Seasonal reproduction of female round stingrays (Urobatis halleri): Steroid hormone profiles and assessing reproductive state

General and Comparative Endocrinology 166 (2010) 379–387

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Seasonal reproduction of female round stingrays (Urobatis halleri): Steroid hormone profiles and assessing reproductive state Christopher G. Mull *, Christopher G. Lowe, Kelly A. Young Department of Biological Sciences, California State University Long Beach, Long Beach, CA 90840, USA

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Article history: Received 8 September 2009 Revised 2 December 2009 Accepted 9 December 2009 Available online 14 December 2009 Keywords: Round stingray Seasonal reproduction Estradiol Progesterone Estuary Seasonal aggregation

a b s t r a c t This study characterizes the seasonal reproductive cycle of female round stingrays (Urobatis halleri) in an open coastal site at Seal Beach, CA and a protected estuary at the Seal Beach National Wildlife Refuge (SBNWR). Female round stingrays were sampled from August 2004 to July 2006, and assessed for reproductive parameters (GSI, maximum ova diameter, pregnancy status) and sex steroid (estradiol (E2), progesterone (P4) and testosterone (T)) concentrations. E2 and P4 increased at the time of ovulation (June and July) and remained elevated until parturition (October and November); recently partruded females were observed until November. Mature females were absent from Seal Beach in August and September, the same time period that abundance of mature females peaked in the SBNWR. This aggregation of predominantly mature females in the upper reaches of the SBNWR was seasonal, and was observed from April to September. To better understand the aggregation behavior, sex steroid hormones were assayed in SBNWR females. In July and August, E2 and P4 concentrations in females at the SBNWR were 1.5-fold and 2-fold higher, respectively, than concentrations in mature females at Seal Beach, and correlated with elevated water temperature in the estuary. Pregnancy was confirmed in aggregating females by increased levels of E2 and P4 and the presence of developing embryos. Our data suggest that coastal estuaries may play a crucial role in round stingray reproduction, perhaps by providing a thermal refuge for pregnant females during gestation. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction The majority of reproductive studies in elasmobranch species have been conducted on sharks, while batoids remain underrepresented in the literature. Because batoids such as round stingrays (Urobatis halleri), and other rays are a common occurrence in coastal marine communities and are likely to be affected by human activities, there is an increasing need to understand the reproductive biology and population dynamics of these elasmobranchs, ideally using less invasive methods. Few batoid species have been studied, and most reproductive studies have focused on the Atlantic stingray (Dasyatis sabina). The genus Urobatis contains six species of short tail round stingrays found in the western north Atlantic and Eastern Pacific (Compagno et al., 2005). Of the six species, only the round stingray and yellow stingray (Urobatis jamaicensis) have been the subject of reproductive studies (Babel, 1967; Hamlett et al., 1999; Fahy et al., 2007; Mull et al., 2008), though relatively little is known about female reproductive function in these species.

* Corresponding author. Address: Department of Biological Sciences, Simon Fraser University, Burnaby, BC, Canada V5A 1S6. E-mail address: [email protected] (C.G. Mull). 0016-6480/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2009.12.009

Round stingrays are a common nearshore elasmobranch in Southern California (Babel, 1967), where large aggregations can occur in shallow, fine sediment beaches and embayments with low wave action (Allen et al., 2002; Babel, 1967; Hoisington and Lowe, 2005; Vaudo and Lowe, 2006; Lowe et al., 2007). Female round stingrays are viviparous, bearing from one-six pups each season, depending on the size of the mother (Babel, 1967). The predominant form of matrotrophy is aplacental yolk sac and histotroph (Wourms and Demski, 1993). Mating typically occurs from March to June with parturition reported to take place approximately three months later, a rapid gestation period for a viviparous elasmobranch (Babel, 1967; Nordell, 1994). Because both female and male round stingrays are believed to reach sexual maturity at approximately 30 months, and retain their reproductive capability throughout much of their life span, round stingrays have a high fecundity relative to other elasmobranchs (Babel, 1967). This is in contrast to other species of elasmobranchs that only reproduce once every two to three years and only give birth to a single pup (Wourms and Demski, 1993). A common observation among sharks and batoids are aggregations of females during the reproductive period (Economakis and Lobel, 1998; Hight and Lowe, 2007). These aggregations may play some role in reproduction although no direct evidence has supported this hypothesis. Elasmobranch aggregations are often

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observed in shallow water that is warmer than surrounding areas, and pregnant females may exhibit thermotaxis as a way to increase the rate of embryo development and reduce gestation time (Economakis and Lobel, 1998; Wallman and Bennett, 2006), although this has never been directly tested. With a high fecundity and ideal habitats extremely dense ray aggregations can result, such as observed in Seal Beach, CA. High densities of round stingrays have been observed at Seal Beach near a thermal outfall at the mouth of the San Gabriel River (Babel, 1967; Hoisington and Lowe, 2005; Vaudo and Lowe, 2006; Lowe et al., 2007) and appear be correlated with warmer water temperatures and low surf conditions (Hoisington and Lowe, 2005). Although water temperatures near the San Gabriel River outfall are higher than surrounding areas, they are highly variable, and can change up to 10 °C in a single tidal cycle (Babel, 1967; Hoisington and Lowe, 2005). While round stingrays are present at Seal Beach throughout the year, abundance varies with season and the residence time of individuals is approximately 2 weeks (Vaudo and Lowe, 2006). Interestingly, during six years of monthly sampling at Seal Beach no evidence of mating activity (e.g., fresh female mating scars) was ever observed (Hoisington and Lowe, 2005; Vaudo and Lowe, 2006; Lowe et al., 2007; Mull et al., 2008). Prior to this study, the effects of photoperiod and water temperature on sex hormones during the reproductive cycle in round stingrays had not been investigated, and the possibility of a bimodal or dual breeding season had not been fully examined in large aggregations of round stingrays. Bimodal distribution of ovulation has been suggested for certain populations of round stingrays, and is hypothesized to result from a portion of ‘‘out of phase” females (Babel, 1967). Yellow stingrays (U. jamaicensis) exhibit a dual reproductive season with females ovulating twice each year, and pregnant females present year-round (Fahy, 2007). In addition to round stingrays, double reproductive seasons have also been sug-

gested for thorny stingrays (Dasyatis centroura) (Capapé, 1993) during years with optimal conditions; however, these hypotheses regarding plasticity have yet to be fully examined. We hypothesized (1) that round stingrays in Seal Beach, CA, would exhibit a single reproductive event each year, (2) the timing of reproductive events would be correlated with changes in steroid hormone concentrations, and (3) changes in steroid hormone concentrations would be correlated with changes in temperature and photoperiod. To address these hypotheses, we sampled round stingrays at Seal Beach, CA, monthly for 16 months to assess seasonal changes in reproductive physiology. Individuals were assessed for plasma steroid hormone estradiol (E2), testosterone (T), and progesterone (P4) concentrations, gonadal morphology and GSI. In addition, female round stingrays in the Seal Beach National Wildlife Refuge (SBNWR) were sampled for 16 months to examine the demographics, reproductive condition, and seasonality for comparison with the Seal Beach individuals. This allowed us to also examine the potential use of a coastal estuary as a breeding ground by round stingrays. 2. Materials and methods 2.1. Field collection Round stingrays were collected monthly at Seal Beach, CA, from August 2004 to July 2006. Rays were captured using a large (3 m  10 m) beach seine near the mouth of the San Gabriel River (33°440 N, 118°060 W) (Fig. 1). All rays captured were sexed and measured for disc width. In addition, a subset of adult female rays was brought back to California State University, Long Beach (CSULB). These rays were then re-measured, weighed, and euthanized via immersion in a seawater ice slurry, in accordance with approved CSULB IACUC protocol # 212. Blood samples were taken

Fig. 1. Overview map of the sampling sites (N) in Seal Beach, CA and the Seventh Street pond of the Seal Beach National Wildlife Refuge (SBNWR). j in inset shows the location of Seal Beach and the SBNWR in California. Gray indicates land areas.

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through cardiac puncture, and blood was placed into heparinized vials and centrifuged for 5 min at 5000g (see Mull et al., 2008). The plasma was removed and stored at 85 °C until assayed. Ovaries were removed, weighed, and placed into 10% neutral buffered formalin for nine days and transferred to 70% ethanol until paraffin embedding. A gonadal somatic index (GSI = gonad mass (mg)/total mass (mg)  100) was established for each individual. Sea surface temperatures were collected from the National Data Buoy Center (NDBC) buoy 46230 off of Huntington Beach, CA (33°370 2300 N 118°000 4300 W) and station OHBC1-9410660 off of San Pedro, CA (33°430 1200 N 118°160 1800 W). Sea surface temperatures were averaged between both stations to approximate water temperatures in the Seal Beach area. While these data do not represent exact sea floor water temperatures from Seal Beach, CA, they do approximate regional differences in temperature. Previous research has shown that round stingrays can move great distances (>60 km) from Seal Beach (Vaudo and Lowe, 2006), and would be subject to a wide range of seasonal seafloor water temperature fluctuations (Hoisington and Lowe, 2005). 2.2. Histology A single 2 mm thick cross-section was removed from the center of each ovary. Tissue samples were dehydrated in a graded ethanol series (70–100%), cleared in xylenes, and infiltrated and embedded in paraffin. Paraffin blocks were sectioned at 6 lm on a Microm HM 315 rotary microtome (Microm International, Walldorf, Germany), and sections were mounted on superfrost plus slides (Fisher Scientific, Hampton, NH). Mounted sections were stained using hematoxylin and eosin for histological analysis (Mull et al., 2008). 2.3. Ovary anatomy Tissue was sectioned by the procedure described above and examined for growth and maturation of oocytes throughout the breeding season. In Atlantic stingrays, temporal periodicity in the ovary is markedly less complex than that observed in the testis, and consists of the production and development of ova. Developing oocytes were measured to determine size of ova during oogenesis. Differences in maximum oocyte diameter between sampling dates were examined using a Kruskal–Wallis analysis followed by Dunn’s post hoc test. At the time of dissection females were categorized based on reproductive state (Pre-ovulatory, Early pregnant, Late pregnant, and Post-partum) as described in U. jamaicensis (Fahy et al., 2007). Pre-ovulatory females were defined as having visible follicles within the ovary, but no development of the uterus. Early pregnant females were defined as having recently ovulated eggs in the uterus, but no visible embryonic development or uterine villi. Late pregnant females were defined as having visibly developing embryos in the uterus, and uterine villi were present. Post-partum females were defined as having no developing embryos in the uterus, but had noticeable uterine development including the presence of uterine villi. 2.4. Sex steroid hormones Plasma concentrations of E2, P4, and T were measured using radioimmunoassay (RIA) kits from Diagnostic Systems Laboratories (Webster, TX). Radioactivity of sample tubes was measured on a Perkin-Elmer Cobra II gamma counter (Packard Instruments Co., Boston, MA). Standard curves were generated for each assay to determine hormone concentration in experimental samples using the four-parameter logistic curve function from Sigma Plot software (SPSS, Inc., Chicago, IL). Plasma analyzed for estradiol was prepared using a triple diethyl ether extraction (Laidley and

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Thomas, 1997) to account for potential assay interferences. Differences among months were analyzed by Kruskal–Wallis test followed by Dunn’s post hoc test. To examine the relationship with water temperature and photoperiod, Spearman’s correlations and stepwise multiple regressions were used. 2.5. Seasonal aggregations in a coastal estuary The utilization of protected estuarine habitat by round stingrays as a mating and pupping ground was also investigated. Rays were collected in the NE corner of the Seventh Street pond of the SBNWR (Fig. 1) using a 4 m long  2 m deep beach seine. During the first summer season all collected rays were sexed and weighed. In addition, disc widths were measured and spines were clipped from each individual to estimate short term recapture rate (Lowe et al., 2007). When available, aborted embryos were also collected, measured, and sexed if possible. During the second summer season, blood samples were collected from the caudal vein of a subset rays, in addition to the above measurements. To sedate rays during blood collection, individuals were placed into tonic immobility in a tray of water so that the spiracles remained submerged and water could be pumped across the gills (Henningsen, 1994). Blood samples were kept in heparinized syringes and placed on ice until they could be centrifuged and prepared according to the method described above. Blood samples were later analyzed using RIA for E2, and P4. Seafloor water temperatures were continuously measured throughout the study NE corner of the Seventh Street pond using a HOBO temperature logger (Onset Computers, Bourne, MA). To examine the differences in steroid hormone concentrations among sampling dates, a Kruskal–Wallis test was used followed by Dunn’s post hoc test. To determine the relationship between water temperature and photoperiod on catch sizes and steroid hormone concentrations, Spearman’s correlations and stepwise multiple regressions were used. 3. Results 3.1. Seasonal distribution Female round stingrays were found at Seal Beach throughout the year, while mature females were absent in August and September (Fig. 2). Female round stingrays were present in the SBNWR from April to September, with the highest abundance in August. Of 428 rays that were sampled throughout the two reproductive seasons study period in the Seventh Street pond of the SBNWR, only two were male, and only 10 were deemed immature, being <160 mm disc width (Fig. 2). The average size of rays sampled did not differ significantly between sampling periods (one-way ANOVA, F = 2.618, df = 7, p > 0.05). 3.2. Female reproductive cycling There was a significant seasonal pattern in GSI throughout the reproductive cycle (F = 2.37, df = 9, p = 0.024). Maximum GSI was observed in June 2005 (1.19 ± 0.08%) followed by a minimum GSI in July 2005 (0.87 ± 0.08%). This change coincided with ovulation in June and July. Oocyte development was characterized by the growth and maturation of distinct cohorts of follicles until ovulation in June and July, when oocytes are approximately 12–13 mm diameter (Fig. 3B). There was a significant difference in maximum egg diameter throughout the season (K = 14.76, df = 8, p = 0.039). Maximum egg diameters were small throughout the late summer and fall (July– October), increased 1.5-fold through the winter and spring (November–April), and peaked in May prior to ovulation in June and July.

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Fig. 2. Length-frequency distribution of female round stingrays from Seal Beach and SBNWR in August and September. Size classes are defined by 10 mm disc width bins. The broken line represents the size at 50% maturity as defined by Babel (1967).

Maximum egg diameters in May were significantly higher than in October–December, around the time of parturition (p < 0.05). 3.3. Pregnancy and gestation Females were categorized by their reproductive state throughout the study period. Pre-ovulatory state females were present

Fig. 3. (A) Gonadosomatic index (GSI), expressed as a percentage of total body weight, of mature female round stingrays collected from Seal Beach throughout the year. (B) The maximum relative egg size (maximum egg diameter (mm)/disc width (mm)) from mature female round stingrays collected from Seal Beach throughout the year. Bars represent mean ± SEM, numbers above bars denote sample size, and letter denote sampling month. NS indicates periods when no sampling occurred and ‘‘0” indicate periods when no rays were captured.

throughout the entire sampling period (Fig. 4). Early pregnant state females were present in June and July throughout both years of the study, composing up to 40% of the females sampled. Late pregnant state females were only present in September of both years. Postpartum state females were observed approximately four months after the peak of ovulation in October and November. The disc widths of aborted embryos from the SBNWR and embryos from pregnant females at Seal Beach were acquired on three separate occasions. Aborted embryos were observed in the SBNWR in August 2005. On 4 August 2005, the average disc width of embryos was 29.1 ± 9.5 SEM mm. By 27 August 2005, the average disc width increased to 42.8 ± 9.4 mm, and by 28 October 2005, was 72.9 ± 10.9 mm disc width. These increases in embryo size were significantly different at each interval (F = 60.47, df = 2, p < 0.001; data not shown).

Fig. 4. Pregnancy state (Fahy et al., 2007) of mature female round stingrays from Seal Beach collected throughout the year. Ovarian females (white bars) were defined as having visible follicles within the ovary, but no development of the uterus. Uterine females (horizontal striped bars) were defined as having recently ovulated eggs in the uterus, but no embryonic development or uterine villi were visible. Embryonic females (diagonally striped bars) were defined as having visibly developing embryos in the uterus, and uterine villi were present. Recently partruded females (black bars) were defined as having no developing embryos in the uterus, but had noticeable uterine development including the presence of uterine villi. The numbers at the top of each bar denote sample size. The letters on the bottom denote sampling month.

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3.4. Reproductive endocrinology While no significant changes in plasma estradiol concentrations from Seal Beach were noted, a trend towards a July peak was observed (K = 16.81, df = 9, p = 0.052) (Fig. 5A). Estradiol concentrations were low (<50 ± 3.9 pg/ml) throughout much of the year, but increased in June, with a 2.5-fold increase to a peak in July. No mature females were captured at Seal Beach in August or September. In October, plasma estradiol concentrations had returned to basal levels. In the SBNWR aggregation, estradiol peaked in June at 747 pg/ml, although this was only from one individual (Fig. 5A). Estradiol concentrations increased 2.5-fold from July through August and September. When included with females from Seal Beach, there was a significant pattern in estradiol concentration among months (K = 22.69, df = 12, p < 0.05). Progesterone in females from Seal Beach changed significantly throughout the year (K = 19.31, df = 9, p = 0.013), with a peak in June at 0.988 ± 0.27 ng/ml (Fig. 5B). Following the June peak progesterone levels quickly returned to baseline levels in July (0.634 ± 0.03 pg/ml). In the SBNWR aggregation, progesterone concentrations increased 3-fold from June to a peak in August at

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1.31 ± 0.17 ng/ml (Fig. 5B). When included with females from Seal Beach, there was a significant pattern in progesterone concentrations among months (K = 43.68, df = 12, p < 0.0001). Plasma concentrations of testosterone in females were significantly higher in April 2005 (9.18 ± 4.96 ng/ml) as compared to all other months (K = 18.26, df = 9, p < 0.05) (Fig. 5C), although this peak was driven by two individuals. Throughout the rest of the year average concentrations never rose above 0.2 ng/ml. There were no significant differences in estradiol concentrations between reproductive states (K = 5.49, df = 3, p = 0.16) (Fig. 6). There was a significant difference in progesterone concentrations between reproductive state (K = 32.94, df = 3, p < 0.0001) (Fig. 6) with concentrations being highest during early late pregnancy (1.08 ± 0.12 ng/ml) (Fig. 6). The high concentrations of progesterone in pre-ovulatory females were driven by a handful of females from Seal Beach in June and July around the time of ovulation. When these females were excluded there was a significant difference between pre-ovulatory and early pregnant females (Mann–Whitney test, U = 161.0, p = 0.01). 3.5. Correlations of abiotic factors and reproductive function There was no significant correlation between estradiol in all females and daylength (Spearman’s correlation, r = 0.11, p = 0.43) (Fig. 7A), change in daylength (Spearman’s correlation, r = 0.23, p = 0.09) (Fig. 7B), or water temperature (Spearman’s correlation, r = 0.18, p = 0.19) (Fig. 7C). There was a significant correlation between progesterone in all females and daylength (Spearman’s correlation, r = 0.38, p = 0.0005) (Fig. 7D), a significant inverse correlation between progesterone in all females and the change in daylength (Spearman’s correlation, r = 0.48, p > 0.0001) (Fig. 7E), and a significant correlation between progesterone in all females and water temperature (Spearman’s correlation, r = 0.62, p < 0.0001) (Fig. 7F). Because there were significant differences in progesterone concentrations between pregnancy states, early and late pregnant females were removed and correlations re-analyzed to ensure that differences in progesterone concentrations were not confounded with pregnancy. Upon re-analysis there was still a significant correlation with daylength (Spearman’s correlation, r = 0.47, p = 0.0002), and water temperature (Spearman’s correlation, r = 0.45, p = 0.0003). Upon re-analysis there was no longer a significant correlation between progesterone concentration and change in daylength (Spearman’s correlation, r = 0.23, p = 0.07). 4. Discussion This study represents the first concurrent assessment of pregnancy, oogenesis, and steroid hormones in female round stingrays

Fig. 5. (A) Estradiol (E2) from female collected in Seal Beach (white bars) and females sampled in SBNWR (black bars) throughout the year. (B) Progesterone (P4) from females collected in Seal Beach (white bars) and females sampled in the SBNWR (black bars) throughout the year. (C) Testosterone (T) from females collected in Seal Beach throughout the year. Bars represent mean ± SEM, numbers denote sample size, and letters denote sampling month.  denotes a significant differences from all other months (p < 0.05),  denotes a significant difference from all other months (p < 0.001).

Fig. 6. Steroid hormones concentrations between pregnancy states of females from Seal Beach and SBNWR. Black bars represent estradiol (E2) and white bars represent progesterone (P4). Bars represent mean ± SEM.  denotes a significant difference from all other pregnancy states (p < 0.01).

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Fig. 7. Seasonal changes in reproductive parameters correlated with changes in photoperiod and water temperature. Estradiol is not correlated with photoperiod (A), is inversely correlated with change in photoperiod (B), and is positively correlated with water temperature (C). Progesterone concentrations are not correlated with daylength (D), or change in daylength (E), but are positively correlated with water temperature (F).

throughout the reproductive cycle. In addition, this study demonstrates a clear annual reproductive cycle, with increased concentrations of estradiol and progesterone from ovulation through gestation, in females from two adjacent habitats. The temporal nature of female aggregations in a coastal estuary noted in this study, suggests that this habitat may play an important role in gestation in round stingrays. We predicted that female round stingrays would exhibit an annual reproductive cycle, correlated with seasonal changes in photoperiod and water temperature. Female round stingrays exhibited an obvious annual cycle based on GSI, with maximum GSI observed in June followed by the minimum in July, reflecting ovulation. Similar to Babel (1967), ovulation was noted in June and July during both years of this study, following a peak in maximum egg diameter in May; however, in the current investigation no evidence of a December ovulation event was observed. At the time of ovulation in June, progesterone concentrations peaked at 0.98 ± 0.27 ng/ml in females from Seal Beach. Similarly, in females

from the SBNWR, progesterone concentrations increased in July to 0.89 ± 0.36 ng/ml, and peaked in August at 1.31 ± 0.17 ng/ml. A slight increase in estradiol was seen at the time of ovulation June although this was not significantly higher than basal levels. In the SBNWR estradiol peaked in June at 747 pg/ml, although this from only from a single female sampled. The surge in progesterone at the time of ovulation from females in Seal Beach and the SBNWR is similar to that seen in other elasmobranch species (Henningsen, 1999; Manire et al., 1995; Rasmussen et al., 1999; Snelson et al., 1997; Tricas et al., 2000). Progesterone remained elevated throughout the embryonic development period, peaking during late pregnancy and then returning to basal levels following parturition. A prolonged increase of progesterone during pregnancy is common in mammals, but to date has only been noted in a handful of elasmobranch species (Callard et al., 1993; Manire et al., 1995). In contrast to the current study, female bonnethead sharks displayed elevated progesterone from pre-ovulation to early pregnancy, but by the time of implantation had returned to basal

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levels (Manire et al., 1995), and in female spiny dogfish progesterone increased at the time of ovulation and peaked during early pregnancy but then returned to basal levels by the beginning of the second year of gestation (Tsang and Callard, 1987). These differences in the progesterone cycles may reflect differences in reproductive cycles (annual vs. biennial) and forms of matrotrophy (yolk only vs. placental analogs vs. yolk-sac placentas). Of note is the timing of the initial surge in progesterone, with significant levels observed in individuals prior to ovulation. In June of 2005, all ten females sampled exhibited high levels of progesterone, despite only three having ovulated. Similarly, female bonnethead sharks show increases in progesterone during the preovulatory phase (Manire et al., 1995). Koob et al. (1986) suggested that progesterone concentrations peaked 24–48 h prior to ovulation in the little skate (Raja erinacea). While the predominant source of post-ovulatory progesterone is the corpus luteum, the observed pre-ovulatory progesterone may be synthesized in granulosa cells. In Squalus acanthias, granulosa cells can secrete significant amounts of progesterone under stimulation of pituitary extracts (Tsang and Callard, 1987, 1992; Callard et al., 1993). There was no significant difference in progesterone concentrations in pre-ovulatory and early pregnant females (Fig. 6), although the high levels in pre-ovulatory females were driven by elevated concentrations in females from June and July. Considering June and July were the observed months of ovulation it is likely these females were displaying the pre-ovulatory surge in progesterone. When these females were removed from the analysis, early pregnant females had significantly higher progesterone concentrations than pre-ovulatory females. While the exact role of progesterone remains unclear, it has been shown to inhibit vitellogenesis (Koob and Callard, 1999; Gelsleichter, 2004) preventing further follicular development until progesterone concentrations decline following parturition. In pregnant Torpedo marmorata progesterone was the only steroid hormone detected in the uterus during reproduction, suggesting it may play a role in histotroph production or composition (Fasano et al., 1992). In addition, increased concentrations of progesterone have been shown to alter the make-up of histotroph in mammals, via altering gene expression (Forde et al., 2009) although this has not been directly explored in elasmobranchs. Regardless, the described pattern of progesterone expression could potentially be utilized as a bioindicator for less invasive reproductive investigations of batoid elasmobranchs, most of which exhibit aplacental matrophy (Van Der Kraak et al., 1993; Wourms and Demski, 1993), although it would need to be verified for other species. Seasonal cycles of testosterone concentrations have been found to vary widely in female elasmobranchs, with some species exhibiting pre-ovulatory surges possibly associated with courtship behavior (Rasmussen and Gruber, 1993; Manire et al., 1995), surges associated with ovulation and fertilization (Tricas et al., 2000), to no seasonal variation (Heupel et al., 1999). Female round stingrays exhibited a significant surge in testosterone concentrations in April, however this was driven by three females with levels 400-fold higher than all other females. The timing would suggest that the increase is associated with courtship behavior (Nordell, 1994) and round stingrays were observed mating at this time (Zahn, personal communication; Jirik, personal communication), however the ephemeral nature of the peak suggests otherwise. There is significant seasonal variation in testosterone concentrations if these females are removed from the analysis. Although a bimodal distribution of ovulation for round stingrays has been proposed (Babel, 1967), we saw no evidence of this during the current study. Ovulation was restricted to June and July during both years, as verified by steroid hormone profiles. It is possible that by sampling in a single location we missed an ovulating subpopulation during the second reproductive event. However, we

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would then expect to see pregnant females at Seal Beach or in the SBNWR through March and April and none were observed. Mating in round stingrays occurs approximately two months prior to ovulation, suggesting females store sperm for a short period of time prior to ovulation and fertilization (Mull et al., 2008). While long term female sperm storage has been demonstrated in some elasmobranchs (Wourms and Demski, 1993) it has yet to be demonstrated in round stingrays, and would be necessary to support a second ovulation event. Additionally, elevated progesterone was observed only from May to October, the previously established pregnancy period (Babel, 1967). While dual reproductive seasons have been suggested for the thorny stingray (Capapé, 1993) during ‘‘ideal” years, this phenomenon has yet to be established in any elasmobranch species. The prior dual ovulation hypothesis was based on limited observations of ovulation as well as measurements of ovarian follicles throughout the year, and the notion that eggs will ovulate at a set size (Babel, 1967). However, it is instead likely that round stingrays exhibit a certain degree of reproductive plasticity. It is possible that during years with warmer water temperature and/or with abundant food resources, large females may ovulate twice each year. The apparent short gestation period, combined with protracted spermatogenesis in males (Babel, 1967; Mull et al., 2008) could support dual breeding seasons in certain years, such as seen in yellow stingrays (U. jamaicensis) (Fahy et al., 2007). In addition to the proposed annual to biennial cycle shift in D. centroura (Capapé, 1993), different individual finetooth sharks (Carcharhinus isodon) from the northern Gulf of Mexico have displayed annual or biennial cycle characteristics in the same year (Driggers and Hoffmayer, 2009). Growth of embryos is rapid throughout the entire four month gestational period. By early August the average disc width of embryos was 29.1 mm. By the end of August the size had increased to 42.3 mm disc width, a growth of approximately 20% of the size at parturition in just three weeks. Some species of Urolophidae (stingarees) and other species of Urobatidae (short tail round stingrays) exhibit a similar rapid embryonic development rate that continues post-partum (White et al., 2001a,b; Fahy et al., 2007; Trinnie et al., 2009). Evidence from the present study suggests a four month gestation period, slightly longer than previously reported (Babel, 1967). This gestational period is relatively fast for a viviparous elasmobranch, when compared to other Urolophids and other aplacental species, for example, Australian Urolophid species have reported gestational rates from 5 to 10 months (White et al., 2001a; White and Potter, 2005; Trinnie et al., 2009). However, the rates suggested by the present study are within the range of gestational rates reported for elasmobranchs, which can vary from 3 months in some in some freshwater stingrays (Charvet-Almeida et al., 2005) to 22 months in the lesser spotted dogfish (Scyliorhinus canicula) (Harris, 1952; Sumpter and Dodd, 1979; Wourms and Demski, 1993), and the chain dogfish (Scyliorhinus rotifer) (Castro et al., 1988). The presence of female round stingrays in coastal estuaries is highly seasonal and possibly linked to water temperature and reproductive condition. Estuarine pond floor water temperature in the NE corner of the Seventh Street pond at the SBNWR is seasonally variable ranging from a low of 10.8 °C in January to a high of 29.3 °C in July. Temperatures averaged 25 ± 1.5 °C from June to September when female round stingrays were most abundant. Of note during these periods of highest abundance in the SBNWR, mature female round stingrays were absent from Seal Beach (Fig. 2), suggesting that a large portion of the female population enters coastal estuaries and marshes during this time. This pattern of female distribution supports the idea of an annual breeding cycle for round stingrays in Southern California. While males were observed in the estuarine ponds, only two were found in the upper reaches of the estuary where mature females were most abundant. The

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presence of predominantly mature females during the warmest portions of the season in both 2005 and 2006 suggest that estuaries may serve as a thermal refuge for pregnant female round stingrays. Similar phenomena have been observed in gray reef sharks (Carcharhinus amblyrhynchos) where females aggregated in water 1–2 °C warmer than adjacent areas during the day (Economakis and Lobel, 1998). This potential thermoregulation may increase the development rate of embryos and reduce gestation time significantly (Economakis and Lobel, 1998; Wallman and Bennett, 2006). A similar phenomenon has been observed in round stingrays in San Diego Bay, where rays aggregate in the upper reaches of the bay where water temperatures are warmer compared with the cooler bay mouth (Allen et al., 2002). This study is the first to verify that these commonly observed female aggregations are in fact composed of pregnant females with high progesterone concentrations, supporting the hypothesis of a thermal gestational benefit. In Atlantic stingrays, captive experiments have demonstrated that pregnant females prefer warmer temperatures than both non-pregnant females and males (Wallman and Bennett, 2006). It is possible pregnant round stingrays are exhibiting thermotaxis, although this was not directly tested in this study. Ambient water temperatures in the SBNWR averaged 25 °C during the summer, when pregnant female rays, as determined by the presence of aborted embryos and higher circulating concentrations of E2 and P4, were most abundant. While water temperature near the San Gabriel River outfall reaches levels similar to those of the SBNWR (Hoisington and Lowe, 2005), ambient water temperature at Seal Beach is more variable, changing up to 10 °C in a single tidal cycle (Hoisington and Lowe, 2005; Vaudo and Lowe, 2006). Therefore, pregnant female round stingrays may move into estuaries in search of a more stable thermal regime, although residence time in the mitigation ponds has not been established. Plasma progesterone concentrations from females in the SBNWR were 2-fold higher than those from females at Seal Beach, and estradiol concentrations from the SBNWR were 1.5-fold higher. Progesterone concentrations were positively correlated with both water temperature and daylength, and negatively correlated with change in daylength. This suggests a temperature influence on steroid hormone concentrations, which has been demonstrated in male round stingrays (Mull et al., 2008) and epaulette sharks (Heupel et al., 1999), but these findings could simply be attributed to pregnancy as gestating females were only found within the SBNWR where water temperatures were significantly higher. Whether higher circulating levels of E2 and P4 could contribute to, or be associated with more rapid gestation or pregnancy state, requires further investigation.

5. Conclusions Our data suggest that round stingrays are annual breeders, mating in the late spring and pupping in the fall, and we found no direct evidence of a dual ovulation peak as had previously been suggested (Babel, 1967). Cycles of steroid hormone levels were associated with important events in the reproductive cycle, such as ovulation and parturition. This study provides the first example of increased progesterone levels throughout the gestational period in round stingrays. With the majority of elasmobranchs populations in decline and a paucity of knowledge of life histories and reproductive strategies, P4 may prove a useful bioindicator for reproductive state in less invasive studies. Coastal estuarine habitat may serve a critical role in the reproduction of round stingrays by providing a stable thermal refuge for females during gestation. Whether there is some reproductive or fitness benefit to this thermotaxy has yet to be determined, although increasing data suggest this may be a common strategy among elasmobranchs and that

coastal estuaries may be a critical component of managing elasmobranch populations.

Acknowledgments The authors thank K. Kelley for his assistance with analyzing RIA data; R. Pounds, J. Bailey and the Seal Beach Lifeguard Dept. for their assistance in collecting rays; R. Schallmann and K. Gilligan of the Seal Beach National Wildlife Refuge for allowing us to sample within the reserve; all of the volunteers who helped with beach seines, especially L. Hale, G. Goodmanlowe, K. Jirik, S. Plank, J. Brusslan, G. McMichael, K. Loke, K. Anthony, H. Zemel, M. Blasius, R. Antes, B. Zitt, E. Jarvis, T. Mason, H. Gliniak, J. Reyes, T. Parker, E. Zahn and all other members of the Young and Lowe Labs. We also thank J. Vaudo and C. Mireles for their assistance in creating a map for this manuscript. We also thank the CSULB Graduate Research Fellowship, Southern California Academy of Sciences, and SCTC Marine Biology Education Foundation for financial assistance.

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