Northeastern Naturalist 50 P.J. Muellman, O. Da Cunha, and C.E. Montgomery 22001188 NORTHEASTERN NATURALIST V2o5l.( 12)5:,5 N0–o5. 51 Crotalus horridus (Timber Rattlesnake) Maternal Scent Trailing by Neonates Peter J. Muellman1, Océane Da Cunha1, and Chad E. Montgomery1,* Abstract - Intraspecific chemical communication among related and unrelated conspecifics may play an important role in social organization and kin recognition within snakes. We monitored the movements of 7 adult Crotalus horridus (Timber Rattlesnake) and 22 of their neonates from May 2008 to October 2010. Our objective was to determine if neonates follow their mothers to suitable den sites in North Central Missouri. Mothers tended to move away from the rookery between parturition and ingress, but neonates stayed in the vicinity of their release site for up to a week after the dispersal of their mother. Despite the loss of radiotransmitters, we were able to follow 6 neonates to ingress: 5 overwintered in the same den as their mother and 1 overwintered in a known den of a conspecific female. Our observations support the hypothesis that Timber Rattlesnake neonates follow their mother or, at the very least, follow conspecifics to suitable den sites in the ir first year. Introduction Parental care, any parental behavior that increases the chance of survival for offspring (Clutton-Brock 1991), is commonly observed in viviparous terrestrial snake species, particularly within the viperids (Green et al. 2002). Parental care in viviparous snakes includes thermoregulation to optimize development during gestation (Gardner-Santana and Beaupre 2009) and offspring attendance following parturition until the first neonatal shed (Graves and Duvall 1995), which may offer protection from predation (Green et al. 2002). Often, these behaviors result in parental investment, or parental care that results in some fitness cost to the parent (Clutton-Brock 1991). Parental attendance is found in 33 species in 14 genera of viperids (Green et al. 2002). Thirteen species of rattlesnakes show this behavior (Green et al. 2002), including Crotalus horridus L. (Timber Rattlesnake) in which individual females were found in association with their litters 7 to 10 days following parturition (Martin 1988, 1990). However, little is known regarding the importance of offspring attendance for imparting information from mother to offspring. In the temperate zones, snakes are at an increased risk of mortality due to unfavorable environmental conditions during winter, particularly if suitable hibernacula cannot be located. Therefore, newborn snakes at higher latitudes are at an increased risk of death during winter because they have no prior knowledge of the location of such hibernacula (Reinert and Zappalorti 1988). In order to locate suitable hibernacula, newborn snakes could search for hibernacula on their own or follow other snakes, including either conspecifics or non-conspecifics, to increase likelihood of 1 Department of Biology, Truman State University, Kirksville, MO, 63501. *Corresponding author - chadmont@truman.edu. Manuscript Editor: Thomas French Northeastern Naturalist Vol. 25, No. 1 P.J. Muellman, O. Da Cunha, and C.E. Montgomery 2018 51 survival (Hirth 1966). In order to increase survivorship of offspring, a female may intentionally provide a scent trail as a guide for her offspring, at the added risk of the trail being detected by potential predators. Such behavior would be considered a form of parental investment. Neonate Timber Rattlesnakes can distinguish between littermates and other conspecifics and will selectively associate with their own littermates in the laboratory when given the opportunity (Brown and MacLean 1983, Clark 2004). In addition, at least some neonate Timber Rattlesnakes will trail their mother to hibernacula during their first fall (Cobb et al. 2005). Some neonate Sistrurus catenatus (Rafinesque) (Eastern Massasauga) also exhibit coordinated movements along their mother’s path, suggesting conspecific pheromone trailing (Jellen and Kowalski 2007). In the laboratory, neonate Eastern Massasaugas will selectively follow the maternal scent trail rather than that of a random adult female conspecific when given the opportunity (Hileman et al. 2015). Conclusions drawn from these observations need to be made with caution due to small sample sizes or because the research was conducted in the laboratory rather than under field conditions. However, taken as a whole, these studies indicate that neonate rattlesnakes can distinguish between relatives and other conspecifics and may use this ability to locate a suitable hibernaculum to overwinter during the first year. Our objective was to further elucidate the post-dispersal interactions between neonate snakes and their mothers under natural field conditions and to determine if neonate Timber Rattlesnakes in North Central Missouri follow their mothers to locate suitable den sites. Material and Methods Study species and field site description The Timber Rattlesnake is a large-bodied member of the family Viperidae that ranges from New Hampshire west to Minnesota, south to northern Florida and southwest Texas. The total historic range encompasses 30 US states (Brown 1993). It is a viviparous species that utilizes communal hibernacula and rookeries (communal birthing sites) in parts of its range (Ernst 1992). Dens utilized are typically south-facing rocky hillsides, though den-site variation does occur depending on latitude (Ernst 1992). Topographic variables, such as slope, aspect, and elevation, are the most important factors defining suitable over-wintering habitat for Timber Rattlesnakes in Arkansas forest habitats (Browning et al. 2005). Rookeries are used by pregnant females to regulate the temperature of the developing offspring (Ernst 1992, Gardner-Santana and Beaupre 2009). We conducted telemetry in Missouri at the Premium Standard Farms (PSF) Scott–Colby facility and at Crowder State Park (CSP). PSF, on the boundary of Daviess and Grundy counties, is a commercial pork production operation with restricted access comprising 5 distinct habitats: open field, open rocky slopes, hardwood forest, riparian, and covered rocky slopes. Rookeries were located within an area of 2.3 ha of open rocky slope habitats characterized by loose boulders, rocks, exposed rock ledges, and scattered trees (Gleditsia triacanthos L. [Honey Locust] Northeastern Naturalist 52 P.J. Muellman, O. Da Cunha, and C.E. Montgomery 2018 Vol. 25, No. 1 and Platanus occidentalis L. [American Sycamore]). CSP, in Grundy County, consists of 774 ha of hardwood forest and open fields with intermittent streams. It is located ~7.5 km west from PSF. The study area within CSP consists of 13 ha of hardwood forest, open field, and abandoned farm infrastructure. Telemetry We opportunistically captured rattlesnakes at PSF from May 2008 to October 2010 and at CSP from May 2009 to October 2010. At each capture site, we recorded location, time, date, and habitat. All snakes were transported back to Truman State University for processing. In the lab, we processed each snake by measuring snout– vent length (SVL; ± 0.1 cm) and tail length (± 0.1 cm) using a squeeze box, recording mass (± 0.1 g) using an electronic balance, and determining sex by cloacal probing. We individually marked each with a PIT Tag and painted rattle segments based on the PIT tag sequence, when possible, to reduce handling in the field. A total of 7 gravid female rattlesnakes were radiotracked during the course of the study, with 2 radiotracked in 2008 in PSF, 1 in 2009 in PSF, and 4 in 2010 (2 in PSF, 2 in CSP). We implanted radiotransmitters (Holohil Systems Ltd, Carp, ON, Canada) following a modified version of Reinert and Cundall’s (1982) protocol. Transmitter mass was less than 2.9% of the smallest female rattlesnake mass. Radiotagged females were released at the site of capture within 3 days and radiotracked 2–3 times per week until we suspected they were close to parturition (August/September). We then captured the radiotagged snakes and brought them to Truman State University until parturition, which was less than 10 days in all cases. Females were housed individually at 25 °C, with a 12:12 light–dark cycle, and provided water ad libitum. After parturition, we processed all neonates in the same manner as described for adults. We selected a subset of neonates from each litter, totaling 22 neonates from 7 litters (Table 1), for transmitter attachment. Following completion of the first shed cycle, we attached the transmitters (Holohil Ltd BD-2HX, 1.65 g) externally on the dorsolateral surface of the body, at ~70% of the SVL, using cyanoacrylic glue (SuperGlue; Cobb et al. 2005). Each female, along with her litter, was released at the site of capture 48 hours after we attached the transmitters to the neonates. We radiotracked all females and neonates 2–3 times per week until transmitter loss or ingress into hibernacula (usually late October). At each relocation, we recorded date, time, location and any behavioral observations or associations between individuals. Results Of the 22 radios we attached to neonates, 16 (73%) fell off of the neonate prior to ingress, enabling us to follow 6 (27%) to ingress. Of those transmitters that did not stay attached, 13 fell off within 5 days of release and 3 were tracked for up to 3 weeks following release. Between release at site of capture and ingress, adult females moved away from the rookery and assumed ambush-foraging postures. Neonates did not move away from the vicinity of their release site for up to a week after the dispersal of their mother. In several cases, neonates remained closely Northeastern Naturalist Vol. 25, No. 1 P.J. Muellman, O. Da Cunha, and C.E. Montgomery 2018 53 associated with siblings, which may have been incidental. Over the course of the next few weeks, neonates, including the 6 tracked to ingress and the 3 partially tracked, made short movements and then began moving in the general direction of the den, roughly following the path of their mother. Locations of tracked neonates did not correspond exactly to previous locations of the mother, which is to be expected because we were not tracking continuously. However, the path of each neonate remained within 20 m of the path of their mother along the distance from release site to den. We tracked 6 neonate rattlesnakes from 3 different litters to ingress in 3 separate dens. Five neonates, from 2 different litters (2 from one litter and 3 from another litter), entered the same den as their respective mothers. The sixth neonate, from a third litter, entered the den of a known conspecific female who had also given birth that season. The den entrance of the conspecific female was within 5 m of the known den of the mother of that neonate. Discussion Females remained with neonates until ecdysis as evidenced by the location of some litters that had started shedding but were still with their mother. Maternal attendance of neonates following parturition has been documented in wild Timber Rattlesnakes (Martin 1990) and captive Timber Rattlesnakes (Brown et al. 1983, Klauber 1956) as well as in other rattlesnake species (Graves and Duvall 1995, Green et al. 2002). Associations between mother and offspring in snakes are usually attributed to maternal attendance, but it appears that Eastern Massasauga neonates play an active role in maintaining this association (Hileman 2015). This maternal attendance-based aggregation may be important in the development of kin discrimination in Agkistrodon piscivorus L. (Cottonmouth; Hoss 2013), and by associating with related or unrelated adult conspecifics, neonates may gain benefits in the form of reduced predation (Hileman et al. 2015). Neonates tended to stay clustered for some time following parturition and dispersal of the mother. Neonate Timber Rattlesnakes are able to discriminate kin from non-kin as evidenced by female Timber Rattlesnakes from the same litter associating with each other more closely than female neonates from a different litter (Clark 2004). Neonate clustering can offer individuals several benefits, including increased predator detection, increased water conservation, and increased thermal stability. Aggregations of Eastern Diamondback Rattlesnake neonates were more likely to flee than lone individuals, suggesting that clustering of neonates may reduce predation (Butler et al. 2005). Neonate aggregations of C. viridis (Rafinesque) (Prairie Rattlesnake) reduce cutaneous water loss by decreasing individual neonate surface area exposed to air (Graves and Duvall 1995). Core temperature of neonate aggregations of C. cerastes cerastes Hallowell (Mojave Desert Sidewinder) was relatively stable in a fluctuating thermal environment (Reis erer et al. 2008). After the neonates in this study dispersed from the site of parturition, they generally followed the path of their mother towards the den. Timber Rattlesnake neonates have been observed in the immediate vicinity (less than 2 m) of conspecifics in the Northeastern Naturalist 54 P.J. Muellman, O. Da Cunha, and C.E. Montgomery 2018 Vol. 25, No. 1 fall (Cobb 2005, Reinert and Zappalorti 1988) including their mother (Cobb 2005). However, it seems that not all Timber Rattlesnakes neonates trailed the mother, suggesting that this behavioral association may be weak and only exhibited by some individuals (Cobb et al. 2005). Eastern Massasauga neonates have also been shown to exhibit coordinated movements along their mothers’ paths (Jellen and Kowalski 2007) The majority (5 of 6) neonates we tracked to ingress ended up in the same den as their mother, and the only exception ended up in the same den as another known female. Our study was complicated by the loss of transmitters, which was similar to problems experienced by Jellen and Kowalski (2007) and Cobb and colleagues (2005). Despite facing common technical difficulties, our results provide additional evidence that neonate temperate pit vipers, specifically Timber Rattlensnakes, follow their mother and at the very least follow conspecifics to find suitable den sites. Whether the female purposefully leaves a scent trail for the neonates to follow or the neonates are simply following a latent trail is unknown but should be examined as a potential form of post-dispersal parental care and investment. Acknowledgements Our project was funded by Truman State University and by Chicago Herpetological Society. Research was conducted under permits from the Missouri Department of Conservation and the Missouri Department of Natural Resources to P.J. Muellman and C.E. 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