Southeastern Naturalist 521 J.D. Konvalina and S.E. Trauth 22001188 SOUTHEASTERN NATURALIST 1V7o(3l.) :1572,1 N–5o3. 03 Seasonal Variation of Testicular Tissue in Northern Rough Greensnakes, Opheodrys a. aestivus, from Alabama John D. Konvalina1,* and Stanley E. Trauth2 Abstract - Gamete production is a fundamental component of the reproductive cycle of an organism. Studies dedicated to the testicular cycle in mammals and birds vastly outnumbers those discussing the process in reptiles. To help increase the availability of such knowledge for reptiles, we histologically examined the testicular cycle of Opheodrys a. aestivus (Northern Rough Greensnake) from populations in Alabama. We measured seminiferous tubule diameter and seminiferous tubule epithelial height from 30 specimens. The individuals in our sample exhibited small seminiferous tubule diameters in spring followed by increases in summer. By October, the lumen was mostly empty of sperm because they had migrated to the vas deferens for winter storage. Seminiferous tubule epithelial height was significantly correlated with seminiferous tubule diameter. Using AIC model selection, we compared both additive and interactive models to determine if either seminiferous tubule diameter or season influenced seminiferous tubule epithelial height. We found that only seminiferous tubule diameter was a significant predictor of seminiferous tubule epithelial height. Like other temperate snakes, Northern Rough Greensnakes in Alabama have postnuptial spermatogenesis where sperm are produced in the summer following spring mating. Future studies of this species need to investigate the testicular cycle in other parts of its geographic distribution to see if this monthly pattern is consistent. Introduction The reproductive cycle of an organism can be divided into 4 stages: production of gametes, mating, oviposition, and birth. Gamete production is the most understudied and fundamental of these components. Books have been dedicated to the study of the testicular cycle in mammals and birds (Ewing et al. 1980, Russell et al. 1990), but only 3 reviews cover the process in reptiles (Gribbins 2011; Gribbins and Rheubert 2011, 2014). In some snakes, such as Thamnophis s. sirtalis L. (Eastern Gartersnake), sperm are stored over winter in the vas deferens (Clesson et al. 2002), whereas in others like Carphophis vermis (Kennicott) (Western Wormsnake), sperm are retained within the ductus deferens year-round (Aldridge and Metter 1973). In Zaocys dhumnades (Cantor) (Chinese Ratsnake), sperm are stored in the vas deferens and also the epididymis (Liang et al. 2011). Comparing testis volume to the total mass of the organism can be used to decipher seasonal allocation of resources between somatic growth and reproduction (Moshiri et al. 2014). 1Department of Biology, University of Central Florida, 4000 Central Florida Boulevard, Orlando, FL 32816. 2Department of Biological Sciences, Arkansas State University, PO Box 599, State University, AR 72467. *Corresponding author - jkonvalina@knights.ucf.edu. Manuscript Editor: John Placyk Southeastern Naturalist J.D. Konvalina and S.E. Trauth 2018 Vol. 17, No. 3 522 Changes in season trigger the different testicular cycle phases, with environmental temperatures playing a pivotal role in initiating spermatogenesis (Hawley and Aleksiuk 1976). We can divide the testicular cycle in squamates into 4 phases: maximum activity (all stages of spermatogenesis are active), regression (cells start to die), quiescence (cessation of spermatogenesis), and recrudescence (reinitiation of spermatogenesis) (Hernández-Gallegos et al. 2014). Seasonal differences in testicular stages are noted by measuring interstitial cell nuclear diameters, seminiferous tubule diameters, and epithelial heights. Differences in season have been found in colubrids (Goldberg and Parker 1975), elapids (Shine 1977), homalopsids (Jadhav and Padgaonkar 2011) and viperids (Gribbins et al. 2008). One example of seasonal differences is in the testicular cycle of Agkistrodon piscivorus (Lacépède) (Northern Cottonmouth). It begins in April with recrudescence, followed by peak sperm production in July and August, and ending with the testes regressing in the winter months (Johnson et al. 1982). Aldridge et al. (1990) also noted that in Opheodrys a. aestivus L. (Northern Rough Greensnake), spermatogenesis peaks in July. However, all their specimens were collected from Arkansas, so their data may not represent characteristics of the testicular cycle in other parts of the expansive range of this species. Rough Greensnakes occur widely in the eastern and southeastern United States, from southern New Jersey and Indiana south along the East Coast to Florida and west to central Texas, eastern Kansas and central Oklahoma (Trauth et al. 2004). We addressed this concern in our study by examining seasonal variation in the testicular cycle in Alabama populations of Northern Rough Greensnake. We hypothesized that both season and seminiferous tubule diameter would have a significant effect on seminiferous tubule epithelial height. Thus, we expected to find significant differences in seminiferous tubule epithelial height among seasons and among different seminiferous tubule diameters. We also hypothesized that Northern Rough Greensnakes from Alabama would exhibit post-nuptial spermatogenesis, corroborating what Aldridge et al. (1990) found in Arkansas. Methods We obtained 30 Northern Rough Greensnakes collected in multiple Alabama counties over a period of 18 years (1957–1975) from the Auburn University Museum (AUM) herpetological collection (Table 1). We dissected the testes from each specimen and dehydrated them in a graded series of increasing ethanol solutions (50–100%) and embedded them in paraffin following the methods of Presnell and Schreibman (1997). We serially sectioned the Paraffin-tissue blocks into ribbons 10 μm in thickness using a rotary microtome and affixed the ribbons, as they were floating on a 2% formalin solution, to microscope slides by using Haupt’s adhesive. We stained slides with hematoxylin and eosin for general cytology. We used a Nikon Eclipse 600 epi-fluorescent light microscope with a Nikon DXM 1200C digital camera (Nikon Instruments Inc., Melville, NY, USA) for photomicroscopy. For each specimen, we randomly selected 20 seminiferous tubules and measured seminiferous tubule diameter and seminiferous tubule epithelial height. Seminiferous tubule diameter was defined as the lumen plus the epithelium at the widest point Southeastern Naturalist 523 J.D. Konvalina and S.E. Trauth 2018 Vol. 17, No. 3 of the tubule. We averaged the 20 measurements to give each individual an average seminiferous tubule diameter and average seminiferous tubule epithelial height. We used a mixed model to test for significant differences in seminiferous tubule diameter and seminiferous tubule epithelial height among samples from different months. Then, we used a post hoc test (extension of Tukey for mixed models) to identify the significant differences among months. Due to small monthly sample sizes, we collapsed the months into 3 seasons: spring (April and May, n = 18), summer (June, n = 5) and fall (September and October, n = 7). ). Each season was normally distributed, but variances were not equal among seasons. Therefore, we performed a non-parametric Kruskal-Wallis test followed by a Pairwise Wilcoxon rank sum test to identify differences in seminiferous tubule diameter and seminiferous tubule epithelial height among seasons. Next, we used a Pearson correlation test to look for a correlation between seminiferous tubule diameter and seminiferous tubule epithelial height. Table 1. Opheodrys a. aestivus (Northern Rough Greensnake) specimens from Alabama used for light microscopy. All sections taken were of the testis and prepared by Stanley E. Trauth. Month Date Alabama county April 9-Apr-59 Lee April 10-Apr-63 Russell April 29-Apr-66 Calhoun April 24-Apr-67 Lee April 13-Apr-68 Dale April 18-Apr-68 Butler April 04-Apr-70 Lee April 13-Apr-73 Baldwin May 19-May-43 Lee May 4-May-57 Walker May May-61 Lee May May-61 Lee May 12-May-68 Cherokee May 19-May-68 Lee May 4-May-69 Marshall May 19-May-69 Barbour May 10-May-75 Dallas May 12-May-75 Randolph June 25-Jun-49 Lee June 22-Jun-57 Cleburne June 22-Jun-57 Cleburne June 12-Jun-65 Mobile June 6-Jun-66 Morgan September 30-Sep-67 Lauderdale September 2-Sep-70 Covington September 27-Sep-70 Elmore October 12-Oct-61 Shelby October 1-Oct-67 Barbour October 12-Oct-67 Russell October 8-Oct-68 Macon Southeastern Naturalist J.D. Konvalina and S.E. Trauth 2018 Vol. 17, No. 3 524 Finally, we tested 4 linear models to determine what factors influenced seminiferous tubule epithelial height. The first model was seminiferous tubule epithelial height as function of seminiferous tubule diameter, the second was seminiferous tubule epithelial height as function of season, the third was seminiferous tubule epithelial height as a function of seminiferous tubule diameter plus season, and the final model was seminiferous tubule epithelial height as a function of seminiferous tubule diameter plus season plus an interaction between those 2 terms. We used AIC to determine which model best fit the data. We performed all statistical analyses at a 5% significance level using the statistical programming software R (R Development Core Team 2014). Results There were significant differences in seminiferous tubule diameter among months (χ2 = 289.64, df = 4, P < 0.001). Seminiferous tubule diameter was significantly different between April and June (z = 3.15, P = 0.014), April and September (z = 3.97, P less than 0.001), May and June (z = -4.87, P less than 0.001), May and September (z = 5.41, P less than 0.001), and May and October (z = 4.06, P less than 0.001). All other month-tomonth comparisons were not significant (Fig. 1). There were significant differences in seminiferous tubule epithelial height among months (χ2 = 118.63, df = 4, P < 0.001). Epithelial height was significantly Figure 1. (Left) Average seminiferous tubule diameter by month for Opheodrys a. aestivus (Northern Rough Greensnake) from Alabama. The letters above the boxes represent significance levels; if 2 months share at least one letter, they are not significantly different, and if 2 months do not share any letters, they are significantly different. (Right) Average seminiferous tubule epithelial height by month for Northern Rough Greensnakes from Alabama. The symbol above the box for May represents that it is significantly different from June. The black horizontal line within each box represents the median value. The bottom and top of each box represents the 1st and 3rd quartiles, respectively. The “whiskers” above and below the box show the maximum and minimum values. The numbers below the boxes represent sample size (i.e., number of snakes per month). Southeastern Naturalist 525 J.D. Konvalina and S.E. Trauth 2018 Vol. 17, No. 3 different between May and June (z = -2.84, P = 0.04). All other month-to-month comparisons were not significant (Fig. 1). Mean seminiferous tubule diameter was significantly different among seasons (Kruskal-Wallis χ2 = 13.3, df = 2, P = 0.001). Both fall (P = 0.003) and summer (P = 0.01) had significantly greater seminiferous tubule diameters than spring (Table 2, Fig. 2). No significant differences in epithelial height were found among seasons (Kruskal-Wallis χ2 =5.23, df = 2, P = 0.07; Table 2, Fig. 2). There was a positive correlation between seminiferous tubule epithelial height and seminiferous tubule diameter (r = 0.64, P < 0.001; Fig. 3). Model 1 (seminiferous tubule epithelial height ~ seminiferous tubule diameter) was the best model according to AIC (Table 3). The summary (Table 4) and plot (Fig. 4) of model 1 show that seminiferous tubule diameter is a significant factor in predicting seminiferous tubule epithelial height. Season was not included in this model and, therefore, was not a significant factor in predicting seminiferous tubule epithelial height. Figure 2. Average seminiferous tubule diameter (left) and average seminiferous tubule epithelial height (right) by season for Opheodrys a. aestivus (Northern Rough Greensnake) from Alabama. The asterix above the box for spring tubule diameter represents that it is significantly different from summer and fall. Open circles above the boxplots are outliers. Boxplot description is the same as that in Figure 1. Table 2. Mean seminiferous tubule diameter and seminiferous tubule epithelial height by season from Opheodrys a. aestivus (Northern Rough Greensnake) testes from Alabama. 95% confidence intervals are listed in brackets beside each mean. Season n Mean tubule diameter Mean epithelial height Spring 18 114.47 μm [102.58, 126.36] 28.95 μm [25.25, 32.65] Summer 5 155.93 μm [137.29, 174.57] 38.24 μm [26.34, 50.13] Fall 7 161.67 μm [139.68, 183.67] 34.72 μm [29.41, 40.04] Southeastern Naturalist J.D. Konvalina and S.E. Trauth 2018 Vol. 17, No. 3 526 Figure 3. Correlation between seminiferous tubule epithelial height and seminiferous tubule diameter for Opheodrys a. aestivus (Northern Rough Greensnake) from Alabama. The correlation coefficient is 0.64 with a P value of < 0.001. Table 3. AIC table comparing linear models examining the factors contributing to seminiferous tubule epithelial height in Opheodrys a. aestivus (Northern Rough Greensnake) from Alabama.(EH = epithelial height, TD = tubule diameter, and S = season). Models ΔAICc df Weight EH ~ TD 0.0 3 0.89 EH ~ TD + S 4.2 5 0.11 EH ~ TD * S 10.3 7 0.01 EH ~ S 13.0 4 0.001 Southeastern Naturalist 527 J.D. Konvalina and S.E. Trauth 2018 Vol. 17, No. 3 Table 4. Summary of best model (seminiferous epithelial height ~ seminiferous tubule diameter) for explaining the factors that affect seminiferous tubule epithelial height in Opheodrys a. aestivus (Northern Rough Greensnake) from Alabama. Estimate Standard error t value P value Intercept 8.37 5.54 1.51 0.14 Tubule diameter 0.18 0.04 4.37 less than 0.001 Figure 4. Plot of best model (seminiferous tubule epithelial height ~ seminiferous tubule diameter) for explaining the factors that affect seminiferous tubule epithelial height in Opheodrys a. aestivus (Northern Rough Greensnake) from Alabama. Shaded areas represent 95% confidence intervals. Southeastern Naturalist J.D. Konvalina and S.E. Trauth 2018 Vol. 17, No. 3 528 Discussion Seminiferous tubule diameter was small in April and May and then increased significantly in June. Seminiferous tubule diameter was not significantly different in September or October. By October, the lumen was mostly empty of sperm since they migrated to the vas deferens for winter storage. Seminiferous tubule epithelial height followed a similar pattern as tubule diameter, with small values in May, a significant increase in June, followed by little change in diameter in September and October. These similar patterns were reflected by a positive correlation between seminiferous tubule epithelial height and seminiferous tubule diameter. Seminiferous tubule diameter increased significantly from spring to summer and stayed significantly greater in fall. Contrastingly, there were no significant differences in epithelial height among seasons. This contrasts with what Konvalina et al. (2018) found while examining the testicular cycle of Northern Rough Greensnakes in Arkansas. They found that both seminiferous tubule diameter and seminiferous tubule epithelial height began with small values in spring, increased in summer, and decreased in fall. The lack of significant decrease in the fall in the Alabama specimens may be due to uneven sample sizes across the seasons. Our sample was heavily favored toward spring (n = 18), whereas summer (n = 5) and fall (n = 7) had few samples. More fall samples might yield a significant decrease in tubule diameter and epithelial height from the peak in summer. Specifically, adding November samples could accomplish this due to the testis entering the quiescence phase. Tubule diameter, but not season, was a significant predictor of epithelial height. This result contrasts with the findings of Konvalina et al. (2018), who found that both season and tubule diameter significantly affected epithelial height in Arkansas specimens of Northern Rough Greensnake. However, both studies found a peak in tubule diameter in summer, which correlates with peak sperm production. Setting aside the uneven sample-size issues, our results suggest that the seasonal variation in seminiferous tubule diameter and epithelial height is less pronounced in southern populations of Northern Rough Greensnakes. Reproductive physiology studies examining the impact of temperature on variation of testicular tissue in this species could provide insight on such geographic differences and also on the seasonal variation (or lack thereof) that climate change-induced warmer temperatures would produce. Saint Girons (1982) found that the only climatic factor that affects spermatogenesis in male snakes is temperature, where cold winters stop spermatogenesis. Future climate models predict warmer winters (Liu et al. 2017), which may lead to longer periods of sperm production over the course of a year. An extended testicular cycle is possible as long as the snakes can stay active and delay hibernation. If the ecology of the Northern Rough Greensnake is similar to that of the colubrid T. s. parietalis (Say) (Red-sided Gartersnake), then photoperiod likely has little to no impact on the recrudescence of the testes (Hawley and Aleksiuk 1976). 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