The Relationship Between Reproductive Status and Innate Immunity in the Dusky Pigmy Rattlesnake (Sistrurus miliarius barbouri)

The relationship between reproductive status and innate immunity in the dusky pigmy rattlesnake (Sistrurus miliarius barbouri). John Massey, Stetson University Abstract: Energetic tradeoffs often underlie the expression of fitness-related life history traits. It is useful to to understand where energy is being reallocated to, particularly as it pertains allocation away from functions that promote survival (e.g. immune function). Knowledge of innate immune ability can help in the management and prediction of opportunistic infections that arise as a result of a compromised immune system. Specifically, the high energetic costs of pregnancy are thought to contribute to immune function becoming compromised. Pit vipers are particularly troubled by the egregious effects of snake fungal disease. We studied free-ranging dusky pigmy rattlesnakes, Sistrus miliarius barbouri, a species of snake afflicted with an opportunistic fungal disease. Sistrus miliarius barbouri breeds biennially and apportions energy for reproductive purposes during the time in which she is not producing offspring. We took blood samples from individuals found in the field and subsequently conducted a bacteria killing assay, a method of measuring innate immune function. We tested the hypothesis that the energetic tradeoff between reproduction and immune function would result in allocation away from innate immune function in vitellogenic and pregnant females. Introduction: Energetic tradeoffs that result in the sacrifice of certain life-history characteristics for the reinforcement of others remain a point of great contention among biologists (Graham et al., 2011). Previous studies on life history tradeoffs determined that phenotypic variation is determined by whether or not the adaptive trait is significant in prolonging an organism’s fitness (Niewiarowski 2001; Roff 1992; Stearns 1992; Charnov 1993). Phenotypic variation occurs within populations; individuals possessing traits that are favored by natural selection are more likely to survive and produce viable offspring (Darwin 1859; Williams 1966). Given the large amount of energy that must be allocated into reproductive processes in certain species, in energy limited systems energy expended on reproduction may be taken from other fitness-related processes such as metabolism, immune function or activity (Harshman and Zera 2007; Roosenberg and Dunham, 1997; French and Moore, 2008; Gustafsson et al., 1994; Nordling et al., 1998; Williams, 1966). In other species of reptiles, the inherent energetic tradeoff that occurs is mediated by hormones and seasonal environmental cues that influence reproductive strategies (Harshman and Zera 2007; Lind et al., 2010; Martin et al., 2008). An organism's resources must be rationed in the most advantageous way possible to ensure survival to reproductive age. Furthermore, the energetic costs of reproduction and its limiting effect on other metabolically-taxing processes is a phenomenon that has become of particular interest to biologists. Significant energy investments during the egg-yolking process of vitellogenesis (Van Dyke and Beaupre 2011) and pregnancy, as well as the subsequent post-parturition energy deficit, can often be deleterious to an organism’s ability to produce viable offspring (Demas et al., 2011). Ultimately, allocation strategies can be considered as a way to proportion available resources to the most essential processes for survival and production of viable offspring, thereby optimizing the lifetime fitness of an organism. There is an energetic tradeoff between reproduction and immune function (Demas et al., 2011). Vulnerability to pathogens is associated with different stages of reproduction as evidenced in studies of several different organisms. Animals such as snakes, lizards, mollusks and birds exhibit immunosuppression during events of the reproductive cycle (Graham et al., 2011; Sparkman and Palacios, 2009; French and Moore, 2008; Angiletta and Sears, 2000; Cox and Calsbeek, 2010; Petes et al., 2008; Wright et al., 1998; Norris and Evans, 2000). Decreased immune function can lead to a heightened risk of infection and disease (Nelson et al., 2002) while heightened immunity may also come at a high energetic cost to the organism (Lochmiller and Deerenberg, 2000). In Tachycineta bicolor, a species of tree swallow, maternal care of enlarged broods comes at a significant energetic cost to immunocompetence (Ardia 2005). In yet another species of sparrow, Passer domesticus, females caring for broods were found to be immunodeficient when required to feed and care for a considerably oversized brood (Bonneaud et al., 2003). We examined the relationship between immune function and the reproductive cycle of the dusky pigmy rattlesnake, Sistrurus miliarus barbouri. The reproductive behavior of S. m. barbouri in Florida is similar to most other temperate viperids. Sistrus miliarius barbouri is a viviparous lecithotroph and therefore yolks eggs but ultimately gives birth to live young (Rowe et al., 2002; Farrell et al., 1995). One of the reproductive characteristics of viperid species such as S. m. barbouri is that females may only reproduce every other year, with non-pregnant females taking the off-year to conserve energy for the next reproductive cycle (Rowe et al., 2002; Farrell et al., 1995). Since S. m. barbouri will often reproduce biennially, in most cases the innate immune function of vitellogenic, pregnant and post-parturition females can be directly compared to that of non-reproducing females within the same season (Rowe et al., 2002). This pattern of reproduction allows biologists to compare immune function between different reproductive stages and controlling for potential effects of season or year. Immunocompromised snakes are thought to be more susceptible to infection by snake fungal disease (Cheatwood et al., 2003), an affliction that is causing great concern in the herpetological world (Sutherland et al., 2014). Snake fungal disease is becoming a growing problem, as it has been observed in nine states in the continental United States and in seven different snake species as of 2014 (Sutherland et al., 2014). It is theorized that snake fungal disease aided in the disappearance of a population of Crotalus horridus in New Hampshire (Clark et al., 2011). Comparable to other North American pit vipers, S. m. barbouri is affected by snake fungal disease in several debilitating ways. The infection caused by Ohidiomyces ophiidiocola in S. m. barbouri has never been observed in any free-ranging snakes but was usually observed with other diseases when present in other reptiles (Cheatwood et al., 2003). Fungal pigmy rattlesnakes encountered in the field are afflicted with open lesions and field specimens taken for observation have been found to contain granulomas throughout their vital organs (Cheatwood et al., 2003). By using S. m. barbouri as our study organism, we can discover patterns of resource allocation that can potentially be applied to other organisms. Several previous studies examined the relationship between reproductive capacity and immune function in a variety of lizard species including Anolis sagrei (Reedy et al., 2015) and Zootoca vivipara (Bleu et al., 2011). Yet, very little is known about the innate immune function of field-active species of snakes and its relation to reproductive state. Studies of captive A. piscivorus have been conducted with reproductive individuals observed to be immunodeficient compared to their non-reproductive counterparts (Graham et al., 2008). Therefore, the intent of this study is to establish a foundational working knowledge of the energetic tradeoffs between life history components, specifically innate immune function and reproduction, in S. m. barbouri. We hypothesize that allocation to immune function will be lowest when the energetic demands of reproduction are highest. We therefore predict that vitellogenic and gravid females will exhibit compromised immune function when compared to non-reproductive individuals captured in the field. Methods: S. m. barbouri occupies a variety of habitats with the highest population density observed near freshwater marshes (May et al., 1996; Farrell et al., 1995). We sampled blood from 32 non-reproductive, 16 vitellogenic, 33 pregnant and 17 post-parturient S. m. barbouri from January 2015 to December 2015 at Lake Woodruff Wildlife Refuge in Volusia County, Florida. Of the 33 pregnant snakes captured in the field, 15 were placed in cages and observed for maternal care behavior (unpublished data; Lind et al., 2016). We captured each individual with the use of tongs; the snake’s head was gently guided into a clear plastic tube so that we could extract a blood sample from the caudal vein using a heparinized 27 gauge needle. We placed the samples in a 1.5 ml micro-centrifuge tube and stored on ice until returning to the lab, where we centrifuged at 1300 rpms for ten minutes to separate red blood cells from plasma. We micropipetted 30 mL plasma into micro-centrifuge tubes and stored at -80℃ until immunoassay. The sample collection guidelines used in this study have been used frequently in other endocrinological studies (Schuett et al., 2005; Taylor and Schuett, 2004; Graham et al., 2008; Graham et al., 2011). We performed a bacteria killing assay (BKA) to effectively measure the ability of the complement protein present in each sample to lyse the membrane of an introduced pathogenic bacteria. Bactericidal ability is a measure of the percentage of bacteria lysed compared to controls; the higher the bacterial colony count, the more immunosuppressed an individual is (Graham et al., 2011). We added 97.0 microliters of GIBCO CO2 independent culture media and 3.0 microliters of snake plasma to labeled tubes. We suspended a pellet of lyophilized Escheria coli ACCT 837 (Microbiologies, St. Cloud, MN) in 40 mL of phosphate-buffered saline. Twenty microliters of suspension was diluted 1:64 to ensure an average of 75 CFUs per 20 microliters. The tubes were vortexed and then incubated at 30℃ for thirty minutes. We plated 50 microliters of sample in duplicate on agar growth media using glass plating beads. The plates were incubated at 37℃ for approximately 12-16 hours. We counted and averaged each duplicate for comparison to control plates. The BKA was conducted by comparing the number of bacterial colonies resulting from control solutions versus those solutions containing plasma. Mean BKA ability of the fixed factor varied at the five reproductive levels (non-reproductive, vitellogenic, pregnant in field, pregnant in cage, post-parturition) was analyzed using a Kruskal-Wallis non-parametric (one way) test (JMP 9.1, SAS 2010). In most instances, an ANOVA would be conducted but the error distribution violated the assumption of normality. A post-hoc pairwise comparison was subsequently conducted (Dunn’s analysis). These measures were repeated for determining whether or not reproductive status was in any way influenced by mean fungal score. Individuals captured were ranked based on appearance of fungal status with non-fungal snakes receiving a rank of zero and highly fungal snakes receiving a three, with 0.5 incremental increases in between each level of infection. Results: Figure 1. Mean BKA score for non-reproductive (NR; n = 32), post-parturition (POST; n = 17), pregnant in cage (PRE1; n = 18), pregnant in field (PRE2; n = 15) and vitellogenic (VIT; n = 16) individuals (p = 0.002) fitted with standard error bars. Letters over each category are indicative of post-hoc analysis; bars that share the same letter are not significantly different from one another. Figure 2. Mean fungal score for non-reproductive, vitellogenic and pregnant individuals fitted with standard error bars. Discussion: Pregnant (PRE1) and vitellogenic (VIT) females sampled in the field were not significantly immunocompromised compared to non-reproductive (NR) females (Figure 1). These data suggest that our initial hypothesis that pregnant and vitellogenic females would be immunocompromised compared to non-reproductive individuals was not supported. This suggestion is supported by the observation that pregnant females never scored higher than a one when ranked for proliferation of snake fungal disease; they also scored significantly lower when compared to non-reproductive females (Figure 2). While it was made evident by Graham et al., (2011) that there is a reduction in complement protein function during pregnancy in the cottonmouth, A. piscivorus, our results do not support the hypothesis that allocation to innate immune function is lower during pregnancy or vitellogenensis in field-active S. m. barbouri. The reduction in immune function could therefore be contributed to the stresses involved in the movement and handling of A. piscivorus. More research is required to determine whether the same distinction exists in S. m. barbouri, and whether or not this “cage effect” can be quantified is the subject of future research. French and Moore (2008) discussed the tradeoff between reproduction and immune function in a species of tree lizard, Urosaurus ornatus, and determined that individuals were immunocompromised during their most resource-dependent reproductive state. Interestingly, French and Moore (2008) also discovered that there was a difference between immune function in field and lab specimens, and that field female tree lizards were more immunocompromised during vitellogenesis when compared to the other reproductive states. However, this variation did not exist between reproductive stages of tree lizards kept in controlled environments. The differences in immune function could always be attributed to resource availability, as lab-kept tree lizards would have a consistent and steady feed cycle compared to their field counterparts. It might be suggested that a similar effect exists in the S. m. barbouri of this study. There have been several landmark studies of bird immune function and its pertinence in life history tradeoffs (Norris and Evans, 2000). Ardia (2005) established that life history tradeoffs between immune function and reproduction exist in Tachycineta bicolor, a species of tree swallow. An observed characteristic in T. bicolor is that a larger brood size is associated with immunocompromised mothers; more energy is going into maternal care behavior than that of immune system maintenance. This is a stark contrast to what we observed in our study. Since vitellogenic and pregnant field individuals are not immunocompromised then it might be suggested that there is no decrease in susceptibility to disease and no reduction in fitness. In optimizing fitness, snakes may be reluctant to allocate away from immune function. However, given what is known about life history tradeoffs, litter size in S. m. barbouri could indeed be affected by the mother's immune status. This might come at a cost in terms of allocation towards development of offspring. We did not measure this allocation, but we must acknowledge that our data do not suggest that no tradeoff exists. Our study suggests that there is a significant difference in immune function between vitellogenesis and gravid females and their non-reproductive counterparts. Field-active pigmy rattlesnakes do not seem to be immunocompromised during the reproductive stages of vitellogenesis or pregnancy when compared to non-reproductive individuals. At the present time, our findings suggest a life-history tradeoff between innate immune function and reproduction in S. m. barbouri. However, it is difficult to make this assertion because we do not know why caged females are immunocompromised. We are unsure why they are immunocompromised; it could either be because they are in cages or merely because they are in the late stages of reproduction, an energetically-taxing procedure. There is most certainly a need for future work to complement the assertions made during the course of this study, particularly given the fact that this was indeed the first experiment of its kind concerning S. m. barbouri. Future studies might look specifically at highly fungal snakes that were immunocompromised and determine why they were reproductive, as evidenced by PRE2 and POST (Figure 1). Further studies are needed to concretize this assertion. 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