of Oo in soils. Here, we use quantitative PCR to detect Oo in soil samples collected from five snake dens. We compare the detection rates between soils collected from within underground snake hibernacula and associated, adjacent topsoil samples. Additionally, we used microcosm growth assays to assess the growth of Oo in soils and investigate whether the detection and growth of Oo are related to abiotic parameters and microbial communities of soil samples. We found that Oo is significantly more likely to be detected in hibernaculum soils compared to topsoils. We also found that Oo was capable of growth in sterile soil, but no growth occurred in soils with an active microbial community. A number of fungal genera were more abundant in soils that did not permit growth of Oo, versus those that did. Our results suggest that soils may display a high degree of both general and specific suppression of Oo in the environment. Harnessing environmental suppression presents opportunities to mitigate the impacts of SFD in wild snake populations.

A paradigm for information needed to protect at-risk species: northern pine snake (Pituophis melanoleucus) in the pine barrens as a case study.

New methods of examining the risk to endangered, threatened and rare species are required to identify vulnerability. A paradigm for examining risk is presented that describes anthropogenic threats, species activities, and vulnerabilities, and uses Northern pine snakes (Pituophis melanoleucus) in the New Jersey Pine Barrens as a case study. The paradigm includes (1) conceptual model of natural, anthropogenic, and interactive stressors, (2) template of the functional attributes of threats from human activities, and (3) template of effects from different human activities. Pine snake behavior throughout the year was used to examine the temporal overlap in high snake vulnerability periods and desired human activities in a shared habitat. New data on autumn behavior of pine snakes are also provided. Passive integrated transponders (PIT tag) tracking technology indicated that the fall basking activity period is both longer in duration, and at a higher intensity than previously presumed. During the autumn, individual snakes moved in and out of dens an average of 6 times over a two-month period. Younger snakes at a small hibernaculum were more active than those at hibernacula with larger and older snakes. The high activity period of pine snakes overlaps with the timing of preferred off-road-vehicle (ORV) use, controlled burns, and other human activities, increasing snake vulnerability, potentially causing behavioral disruptions, injury, and death. The conceptual model illustrating relationships between attributes of human activity and effects may be utilized to determine risks to other listed species, and those of special concern in different habitats. This paradigm also provides managers with template tools to assess risks to species that may also be used to provide information to the public.

Hatchling survival to breeding age in Northern Pine Snakes (Pituophis melanoleucus) in the New Jersey Pine Barrens: Human effects on recruitment from 1986 to 2017.

To conserve threatened/endangered species, we need to understand the factors contributing to reproductive success and recruitment to reproductive stage. Obtaining this information is difficult for snakes because they are secretive, are not easy to locate at the same stage each year, and are sometimes sparsely distributed. We determined nest fate, hatchling growth and survival to age 5 years, and recruitment to breeding age of Northern Pine Snakes (Pituophis melanoleucus) in New Jersey Pine Barrens from 1986 to 2017. Pine Snakes are 'threatened' in New Jersey and in other states, and are at risk because of increased human population, habitat loss, predation, and poaching. Age of first-breeding was 4-years, based on snout-vent length of gravid and laying females, and snout-vent length of females followed as hatchlings to 5-years. Mean clutch size (+ 1 SE) was 9.5 + 0.3 (N = 53). The annual percent of nests in which eggs hatched averaged 25% (N = 288 nests), and varied among 5-year periods (5% to 30%/year). Of lab-reared hatchlings released into natal nests (N = 90), 26% (2015) and 32% (2016) reached hibernacula excavated in 2016 and 2017. The sex ratio of hatchlings reaching hibernation sites (N = 181) between 1986 and 2015 was skewed toward females (74/106, 59% females), and varied among 5-year periods (47-75% females). Once hatchlings reached a hibernaculum, there was a sex-related difference in survival. For hatchlings reaching a monitored hibernaculum, survival to 3-years was 35% in females and 40% in males, and to 4-years was 25% in females and 33% in males. Using these data, only 10% of females reached 3 years (first possible breeding age), and 7% survived to 4-years. Methodological problems with determining survival rates during these early critical years are discussed.

Arsenic, Cadmium, Chromium, Lead, Mercury and Selenium Concentrations in Pine Snakes (Pituophis melanoleucus) from the New Jersey Pine Barrens.

Top trophic level predators are at risk from bioaccumulation of heavy metals from their prey. Using nondestructively collected tissues as a method of assessing metal concentrations in snakes is useful for populations that are threatened or declining. This paper reports concentrations of arsenic (As), cadmium (Cd), chromium (Cr), lead (Pb), mercury (Hg), and selenium (Se) in tissues of Northern pine snakes (Pituophis melanoleucus) from the New Jersey Pine Barrens, a relatively pristine, undisturbed habitat. We also determined if skin is an appropriate indicator of internal concentrations and identified the factors (tissue, year of collection, length, sex) that might explain variations in metal concentrations. Because they can grow to 2-m long and live for 25 years, we suggest that these snakes might accumulate heavy metals. Multiple regression models were significant, explaining 16% (lead) to 61% (mercury) of variation by tissue type. For mercury and chromium, size also was significant. The highest concentrations were in liver and kidney for all metals, except chromium and lead. Mercury concentrations in tissues were within the range reported for other snakes and were below effects concentrations in reptiles. The concentrations in skin were correlated with all internal tissues for mercury and for all internal tissues except heart for cadmium. These data show that shed skin can be used as an indicator of metals in pine snakes and that, at present, concentrations of heavy metals in this population are within the range of those found in other snake species from uncontaminated sites.

Hibernation Site Philopatry in Northern Pine Snakes (Pituophis melanoleucus) in New Jersey.

Northern Pine Snakes (Pituophis melanoleucus) are one of the few snakes that spend the winter in underground hibernacula that they excavate. We report the use of hibernacula by Pine Snakes from 1986 to 2012 in the New Jersey Pine Barrens. We determined whether philopatry to a specific hibernaculum varied as a function of age, sex, and location of the hibernaculum. Three hibernacula were occupied nearly continuously for 27 yr by 1 to 27 snakes each year. With known-age snakes (N = 120), captured mainly as hatchlings and 2-yr-olds, we found that 23% were always philopatric. Philopatry was related to age of last capture, sex, and capture location. Philopatry was higher for 1) females compared with males, 2) snakes at two solitary hibernacula compared with a hibernaculum complex, and 3) snakes 6 yr old or younger, compared with older snakes. Of hatchlings found hibernating, 24% used the same hibernation site the next year, and 38% were located at year 4 or later. The number of snakes that always used the same hibernation site declined with the age of last capture. Snakes that entered hibernacula as hatchlings were found more often than those that entered as 2-yr-olds. For the seven snakes that were 14 yr or older, females were found 64- 86 % of the time, whereas males were found 15 to 50% of the time. Understanding the behavior and habitat requirements of snakes during different seasons is central to life-history analysis and for conserving viable populations.

Fidelity of northern pine snakes (Pituophis m. melanoleucus) to natural and artificial hibernation sites in the New Jersey Pine Barrens.

Environmental managers require information on whether human-made hibernacula are used by rare snakes before constructing large numbers of them as mitigation measures. Fidelity of northern pine snakes (Pituophis m. melanoleucus) was examined in a 6-year study in the New Jersey Pine Barrens to determine whether they used natural and artificial hibernacula equally. Pine snakes used both artificial (human-made) and natural (snake-adapted) hibernacula. Most natural hibernacula were in abandoned burrows of large mammals. Occupancy rates were similar between natural and artificial hibernacula. Only 6 of 27 radio-tracked snakes did not shift hibernacula between years, whereas 78% shifted sites at least once, and fidelity from one year to the next was 42%. For snakes that switched hibernacula (n = 21), one switched among artificial hibernacula, 14 (65%) switched among natural hibernacula, and 6 (29%) switched from artificial to natural hibernacula. Data indicate that most pine snakes switch among hibernacula, mainly selecting natural hibernacula, suggesting that artificial dens are used, but protecting natural hibernacula should be a higher conservation priority.