1. Joshua J. Sweet, and Amanda M. Roise Department of Biology and Amphibian Growth Project, Minot State University An Analysis of Sex Differences in Cloacal and Skin pH in Ambystoma mexicanum (axolotl) Salamanders Acknowledgments. This project was supported by NIH Grant Number P20RR-016741 from the INBRE program of NCRR. Dr. Christopher Beachy showed us how to determine sex by dissection and provided statistical expertise. D.M. Sever from the Department of Biological Sciences, Southeastern Louisiana University provided advice on experimental design. Definitive salamander sex assignment is typically a destructive process involving dissection. Non-destructive methods exist, but such methods are often qualitative, inconclusive, and require expertise (Ollivier and Welsh, 2003). This study was initiated in search of a simple, quantitative, non-destructive method of determining salamander sex to enhance biomonitoring of declining species. We tested the hypothesis that sexes differ in cloacal pH in salamander. pH differs between male and female reproductive anatomy in other tetrapods. The human, bovine, and rat vaginal pH levels are <5, 7.0, and 4.5 respectively (Bernardis et al., 1998; Schilling and Zust, 1968; Suarez and Pacey, 2006). This differs significantly from the male reproductive tracts which are 6.9-7.4, 5.8, and 6.9 respectively (Carr et al., 1985; Suarez and Pacey, 2006). Our null hypothesis was that no such differences would be observed between salamander sexes. INTRODUCTION METHODS AND PROTOCOL REFERENCES Bernardis, F.D., F.A. Mühlschlegel, A. Cassone, W.A. Fonzi. 1998. The pH of the Host Niche Controls Gene Expression in and Virulence of Candida albicans. Infection and Immunity 66:3317-3325. Carr, D.W., M.C. Usselman, T.S. Acott. 1985. Effects of pH, Lactate, and Viscoelastic Drag on Sperm Motility: A Species Comparison. Biology of Reproduction 33:588- 595. Levine, N., H. Kelly. 1978. Measurement of pH in the Rat Epididymis In Vitro. Journal of Reproduction and Fertility 52:333-335. Ollivier, L., H.H. Welsh Jr. 2003. Determining Sex and Life Stage of Del Norte Salamanders From External Cues. Northwestern Naturalist 84:129-134. Schilling, E., J. Zust. 1968. Diagnosis of Oestrus and Ovulation in Cows by pH-Measurements Intra Vaginam and by Apparent Viscosity of Vaginal Mucus. Journal of Reproduction and Fertility 15:307-311. Suarez, S.S., A.A. Pacey. 2006. Sperm Transport in the Female Reproductive Tract. Human Reproduction Update 12:23-37. RESULTS Grow salamanders in captivity and maintain in coolers at 20°C. Feed salamanders with shrimp as tadpoles and blood worms as adults feeding ad libitum. Begin pH testing when salamanders are at or near maturity but not sexually active. The water pH was closest to neutral with pH 6.86 as the lowest and 6.99 as the highest mean value. The skin pH was furthest from neutral with 6.15 as the lowest and 6.25 as the highest mean value. The cloacal means exhibited a pH just above skin pH at ~ 6.31 for both sexes. All category means exhibited a pH of less than 7.0. Remove one salamander from its container and obtain cloacal, skin and water pH with 5 different ranges of pH paper: 1 – 12, 4.8 – 6.7, 5.2 – 6.6, 5.6 – 6.8, 6.0 – 8.0. Replace salamander and repeat process for 31 remaining specimens. Salamanders were sexed definitively by dissection. Females were identified by the presence of ova and oviduct. Males were identified by the presence of testis lobe and fat body. ova oviduct ♀ fat body Testis lobe ♂ 1) 2) 3) The least mean pH difference between sexes was exhibited in the cloaca which differed by only ~ 0.001. The greatest difference was present in the water pH levels which differed by ~ 0.126. Mean skin pH between males and females differed by ~ 0.105. Analysis of Means Conclusions Cloacal pH exhibited the least mean difference. This is likely due to similarity of function of the cloaca between sexes. In both sexes the cloaca is used for reproduction as well as excretion of nitrogenous waste and fecal matter. These salamanders were not sexually active. It is plausible that cloacal pH tests produced measurement relevant to only the currently active processes of the cloaca (excrement) which would be the same between sexes. T-tests indicate no significant difference between female and male pH. There was greater pH variation within a sex than between sexes. T-testing within sex revealed significant difference between cloacal and skin pH of female salamanders only. This suggests a possible means of identifying female salamanders by pH and discriminating between sex. The male sample size, however, was 32% smaller than the female sample size. This difference in sample size could influence the T-score. Reducing the female sample size by 33%, however, still returns significant difference (T-stat = 1.86; cut-off = 1.78). In future analyses, sample sizes should be increased. In regards to the raw data, some of our expectations were met. We expected the water pH to be slightly acidic. This is due to under water respiration. As the salamanders release CO2 into the water it is quickly converted to H2CO3 which dissociates into H2 + CO3 and lowers the water pH. The skin should, therefore, be expected to show the greatest acidity as the skin in general (the primary respiratory surface) is where this process would be most active. This is demonstrated by our findings. These results, which seem consistent with what is observed in nature, warrant continued analysis of pH differences between and within sexes. Future analyses should be broadened to include larvae, metamorphosed but immature, and sexually active individuals along with the original categories. 1.731.77 T-Distribution Our total sample size was 32 salamanders. After sexing definitively by dissection, the female total was 19. The male total was 13. Our significance criterion was α = 0.05. The graphs below show T-scores (bars) compared to their significance cut-offs (lines). These T-stats (left) were based on one population: female or male. The Female T-score (2.20) exceeds its significance cut-off (1.73). Male T- score (0.81) does not exceed its cut- off (1.78). We conclude that the pH difference between cloaca and skin was significant in females only. These T-stats (right) were based on two populations: female and male. None of the categories exceeded the cut-off of 1.70 (cloaca = 0.01; skin = 1.65; water = 1.59). We conclude that the pH of cloacae, skin and water were not significantly different between male and female salamanders. 1.73 1.78 1.70