N27 2019 Southeastern Naturalist Notes Vol. 18, No. 3 J.Y. Lamb Skin Sloughing and Sperm Cap Loss during Courtship in Dusky Salamanders (Genus Desmognathus) Jennifer Y. Lamb* Abstract - Many species of amphibians and reptiles shed and sometimes consume all or part of the slough (dermatophagy). However, detailed accounts of these behaviors are lacking. The potential relationship between sloughing and social behavior in amphibians is largely unexplored. Herein, I describe 4 observations of shedding and 1 of dermatophagy during courtship for 2 species of lungless salamanders (Family Plethodontidae): Desmognathus valentinei (Valentine’s Southern Dusky Salamander) and Desmognathus conanti (Spotted Dusky Salamander). Two of the salamanders were recently inseminated females and each lost a sperm cap during shedding. I explore multiple hypotheses for the potential relationship between courtship and shedding in lungless salamanders, including the possibility that shedding serves as a means of cryptic female choice. Descriptions of skin shedding exist for at least 7 families of salamanders and 13 families of frogs and toads, but shedding likely occurs across other amphibian taxa (Larsen 1976, Weldon et al. 1993). The specific behaviors that are used to remove the detached outer layer of the epidermis (i.e., stratum corneum) are well documented for several species, including Plethodon cinereus (Green) (Eastern Red-backed Salamander; Bartlett 1952), Amphiuma tridactylum (Cuvier) (Three-toed Amphiuma; Fontenot and Pojman 2016), and Bufo bufo (L.) (Common Toad; Larsen 1976), as are instances of dermatophagy (i.e., consuming part or all of the sloughed skin; Fontenot and Pojman 2016, Weldon et al. 1993). Hormones control the process of shedding (for a review see Larsen 1976), and the frequency of shedding differs between and within species, as well as with environmental conditions such as temperature, humidity, and or photoperiod (Cramp et al. 2014, Meyer et al. 2012). There are several potential explanations for why amphibians shed and sometimes eat their skin. Amphibian skin may be more vulnerable to abrasion and damage than that of other tetrapods because it lacks a thick layer of keratin and may require more frequent renewal in order to function optimally (Wells 2010). Shedding may also be a means of responding to or defending against infection by epidermal pathogens (e.g., certain prokaryotes and fungi; Cramp et al. 2014, Meyer et al. 2012, Ohmer et al. 2017). Subsequent instances of dermatophagy may be a way to reclaim nutrients or energy in the shed skin, eradicate parasites or pathogens from the individual’s immediate surroundings, or avoid detection by predators through chemical cues (Mitchell et al. 2006, Weldon et al. 1993). Certain social interactions, such as courtship and mating, bring individual amphibians into direct contact, which can result in damage to the skin (e.g., through abrasion; Houck and Arnold 2003) or in colonization of the surface of the skin by foreign microbes (Rowley and Alford 2007). Despite increased risk to the skin during these kinds of social interactions, the potential link between shedding and social behavior in amphibians is relatively unexplored. There are only a few detailed observations of shedding during prolonged social encounters (Larsen 1976). In multiple species of tongueless frogs (Family Pipidae), males slough and consume their skin during periods of high sexual activity (Larsen 1976). In Hymenochirus boettgeri (Tornier) (Zaire Dwarf Clawed Frog), males shed just prior to, *Department of Biology, St. Cloud State University, 720 4th Avenue South, St. Cloud, MN 56301- 4498; jylamb@stcloudstate.edu. Manuscript Editor: Karen Powers Notes of the Southeastern Naturalist, Issue 18/3, 2019 2019 Southeastern Naturalist Notes Vol. 18, No. 3 N28 J.Y. Lamb during, or immediately after mating (Rabb and Rabb 1960, 1963). There is also at least 1 other published example of these behaviors occurring together in the family of true frogs (Family Ranidae)—Lithobates temporaria (L.) (Common Frog; Fischer-Sigwart 1897). Descriptions of shedding during courtship trials involving caudates exist for 2 species of lungless salamanders (Family Plethodontidae): Plethodon montanus (Highton and Peabody) (Northern Gray-Cheeked Salamander; Organ 1958) and Desmognathus fuscus (Rafinesque) (Northern Dusky Salamander; Wilder 1923). Here, I describe 4 instances of shedding, including 1 that involved dermatophagy, during the courtship of 2 species of plethodontids, Desmognathus conanti (sensu lato) (Rossman) (Spotted Dusky Salamander) and D. valentinei (Means et al.) (Valentine’s Southern Dusky Salamander). I observed these behaviors during 2 courtship studies: one testing for sexual isolation between the Spotted Dusky Salamander and Valentine’s Southern Dusky Salamander (Lamb 2017) and an unpublished study examining sexual isolation between different mitochondrial lineages of the Spotted Dusky Salamander (Lamb 2016). In each study, salamanders remained in environmental chambers prior to and during courtship encounters, as described in Lamb (2017). I observed the behavior of salamanders during the nocturnal portion of their daily cycle via 30-sec time-lapse photos taken with GoPro™ HERO3 cameras. In each of the 4 observations, only female salamanders shed, and in 2 of the 4 instances, females lost a sperm cap from their cloaca as a consequence of skin sloughing. I first observed shedding for both species of dusky salamanders in the summer of 2015 and among individuals that were a part of the Lamb (2017) study. On 23 April 2015, timelapse photos captured a female Spotted Dusky Salamander shedding. This female, collected from the Pearl River drainage in Marion County, MS, was paired with a male Valentine’s Southern Dusky Salamander. In this case, the female turned her body in a small, clockwise circle, fully revolving once, to completely remove the shed skin. Neither the female nor the male consumed the slough. After the female moved away, the shed skin was visible on the substrate of the arena via the time-lapse footage (Supplementary Video 1; see Supplemental File 1, available online at http://www.eaglehill.us/SENAonline/suppl-files/s18-3-S2546- Lamb-s1, and for BioOne subscribers, at https://dx.doi.org/10.1656/S2546.s1). On 1 May 2015, a female Valentine’s Southern Dusky Salamander shed during a courtship encounter with a male Spotted Dusky Salamander (Pascagoula River drainage, Forrest County, MS). This female did not turn in a circle during the process. When the shed skin had gathered at the middle of the female’s tail, she reached her head back towards her tail and appeared to contact it with her rostrum. In this second observation, the shed skin was not visible on the substrate in later time-lapse photos, and the shed was not present in the courtship arena the following morning. The lack of a shed in the arena suggests that this female Valentine’s Southern Dusky Salamander likely consumed the slough. It was difficult to accurately determine how long it took each individual to complete the shedding process because the onset of shedding is not apparent from the time-lapse footage. However, only a few minutes passed between the point at which a roll of skin was visible on a female to the conclusion of shedding. In both of these courtship trials, the pair of heterospecific salamanders engaged in preliminary pheromone transfer behaviors (i.e., head rubbing and body contact), but courtship did not proceed beyond that point (Lamb 2017). On 2 other occasions, I observed shedding during courtship trials between Spotted Dusky salamanders that resulted in the loss of a sperm cap from the cloaca of a recently inseminated female. On 4 May 2015, time-lapse photos documented courtship between a female from the Red River in Rapides Parish, LA, and a male from the Pascagoula River in N29 2019 Southeastern Naturalist Notes Vol. 18, No. 3 J.Y. Lamb Simpson County, MS. After insemination at ca. 1219 h, the couple separated in the arena and did not engage in pheromone transfer or other courtship behaviors. At 0212 h, the female shed, and her sloughed skin was visible on the substrate in time-lapse photos. At 0227 h, the male began to court the female for a second time, exhibiting behaviors that included orientation, pursuit, and body contact as described in Lamb (2017). The female consistently moved away from the male until the end of the trial (Supplementary Video 2; see Supplemental File 2, available online at http://www.eaglehill.us/SENAonline/suppl-files/ s18-3-S2546-Lamb-s2, and for BioOne subscribers, at https://dx.doi.org/10.1656/S2546. s2). After the conclusion of the trial, I found and photographed the sloughed skin and sperm cap left behind by the female (Fig. 1). I also confirmed that no sperm cap was present in the female’s cloacal opening. On 16 May 2016, I filmed a male and female that were collected from the same site in the Tombigbee River Drainage in Winston County, MS. Spermatophore deposition and insemination occurred at ca. 2230 h. The female began shedding at ca. 0215 h but did not complete the process until after filming had ended for that trial. I did not observe the shed on the substrate of the arena in time-lapse photos, but did photograph the shed and attached sperm cap later that morning (Fig. 2). These descriptions add 2 species of amphibians to the list of those that shed their skin during or soon after social interactions. There are multiple hypotheses that could explain these observations. First, there may be no relationship between shedding and social interactions in lungless salamanders, and these instances represent coincidence. I only observed sloughed skins or individuals shedding their skin in 4 of the 62 courtship pairings across Figure 1. Shed skin and sperm cap (white) from a female Spotted Dusky Salamander from the Red River in Rapides Parish, LA. Photo taken with an Olympus E620 DSLR camera. PaperMate Comfort- Mate Ultra 1.0M pen is included for scale. 2019 Southeastern Naturalist Notes Vol. 18, No. 3 N30 J.Y. Lamb both studies. However, it is also possible that I have underestimated the frequency of shedding in courting pairs of salamanders. If dermatophagy occurred among the pairs that were not filmed, there would be no evidence to indicate that shedding took place. Several individuals were housed for more than 1 y as part of these courtship studies, and although I did not observe evidence of shedding in individual containers, it is likely to have occurred. Still, I cannot compare the frequency of molting between individuals that were actively courting and those which were not because I did not closely monitor salamanders when they were being housed individually. A competing hypothesis is that social interactions between lungless salamanders can trigger the process of shedding. Skin sloughing associated with courtship or reproduction may be a byproduct of increased activity of the pituitary gland during mating (Larsen 1976, Rabb and Rabb 1960). Shedding during courtship could also be a response to damage done to the surface of the skin during particular behaviors. Many plethodontid salamanders, including Dusky Salamanders, exhibit elaborate courtship sequences that involve several behaviors wherein the male uses specialized, elongate premaxillary teeth to scratch the dorsal surface of the skin of the female (Houck and Arnold 2003, Palmer et al. 2007). The amount of abrasion may be sufficient to trigger sloughing in some female salamanders. Alternatively, shedding may be triggered in response to the transmission of foreign microbes as a result of direct contact between individuals during courtship. Prolonged reproductive behaviors in some frogs are thought to contribute to an increase in disease transmission (Rowley and Alford 2007), and laboratory trials have indicated that microbial abundance on the surface of the skin is significantly lower after sloughing i n anurans (Cramp et al. 2014, Figure 2. Shed skin and sperm cap (white) from a female Spotted Dusky Salamander from Tombigbee River Drainage in Winston Cunty, Mississippi. Photo taken with an iPhone 7. N31 2019 Southeastern Naturalist Notes Vol. 18, No. 3 J.Y. Lamb Meyer et al. 2012). Further, the frequency of skin shedding in some species increases after exposure to pathogens like the chytrid fungus (Batrachochytrium dendrobatidis; Ohmer et al. 2017). My observations are not the first to note the loss of some or all of a sperm cap during shedding in plethodontids, but published descriptions primarily involve male salamanders. Organ (1958) noted that a spermatophore was dislodged from the cloaca of a male Northern Gray-Cheeked Salamander immediately after shedding. In the same study, Organ found a spermatophore adhered to a slough in a container housing both males and females, which they suggest likely belonged to a male as the spermatophore was “normally formed” and not a sperm cap. Organ and Lowenthal (1963) noted that spermatophores were lost during shedding among male Eastern Red-backed Salamanders and Plethodon yonahlossee (Dunn) (Yonahlossee Salamander), but they did not consider these males to be actively courting (Organ and Lowenthal 1963). In the only prior publication of sperm cap loss in females, Wilder (1923) noted that the cap from an inseminated Northern Dusky Salamander was “detached by adhering to the moult layer”. Each of the hypotheses discussed previously could also apply to Wilder’s (1923) and my observations of skin shedding and subsequent sperm cap loss in female Dusky Salamanders. However, another explanation may be that female lungless salamanders shed sperm caps as a method of cryptic female choice. Cryptic female choice (CFC) occurs when a female uses behavioral, morphological, or physiological mechanisms to affect a male’s reproductive success after insemination (Eberhard 1996, Firman et al. 2017). If these observations represent CFC, then it is a type of sperm ejection or rejection which, though recorded in some invertebrates and vertebrates with internal fertilization (Eberhard 1996), has not been documented in amphibians. My observations of shedding and sperm cap loss among female Dusky Salamanders are limited, but they do meet 2 of the 5 conditions for demonstrating CFC described by Eberhard (1996). First, females vary in their response to males. Conspecific males from 3 other courtship trials successfully inseminated the female Spotted Dusky Salamander from the Tombigbee River Drainage in Mississippi, and she did not subsequently shed any of their sperm caps. I used the female from the Red River in Louisiana in the single courtship trial described here and so I cannot comment about differences in her responses to other males. Second, this variation in female response likely resulted in differences in reproductive success between conspecific males. Sperm caps can remain visible in the cloacas of female lungless salamanders for more than 14 h after insemination (Verrell 1991).The loss of a sperm cap within a period of 5 h, as I observed here, may have prevented some sperm from reaching sperm-storage areas within the cloacas of these females because sperm caps may require additional time to release their gametes. More data and descriptive accounts are needed in order to determine whether there is a definitive relationship between social interaction and shedding in lungless salamanders. Fortunately, those data may not be difficult to collect for this group. Plethodontids are easily maintained in large numbers in captivity (Arnold et al. 1993) and researchers could readily observe shedding through time-lapse photography, or potentially during regular inspections of enclosures. If dermatophagy is common, however, then time-lapse photography may be the most effective method of studying the potential correlation between these behaviors. If there is no relationship between courtship and shedding in lungless salamanders, then Wilder’s (1923) and my observations of sperm cap loss in female Dusky Salamanders indicate that the reproductive investment of a male can be haphazardly discarded by a female. If this is the case, then selection may favor the evolution of other behaviors associated with 2019 Southeastern Naturalist Notes Vol. 18, No. 3 N32 J.Y. Lamb successful reproduction such as mate guarding (Deitloff et al. 2014) and multiple, successive matings between individuals. Acknowledgments. These data were collected as part of other studies funded by a National Science Foundation (NSF) Graduate Research Fellowship under Grant No. 0940712 and an NSF “Molecules to Muscles” GK-12 Fellowship Award No. 0947944 through the University of Southern Mississippi. I am grateful to the J. Schaefer Lab for use of their GoPro™ HERO3 cameras. I also thank anonymous reviewers and B. Morris for their editorial assistance, as well as S. Woodley for our discussions pertaining to this manuscript at the 2016 Special Highland Plethodontid Conference. The Mississippi Department of Wildlife, Fisheries, and Parks (Permits 0422131 and 0422141) provided scientific collection permits. 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