Terence M. Farrell

Proteomic analysis of ontogenetic and diet-related changes in venom composition of juvenile and adult Dusky Pigmy rattlesnakes (Sistrurus miliarius barbouri)

Snake venom proteins show high levels of variation at the level of the individual yet the environmental and molecular mechanisms that generate this diversity remain unclear. Here we report the results of a controlled feeding experiment combined with proteomic analyses of periodically collected venom samples to assess the roles of ontogenetic and diet-related effects on venom composition of captive juvenile and adult Dusky Pigmy rattlesnakes (Sistrurus miliarius barbouri). Juvenile snakes fed from birth with mice, lizards, or frogs showed little evidence for an ontogenetic shift in venom composition from 5 to 26 months in terms of substantial changes in the relative abundance of major classes of venom toxins. However, there were fine-scale changes in the relative abundance of D49-PLA₂ 15, PI-SVMPs, and PIII-SVMP 28, and a decline in the abundance of other PIII-SVMPs. Although juveniles raised on different prey exhibited distinct relative toxin compositional change rates, at 26 months old, their venoms showed similar patterns of protein composition suggesting little effect of diet on the overall make-up of venom in snakes this age or younger. In contrast, adult females raised on different prey over a 26 month period showed prey-related changes in the relative abundance of major protein families from initial to final samples. Specifically, mouse-fed females showed substantial increases in the relative abundance of total PLA₂s and serine proteinases of 95% and >100%, respectively, whereas comparable values for lizard- (42% and -22%) and frog-fed females (2% and 11%) were distinctly smaller in magnitude. Venom from adult snakes fed on different prey also showed distinct changes in the abundance of PLA₂ molecules 15, 19a, and 19, which were, respectively, (>100%, 33%, 63%), (>100%, 0%, 35%), and (71%, 20%, -4%) for the mice-, lizard-, and frog-diet. Venom from snakes raised on frogs contained a small (1.1%) but consistent amount of a PLA₂ molecule (15a) not present in snakes fed on mice or lizards. This work provides evidence that venom composition is somewhat plastic in both juvenile and adult S. m. barbouri and that, at least in adults, prey consumed may influence the relative abundance of possibly functionally-distinct classes of venom proteins.

REPRODUCTION IN THE RATTLESNAKE, SISTRURUS MILIARIUS BARBOURI, IN CENTRAL FLORIDA

-We studied reproduction in female pigmy rattlesnakes in a subtropical area. Twenty-six females were either captured gravid and held until parturition or found in the field with their litters. Over 92% of the snakes gave birth in August. Individuals with large clutch masses gave birth earlier in the season than individuals with small clutch masses. Mean clutch size was 5.88 (range 2-11). Litter size and clutch mass were significantly correlated with female SVL. There was significant variation in offspring size among females, but this variation was not a result of larger females having larger young, or the young being smaller in larger litters. Of 19 females whose reproductive condition was determined in the summers of both 1992 and 1993, 32% were not gravid in either year, 42% were gravid in one of the two years, and 26% were gravid in both years. We hypothesize that annual reproduction in some females is a result of the year-round activity of S. miliarius in central Florida. Journal of Herpetology, Vol. 29, No. 1, p . 21-27, 1995 Copyright 1995 Society for the Study of Amphibians and Reptiles Reproduction in the Rattlesnake, Sistrurus miliarius barbouri, in Viperids typically exhibit a suite of life history characteristics, including slow growth, which results in delayed maturation and low frequency of reproduction (LFR) by adult females (Dunham and Miles, 1985). In many viperids most adult females reproduce every other year (reviewed by Seigel and Ford, 1987; Dunham et al., 1988) and in some species females reproduce only every third or fourth year (Macartney and Gregory, 1988; Brown, 1991; Martin, 1993). It is not clear why viperids lack t e resources for frequent reproduction (Seigel and Ford, 1987). Shine (1991) suggests that sitand-wait predators, including most viperids, will tend to have low foraging success compared to snakes that actively search for prey. Some costs of reproduction resulting from viviparity may be independent of fecundity. For example, gravid females may need to move to basking sites with low prey availability to accelerate development of young. These fecundity-independent costs of reproduction may re21 This content downloaded from 207.46.13.169 on Sat, 01 Oct 2016 05:31:40 UTC All use subject to http://about.jstor.org/terms T. M. FARRELL ET AL. sult in LFR (Bull and Shine, 1979). Shine (1980) also suggested that sit-and-wait predators are less likely to be detected by their own predators and therefore have the high survivorship rates that are needed to make delayed maturity and infrequent reproduction a potentially successful life history. Another factor that may result in LFR is short activity periods imposed by climatic factors. Thoroughly studied viperid populations have been most frequently found in high latitude or high altitude sites (e.g., Macartney and Gregory, 1988; King and Duvall, 1990; Brown, 1991; Madsen and Shine, 1993; Martin, 1993), where cold weather results in prolonged periods of hibernation. Martin (1993) reviewed evidence that indicates the length of the yearly activity period in Crotalus horridus influences both the age of first reproduction and reproductive frequency. Similarly, Larsen et al. (1993) found that Thamnophis sirtalis, an active foraging colubrid, exhibited LFR near its northern distributional limit. If viviparity and foraging mode are the major determinants of life history characteristics in viperids, then subtropical and tropical species should often exhibit LFR, just as in temperate species. If, however, the length of the activity season is an important factor then we expect that viperid populations in warm regions will exhibit LFR less often. Unfortunately, we know very little about reproductive frequency and other life history traits of viperids in subtropical and tropical regions. The pigmy rattlesnake has a broad range, including subtropical areas, and is fairly common in much of its range. However, few studies have addressed its life history (Ernst, 1992). In this study we present a detailed examination of reproductive parameters of Sistrurus miliarius barbouri, including (a) data from a large sample of females encompassing the full range of sizes at which these snakes reproduce, and (b) data on the frequency of reproduction in females. In central Florida, S. miliarius can be found in a variety of habitats, ranging from drier pine uplands to seasonally flooded hardwood forests. In this subtropical region rattlesnakes are active above ground throughout the year. The highest density populations (>40 rattlesnakes/ ha, unpubl. data) are found in mesic habitats located near freshwater marshes where they feed primarily on lizards (Scincella laterale, Eumeces inexpectatus, Anolis carolinensis) and frogs (Hyla spp. and Rana utricularia). MATERIALS AND METHODS Our study sites were four tracts of mesic-hydric forest dominated by live oak, sabal palmetto, red maple, and scattered slash pines. The sites were along the floodplain of the St. Johns River in Volusia County, Florida and were seprated by <4 km. We visually searched for S. m. barbouri in our study sites from 17 February 1992 to 31 August 1993. We individually marked snakes when they were first captured by scale clips (February-July 1992) or PIT-tags (after August 1992). For each snake, we measured weight using spring-loaded Pesola scales, and snoutvent length (SVL) in a squeeze box (Quinn and Jones, 1974). The reproductive condition of each snake was determined by palpation. Data were collected in the field and the snakes were rel ased at the site of capture within approximately 15 min of capture. To study reproduction more fully we held some gravid females captive until they gave birth. They were collected by visually searching the study sites in the morning or late afternoon. We placed cages in one hammock to hold gravid females until birth. The cages measured 0.6 m x 0.6 m x 0.25 m (1 x w x h) and were made of wood framing surrounded by aluminum screen (mesh size = 1 mm x 1 mm). A layer of dirt and leaf litter was placed in the cages along with a length of PVC pipe (5 cm diameter, 30 cm long) and a piece of oak or pine bark to provide shelter. The cages were exposed to direct sunlight for much of the day to allow females to bask. A palm frond was placed on each cage to provide shade. Once placed in a cage, each gravid snake was hecked daily until it gave birth. Snakes had access to water but were not fed. After parturition, the female and young were brought into the laboratory for measurement of mass and length. The data were analyzed using Systat (Wilkinson, 1988). Since many correlations were carried out in this analysis, we used a sequential Bonferroni technique (Rice, 1989) to ensure the probability of Type I error was less than 0.05 for the correlation matrix involving reproductive variables (Table 1).

AN OUTBREAK OF FUNGAL DERMATITIS AND STOMATITIS IN A FREE-RANGING POPULATION OF PIGMY RATTLESNAKES (SISTRURUS MILIARIUS BARBOURI) IN FLORIDA

Between September 1997 and March 1998, a severe skin, eye, and mouth disease was observed in a population of dusky pigmy rattlesnakes (Sistrurus miliarius barbouri), at the Lake Woodruff National Wildlife Refuge in Volusia County, Florida (USA). Three affected pigmy rattlesnakes were submitted for necropsy. All snakes had severe necrotizing and predominantly granulomatous dermatitis, stomatitis, and ophthalmitis, with involvement of the subadjacent musculature and other soft tissues. Numerous fungal hyphae were seen throughout tissue sections stained with periodic acid Schiff and Gomoris methenamine silver. Samples of lesions were cultured for bacteria and fungi. Based on hyphae and spore characteristics, four species of fungi were identified from culture: Sporothrix schenckii, Pestalotia pezizoides, Geotrichum candidum (Galactomyces geotrichum), and Paecilomyces sp. While no additional severely affected pigmy rattlesnakes were seen at the study site, a garter snake (Thamnophis sirtalis) and a ribbon snake (Thamnophis sauritis) with similar lesions were found. In 1998 and 1999, 42 pigmy rattlesnakes with multifocal minimal to moderate subcutaneous masses were seen at the study site. Masses from six of these snakes were biopsied in the field. Hyphae morphologically similar to those seen in the severe cases were observed with fungal stains. Analysis of a database representing 10,727 captures in previous years was performed after the 1998 outbreak was recognized. From this analysis we determined that 59 snakes with clinical signs similar to those seen during the 1998 outbreak were documented between 1992 and 1997. This study represents the first documented report of a mycotic disease of free-ranging snakes.

Seasonal Abundance and Activity of a Rattlesnake (Sistrurus miliarius barbouri) in Central Florida

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PIGMY RATTLESNAKES USE FROG-DERIVED CHEMICAL CUES TO SELECT FORAGING SITES

We performed a field experiment to test the hypothesis that pigmy rattlesnakes select foraging locations based on the presence of prey odor. We established six pairs of parallel transects, each 25 m long; the experimental transect was sprayed with distilled water in which leopard frogs had been housed for 48 h, and the parallel control transect was sprayed only with distilled water. The transects were then censused for the presence of pigmy rattlesnakes at 24-h intervals for the next 96 h. More snakes were found along the experimental transects at all times, and significantly more (P = 0.019, binomial test) were found at 72 h after spraying. Our results indicate that prey odor is at least one of the factors involved in foraging site selection by these sit-and-wait predators.

Snake fungal disease: an emerging threat to wild snakes

Since 2006, there has been a marked increase in the number of reports of severe and often fatal fungal skin infections in wild snakes in the eastern USA. The emerging condition, referred to as snake fungal disease (SFD), was initially documented in rattlesnakes, where the infections were believed to pose a risk to the viability of affected populations. The disease is caused by Ophidiomyces ophiodiicola, a fungus recently split from a complex of fungi long referred to as the Chrysosporium anamorph of Nannizziopsis vriesii (CANV). Here we review the current state of knowledge about O. ophiodiicola and SFD. In addition, we provide original findings which demonstrate that O. ophiodiicola is widely distributed in eastern North America, has a broad host range, is the predominant cause of fungal skin infections in wild snakes and often causes mild infections in snakes emerging from hibernation. This new information, together with what is already available in the scientific literature, advances our knowledge of the cause, pathogenesis and ecology of SFD. However, additional research is necessary to elucidate the factors driving the emergence of this disease and develop strategies to mitigate its impacts. This article is part of the themed issue ‘Tackling emerging fungal threats to animal health, food security and ecosystem resilience’.

The Effects of Age and Light Level on Foraging Posture and Frequency of Caudal Luring in the Rattlesnake, Sistrurus miliarius barbouri

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Defensive Behavior of Free-Ranging Pygmy Rattlesnakes (Sistrurus miliarius)

Abstract Sistrurus miliarius frequently bites humans in the southeastern United States. We used a large population of S. miliarius in central Florida to investigate the importance of several factors on the defensive behavior of this species. Upon detection of a snake, we tapped the snout of the snake with a gloved hand. We recorded whether the snake struck or fled. Our large sample size (N = 336) allowed us to make strong conclusions regarding the defensive behavior of this rattlesnake species. Overall, only 27 snakes (8%) bit the glove indicating that this species is rather nonaggressive. Initial posture was the only factor that influenced striking behavior; uncoiled snakes struck significantly more than coiled snakes. Fleeing behavior was affected by three variables: sex; whether the snake was about to shed; and initial posture. Females fled more often than males, as did snakes about to shed. Initial posture is the only factor that affected both response variables, and uncoiled individuals were more likely to flee than coiled snakes.

Environmental and physiological correlates of the severity of clinical signs of snake fungal disease in a population of pigmy rattlesnakes,Sistrurus miliarius

In free-ranging pigmy rattlesnakes, clinical signs of snake fungal disease varied seasonally and were negatively correlated with energetic status and mean air temperature. Severely infected snakes were in poor body condition but did not show deficits in innate immune function. Innate immunocompetence varied seasonally, but not in association with costly life-history stages.

Experimental Manipulation of Tail Color Does Not Affect Foraging Success in a Caudal Luring Rattlesnake

Abstract Snakes that exhibit caudal luring typically have a conspicuously colored tail tip. We conducted a field experiment to test the adaptive significance of tail color. A sample of 169 neonate Pigmy Rattlesnakes (Sistrurus miliarius) was treated by coloring their yellow tails with either black pigment similar to their cryptic body color or yellow ink as a procedural control. Snakes were released and recaptured over the next three months. Tail color manipulation did not significantly influence the proportion of snakes with palpable food. Also, the treatments did not affect growth in SVL or mass. These findings suggest that snakes with cryptic tails did not experience reduced prey capture. The presence of conspicuous tail coloration in this species may be explained by phylogenic inertia, selective benefits of small magnitude, or by habitat-related differences in prey composition or abundance.