TY - JOUR
AU1 - Wheeler, Carolyn, R.
AU2 - Novak, Ashleigh, J.
AU3 - Wippelhauser, Gail, S.
AU4 - Sulikowski, James, A.
AB - Abstract The Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus) is a long-lived, anadromous fish species ranging from Labrador, CA to Florida, USA. In the Saco River, located in the Gulf of Maine, this species was not present during a survey study ending in 1982, but was found inhabiting the estuary in 2007. Although the reason for the return of this sturgeon to this river system remains unknown, research on basic life-history information is necessary to facilitate the conservation of this federally protected species. Given the conservation status of the species, the present study used circulating sex steroid hormones to determine the sex of 288 Atlantic sturgeon captured between 2012 and 2014 in the Saco River estuary located in the Gulf of Maine. Overall, the sex was determined for 93% of Atlantic sturgeon sampled. Mean hormone values were similar to other Atlantic sturgeon reproductive studies. The findings indicate the validity of sex steroid hormones as a singular method for sex determination in wild Atlantic sturgeon. Results also indicated a likely 1:1 (male:female) sex ratio in the system, except in 2014 when a 1:3 ratio was observed. It is not believed that the Saco River estuary is used for spawning, as several impassable dams block access to spawning habitat. However, this area might provide crucial foraging for growth and development of juveniles and a habitat for adults forgoing spawning. Introduction The Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus) is a large, k-selected, anadromous fish ranging from Labrador, CA to Florida, USA [Atlantic sturgeon Status Review Team (ASSRT), 2007]. This species was highly prized for its caviar and flesh, initiating a robust fishery for Atlantic sturgeon, peaking at 3350 metric tons in 1870 (Smith and Clungston, 1997; Secor and Waldman, 1999). However by 1901, the fishery was harvesting only 10% of its peak levels largely owing to the species’ aforementioned life-history traits (Smith and Clungston, 1997). After ongoing declines in the population, the fishery was finally terminated in 1998 (Atlantic States Marine Fisheries Commission, 1998). While commercial targeting of Atlantic sturgeon is currently prohibited (Atlantic States Marine Fisheries Commission, 1990), populations are threatened by other factors, such as habitat loss from damming (i.e. blocking passage to spawning grounds; ASSRT, 2007), poor water conditions (i.e. decreasing dissolved oxygen concentrations from pollutants; Niklitschek and Secor, 2005) and incidental catch in other fisheries (ASSRT, 2007). Given this and the failure of populations to rebound despite a prohibited status, Atlantic sturgeon were listed in 2012 under the federal Endangered Species Act (ESA). This listing designated populations in the Gulf of Maine (GOM) Distinct Population Segments (DPS) as threatened and DPS units in the southern extent of the range (New York Bight, Chesapeake Bay, Carolina and Southern Atlantic) as endangered (ASSRT, 2007). An essential component for assessing and managing fish populations is determining species reproductive parameters, such as when sexual maturity occurs, the timing of seasonal cycles and locations of spawning aggregations (e.g. Walker, 2005). These data are important for outputs such as generating population models used to estimate sustainable harvest levels and assessing risk of extinction or recovery by the species (Walker, 2005). However, these types of data are poorly characterized for the majority of systems inhabited by Atlantic sturgeon because of their large size, high mobility, depleted stock status, undetermined sex-specific genes and lack of external sexual dimorphism (Scott and Crossman, 1973; ASSRT, 2007; Keyvanshokooh and Gharaei, 2010). Thus, the acquisition of this information has largely been through lethal or sublethal techniques, such as gross dissections (e.g. Barannikova et al., 2004) and endoscopy to view and collect gonadal tissue for histological analysis (e.g. Petochi et al., 2011). Although these techniques are widely accepted and implemented in aquaculture practices, they are far less suitable for wild populations that are severely depleted or endangered (Boreman, 1997; Kynard and Kiiffer, 2002; Colombo et al., 2004). Thus, given the stock status and recent listing of the species, the development and use of effective non-lethal methods for Atlantic sturgeon are needed to inform management decisions without further compromising the population of this protected species. Previous studies have successfully used circulating reproductive hormones [i.e. testosterone (T) and 17β-estradiol (E2)] to identify sex and reproductive status in captive (e.g. Amiri et al.,1996a, b; Semenkova et al., 2002; Barannikova et al., 2004; Davail-Cuisset et al., 2011) and wild sturgeon (e.g. Van Eenennaam et al., 1996; Ceapa et al., 2002; Webb et al., 2002; Heise et al., 2009). For example, T is an important androgen contributing to maturation in males and females (Cuisset et al., 1995; Moberg et al., 1995; Van Eenennaam et al., 1996; Amiri et al., 1996a, b; Webb et al., 2002), whereas E2 has been shown to increase during vitellogenesis and decrease during the terminal stages of oocyte maturation in females (Amiri et al., 1996b; Doroshov et al., 1997; Webb et al., 2002). Thus, by using a ratio of T and E2 concentrations, the sex of sturgeon can be determined (Craig et al., 2009). For example, significant differences in androgen and E2 concentrations were observed between sex and reproductive phases (i.e. ovulating, gravid and spent females) in Atlantic sturgeon sampled within the Hudson River (Van Eenennaam et al., 1996). In addition, Heise et al. (2009) identified the sex of Gulf sturgeon (Acipenser oxyrinchus desotoi) by E2 concentrations that clustered into a low and high group (<0.5 and >8 ng ml−1, respectively). Likewise, Craig et al. (2009) found it possible to use these reproductive hormones to identify sex in lake sturgeon (Acipenser fulvescens), but noted the need for a species-specific analysis for more accurate results. Finally, many hormone-based studies have been paired with other sex-determining methods (i.e. gross dissection and endoscopy) to valid hormone profiles (Webb et al., 2002). These data collectively have given rise to sets of accurate functions (up to 95% accurate) using T and E2 concentrations to determine sex (Webb et al., 2002), providing analysis techniques for future fully non-lethal studies. For Atlantic sturgeon, reproductive research has typically been conducted within large river systems with documented spawning populations, such as the Hudson (Van Eenennaam et al., 1996; Kahnle et al., 1998, 2005) and Delaware Rivers (Secor and Waldman, 1999; Secor, 2002). Watersheds where access to freshwater habitat is limited tend to be overlooked because they are not thought to hold reproductive significance for this species (ASSRT, 2007). The Saco River, located in the GOM DPS, is the fourth largest river in Maine (Furey and Sulikowski, 2011); however, it is not considered an historical spawning site for Atlantic sturgeon because of the impassable Cataract Falls and a dam at river kilometre (rkm) 10 constructed about 1682 (ASSRT, 2007; US Army Corps of Engineers, 2013), which limits access to freshwater. As a result of heavy fishing pressures within the GOM near the turn of the 20th century (Atlantic States Marine Fisheries Commission, 1990), Atlantic sturgeon were not present in the Saco River Estuary (SRE) during a survey study from 1979 to 1982 (Reynolds and Casterlin, 1985). However, after a considerable absence from the SRE, an Atlantic sturgeon was captured in 2007 during a routine trawl survey (Furey and Sulikowski, 2011). This finding initiated a study aiming to investigate the possible resurgence of the species. Gillnet sampling conducted by Little (2013) from 2008 to 2011 resulted in an mean catch per unit effort of 7.26 Atlantic sturgeon per hour, comparable to that reported for the Kennebec system for the period 1998–2000 (ASSRT, 2007; catch per unit effort = 7.43). This high catch per unit effort and the limited available life-history information created an opportunity to use circulating steroid sex hormones to assess the reproductive status and sex ratio of Atlantic sturgeon captured within the SRE. Simultaneously, the importance of the system to the recovery of the species could be assessed. Materials and methods Capture Atlantic sturgeon were collected between May and November from 2012 to 2014. Sampling was conducted primarily in the mouth of the SRE between the two jetties that extend ~2.3 rkm into Saco Bay (Brothers et al., 2008). Specifically, sampling occurred with bottom gillnets (15.2 or 30.5 cm stretched mesh × 2 m height × 100 m long) fished at slack low tide perpendicular to the jetties for 15 min. Following capture, fish were carefully removed from the gillnet and transferred to a net-pen (2.1 m × 0.9 m × 0.9 m), which was submerged in river water reflecting the ambient condition of the SRE. Sampling procedure This project was part of a larger study investigating Atlantic sturgeon in the SRE, where a standard protocol of sampling was followed (Kahn and Mohead, 2010). Briefly, sturgeon were removed from the net-pen and the total length, fork length (FL) and head length were measured to the nearest centimetre and the interorbital width to the nearest millimetre. Each Atlantic sturgeon was then scanned with an AVID PowerTracker VIII scanner to check for the presence of an AVID passive integrated transponder (PIT) tag. If no tag was found, a 134.2 kHz PIT tag (model HPT12; Biomark) was inserted into the dorsal musculature at the base of the dorsal fin. Additionally, a US Fish and Wildlife Service T-bar tag was injected opposite the PIT tag in the same location. A 4 ml aliquot of blood was then drawn from the caudal vein using a heparinized 3.17 cm, 22 gauge needle and 7 ml BD Vacutainer tube. The blood samples were stored on ice in the field no longer than an hour until further processing back in the laboratory. After the sampling process commenced, fish were returned to a net-pen and allowed to recover before being released. Finally, in the laboratory, a subsample of each blood sample was analysed for haematocrit (VWR micro-haematocrit capillary tubes), followed by centrifugation at 1242g for 10 min. The plasma was then removed and frozen at −20°C until steroid hormone analysis of circulating levels of testosterone (T) and 17β-estradiol (E2) could be completed. Steroid hormone extraction The steroid hormones T and E2 were extracted from plasma samples following modified protocols from Sulikowski et al. (2016) and Prohaska et al. (2013). Briefly, each sample was spiked with 1000 counts min−1 of the respective tritiated hormone (Perkin Elmer, Waltham, MA, USA) to account for procedural loss and extracted twice with 10 volumes of ethyl ether (ACS grade). The organic phase was evaporated under nitrogen at 37°C, and each sample was reconstituted in phosphate-buffered saline with 0.1% gelatin. Mean extraction recoveries for T and E2 were 78 and 71%, respectively. Radioimmunoassay Plasma concentrations of T and E2 were determined by radioimmunoassay (RIA) following a modified protocol by Tsang and Callard (1987). In this protocol, non-radiolabelled T and E2 stocks (Steraloids Inc., Newport, RI, USA) were prepared into stock solutions at 80 µg ml−1 for T and 6.4 µg ml−1 for E2 in absolute ethanol (ACS grade). The specifics of the radiolabelled steroids, the antibody characteristics and titres are provided by Sulikowski et al. (2004). Radioactivity was detected by a Perkin Elmer Tri-Carb 2900TR liquid scintillation analyser (Waltham, MA, USA). The mean intra-assay coefficients of variance for T and E2 were 10 and 9% and the inter-assay coefficients of variance were 11 and 12%, respectively. Sex determination To provide a cross-examination of data in the present study, sex determination by blood hormone concentrations was performed by incorporating findings of two previous studies that used hormones to assess reproductive condition and determine sex and maturity of wild populations (Van Eenennaam et al., 1996; Webb et al., 2002). For this component of the present study, the mean Atlantic sturgeon androgen (combined values of T and 11-ketotestosterone) and E2 concentrations (126.83 ± 62.52 and 0.33 ± 0.15 ng ml−1, respectively; values from Van Eenennaam et al., 1996) were used as threshold values, but to improve accuracy, the E2 concentration was modified to 0.8 ng ml−1 (three standard deviations above the reported mean; Van Eenennaam et al., 1996). Therefore, an individual in the present study was considered a male if E2 concentrations were ≤0.8 ng ml−1 or if testosterone concentrations were ≥126 ng ml−1. If neither of these conditions was satisfied, the individual was classified as a female. The second method was a discriminant function analysis by Webb et al. (2002) that was generated from white sturgeon (Acipenser transmontanus) using T and E2 as predicting factors of sex. As such, the following equations from Webb et al. (2002) were applied to the SRE Atlantic sturgeon hormone values: Female = −1.6727 + 2.3678(log10T) − −3.5783(log10E2) Male = −5.2972 + 5.2524(log10T) – 7.5539(log10E2) These functions predict the sex of an individual when logarithmically transformed T and E2 values are input into each equation. The equation with the higher product then determines the sex (Webb et al., 2002). A sturgeon was definitively sexed when both RIA evaluation methods were in accordance, and subsequently, individuals with conflicting sex were categorized as unidentified. To assess hormonal differences according to sex and size class, mean T and E2 were compared between juveniles and adults for each sex (excluding unknown individuals). Owing to non-normality and equal variance within the hormone data, a Kruskal–Wallis one-way ANOVA was performed, followed by Dunn's pairwise multiple comparison. Furthermore, owing to the large number of fish sampled in this study, a sex-predictive model for application to other Atlantic sturgeon populations was modelled. The T and E2 values from fish in the known RIA category were fitted in a multiple logistic regression with forward selection (selection criterion = 0.10), with the consequent model predicting the probability of a female Atlantic sturgeon. A χ2 test of homogeneity was used to assess the male-to-female ratio in the SRE of Atlantic sturgeon in the known category. The sex ratio was compared between 2012, 2013 and 2014 to determine whether annual shifts existed. Then, a series of Fisher's exact tests were used to assess the ratio by maturity derived from fork length [for late-stage juvenile (63–134 cm FL) and adult (135–190 cm FL); Bain, 1997] and by the seasonal groupings of spring (April–May), summer (June–August) and autumn (September–November) for each year. The Kruskal–Wallis one-way ANOVA and the multiple logistic regression were analysed using Systat 13 (Systat Software, San Jose, CA, USA), whereas the χ2 and Fisher's exact test analyses were performed using R 2.15.2. All statistical analyses were considered significant at α ≤ 0.05. Results The mean haematocrit value from all blood samples was 29 ± 0.5%. Sex was determined for 267 of 288 (93%) Atlantic sturgeon sampled in the present study. When sex determination was assessed by late-stage juveniles (63–134 cm FL) and adults (135–190 cm FL), as defined by Bain (1997), sex was determined in 92 and 97% of Atlantic sturgeon, respectively. Of the individuals with unidentified sex, 67% were classified as juveniles. The mean fork length of unidentified fish was 122 ± 5 cm. Evaluation of RIA hormone values of T in the Dunn's pairwise comparison revealed that juvenile male and female Atlantic sturgeon were not significantly different from one another (P = 1.00; Fig. 1A). However, E2 was found to be significantly higher in female juvenile sturgeon than in male juveniles (P < 0.001; Fig. 1B). In adult sturgeon, males and females had significantly higher levels of T (P = 0.031; Fig. 1A) and E2 (P < 0.001; Fig. 1B), respectively, over all other groupings. When T concentrations were compared between juvenile and adult sturgeon, the adult mean was significantly higher in males (P < 0.001; Fig. 1A) as well as in females (P = 0.46; Fig. 1B). The E2 concentrations were not significantly different between juvenile and adult females (P = 0.054; Fig. 1B) or between juvenile and adult males (P = 1.00; Fig. 1B). Figure 1: Open in new tabDownload slide Mean ± SEM testosterone (A) and E2 (B) for late-stage juvenile (63–134 cm fork length) and adult (135–190 cm fork length) Atlantic sturgeon. Means were significantly different at α ≤ 0.05, and are denoted with different letters. Figure 1: Open in new tabDownload slide Mean ± SEM testosterone (A) and E2 (B) for late-stage juvenile (63–134 cm fork length) and adult (135–190 cm fork length) Atlantic sturgeon. Means were significantly different at α ≤ 0.05, and are denoted with different letters. Finally, the model fit (d.f. = 2, P < 0.0001) from the logistic regression found both T and E2 to be significant predictors of Atlantic sturgeon sex in the SRE. The relationship between the two hormones is represented by the following equation: P(female)=e−12.9308+(16.9520∙E2)−(0.0798∙T)1+e−12.9308+(16.9520∙E2)−(0.0798∙T) When all RIA results from 2012−2014 were pooled, the results indicated an overall sex ratio of 33% males, 60% females, and 7% unidentified sturgeon occurred within the sampled SRE Atlantic sturgeon. The χ2 test of homogeneity of the sex ratio between 2012 and 2013 showed no significant difference (χ2 = 5.559, d.f. = 2, P = 0.062; Table 1), and also indicated a likely 1:1 ratio (male:female; 2012 and 2013, 41% male, 50% female and 9% unidentified). However, 2012 and 2013 each showed a significant shift in sex ratio in comparison to 2014 (2012, χ2 = 9.423, d.f. = 2, P = 0.009; and 2013, χ2 = 14.896, d.f. = 2, P = 0.001; Table 1) attributable to an increase in the number of females present in the SRE (2014, 23% male, 72% female and 4% unidentified). No significant difference between the overall sex ratio for juvenile and adult Atlantic sturgeon for any sampling year was observed via Fisher's exact test (2012, P = 0.808; 2013, P = 1.000; and 2014, P = 0.682; Table 2), indicating that the female skewed ratio in 2014 existed for both juveniles and adults alike. Table 1: Counts and percentages of males, females and unidentified Atlantic sturgeon for the entirety of the study and for individual years Sex . Total . 2012a . 2013b . 2014a,b . n . Percentage . n . Percentage . n . Percentage . n . Percentage . Male 96 33.5 30 44.9 36 38.7 30 23.4 Female 171 59.6 35 52.2 44 47.3 92 71.9 Unidentified 21 6.9 2 2.9 13 14.0 6 3.9 Sex . Total . 2012a . 2013b . 2014a,b . n . Percentage . n . Percentage . n . Percentage . n . Percentage . Male 96 33.5 30 44.9 36 38.7 30 23.4 Female 171 59.6 35 52.2 44 47.3 92 71.9 Unidentified 21 6.9 2 2.9 13 14.0 6 3.9 Letters represent statistical differences in sex ratios compared by χ2 contingency tables. Table 1: Counts and percentages of males, females and unidentified Atlantic sturgeon for the entirety of the study and for individual years Sex . Total . 2012a . 2013b . 2014a,b . n . Percentage . n . Percentage . n . Percentage . n . Percentage . Male 96 33.5 30 44.9 36 38.7 30 23.4 Female 171 59.6 35 52.2 44 47.3 92 71.9 Unidentified 21 6.9 2 2.9 13 14.0 6 3.9 Sex . Total . 2012a . 2013b . 2014a,b . n . Percentage . n . Percentage . n . Percentage . n . Percentage . Male 96 33.5 30 44.9 36 38.7 30 23.4 Female 171 59.6 35 52.2 44 47.3 92 71.9 Unidentified 21 6.9 2 2.9 13 14.0 6 3.9 Letters represent statistical differences in sex ratios compared by χ2 contingency tables. Table 2: Counts and percentages of males, females and unidentified Atlantic sturgeon for late-stage juvenile (63–134 cm fork length) and adult (135–190 cm fork length) sturgeon sampled each year . Juvenile . Adult . n . Percentage . n . Percentage . A. 2012 Male 14 41.2 16 48.5 Female 19 55.9 16 48.5 Unidentified 1 2.9 1 3.0 B. 2013 Male 25 39.0 11 37.9 Female 30 46.9 14 48.3 Unidentified 9 14.1 4 13.8 C. 2014 Male 18 25.4 12 21.1 Female 49 69.0 43 75.4 Unidentified 4 5.6 2 3.5 . Juvenile . Adult . n . Percentage . n . Percentage . A. 2012 Male 14 41.2 16 48.5 Female 19 55.9 16 48.5 Unidentified 1 2.9 1 3.0 B. 2013 Male 25 39.0 11 37.9 Female 30 46.9 14 48.3 Unidentified 9 14.1 4 13.8 C. 2014 Male 18 25.4 12 21.1 Female 49 69.0 43 75.4 Unidentified 4 5.6 2 3.5 No statistically significant differences were found between juvenile and adult percentages in any given year. Table 2: Counts and percentages of males, females and unidentified Atlantic sturgeon for late-stage juvenile (63–134 cm fork length) and adult (135–190 cm fork length) sturgeon sampled each year . Juvenile . Adult . n . Percentage . n . Percentage . A. 2012 Male 14 41.2 16 48.5 Female 19 55.9 16 48.5 Unidentified 1 2.9 1 3.0 B. 2013 Male 25 39.0 11 37.9 Female 30 46.9 14 48.3 Unidentified 9 14.1 4 13.8 C. 2014 Male 18 25.4 12 21.1 Female 49 69.0 43 75.4 Unidentified 4 5.6 2 3.5 . Juvenile . Adult . n . Percentage . n . Percentage . A. 2012 Male 14 41.2 16 48.5 Female 19 55.9 16 48.5 Unidentified 1 2.9 1 3.0 B. 2013 Male 25 39.0 11 37.9 Female 30 46.9 14 48.3 Unidentified 9 14.1 4 13.8 C. 2014 Male 18 25.4 12 21.1 Female 49 69.0 43 75.4 Unidentified 4 5.6 2 3.5 No statistically significant differences were found between juvenile and adult percentages in any given year. The analysis by season indicated that the maturity and sex ratio did not change throughout the sampling in 2012 and 2014 (Table 3A and C). However, although the overall proportion of male to female was the same between juveniles and adults in 2013 (Table 2B), there was a significant shift from summer to autumn in the ratio (P = 0.002; Table 3B). Owing to the low samples size (n = 9), data from the spring could not be evaluated in any of the sampling years (Table 3A, B and C). Table 3: Counts and percentages of males, females and unidentified Atlantic sturgeon for seasons from all years sampled Sex . Maturity . Spring* . . Summera . . Autumnb . . . . n . Percentage . n . Percentage . n . Percentage . A. 2012 Female Juvenile 0 0 15 37.5 4 19.1 Adult 0 0 9 22.5 7 33.3 Male Juvenile 0 0 8 20.0 6 28.6 Adult 4 100.0 8 20.0 4 19.0 B. 2013 Female Juvenile 0 0 26 44.1 4 20.0 Adult 0 0 11 18.6 3 15.0 Male Juvenile 1 100.0 19 32.2 5 25.0 Adult 0 0 3 5.1 8 40.0 C. 2014 Female Juvenile 1 25.0 35 39.8 13 43.3 Adult 1 25.0 32 36.4 10 33.3 Male Juvenile 2 50.0 15 17.0 1 3.4 Adult 0 0 6 6.8 6 20.0 Sex . Maturity . Spring* . . Summera . . Autumnb . . . . n . Percentage . n . Percentage . n . Percentage . A. 2012 Female Juvenile 0 0 15 37.5 4 19.1 Adult 0 0 9 22.5 7 33.3 Male Juvenile 0 0 8 20.0 6 28.6 Adult 4 100.0 8 20.0 4 19.0 B. 2013 Female Juvenile 0 0 26 44.1 4 20.0 Adult 0 0 11 18.6 3 15.0 Male Juvenile 1 100.0 19 32.2 5 25.0 Adult 0 0 3 5.1 8 40.0 C. 2014 Female Juvenile 1 25.0 35 39.8 13 43.3 Adult 1 25.0 32 36.4 10 33.3 Male Juvenile 2 50.0 15 17.0 1 3.4 Adult 0 0 6 6.8 6 20.0 A significant difference was found only between summer and autumn of 2013 using Fisher's exact test and is denoted by differing letters. *Low sample size in April–May, precluding a statistical test of the spring category. Table 3: Counts and percentages of males, females and unidentified Atlantic sturgeon for seasons from all years sampled Sex . Maturity . Spring* . . Summera . . Autumnb . . . . n . Percentage . n . Percentage . n . Percentage . A. 2012 Female Juvenile 0 0 15 37.5 4 19.1 Adult 0 0 9 22.5 7 33.3 Male Juvenile 0 0 8 20.0 6 28.6 Adult 4 100.0 8 20.0 4 19.0 B. 2013 Female Juvenile 0 0 26 44.1 4 20.0 Adult 0 0 11 18.6 3 15.0 Male Juvenile 1 100.0 19 32.2 5 25.0 Adult 0 0 3 5.1 8 40.0 C. 2014 Female Juvenile 1 25.0 35 39.8 13 43.3 Adult 1 25.0 32 36.4 10 33.3 Male Juvenile 2 50.0 15 17.0 1 3.4 Adult 0 0 6 6.8 6 20.0 Sex . Maturity . Spring* . . Summera . . Autumnb . . . . n . Percentage . n . Percentage . n . Percentage . A. 2012 Female Juvenile 0 0 15 37.5 4 19.1 Adult 0 0 9 22.5 7 33.3 Male Juvenile 0 0 8 20.0 6 28.6 Adult 4 100.0 8 20.0 4 19.0 B. 2013 Female Juvenile 0 0 26 44.1 4 20.0 Adult 0 0 11 18.6 3 15.0 Male Juvenile 1 100.0 19 32.2 5 25.0 Adult 0 0 3 5.1 8 40.0 C. 2014 Female Juvenile 1 25.0 35 39.8 13 43.3 Adult 1 25.0 32 36.4 10 33.3 Male Juvenile 2 50.0 15 17.0 1 3.4 Adult 0 0 6 6.8 6 20.0 A significant difference was found only between summer and autumn of 2013 using Fisher's exact test and is denoted by differing letters. *Low sample size in April–May, precluding a statistical test of the spring category. Discussion The present study used a non-lethal approach to determine the sex, reproductive status and the overall sex ratio of Atlantic sturgeon in the SRE. By using two methods to assess the proportion of T and E2 in each fish, this study was effective at identifying sex in 93% of all sampled Atlantic sturgeon. Furthermore, the 92 and 97% effectiveness of the method in juveniles and adults, respectively, indicates that this method is valuable over multiple life stages in wild fish. Although it was hypothesized that the majority of unidentified fish would be juvenile owing to pre-maturation hormone concentrations, the mean fork length of 121 ± 0.5 cm reveals that size was not a likely factor in sex determination. Testosterone concentrations in this study could not be directly compared with Atlantic sturgeon in the Hudson River because Van Eenennaam et al. (1996) assessed total androgen concentrations (including 11-ketotestosterone). In comparison to T concentrations in male maturing white sturgeon (150 ng ml−1), the mean SRE sturgeon value (49 ± 5.4 ng ml−1) was lower (Webb et al., 2002). However, the adult male T mean in the SRE was higher than means in lake sturgeon (Craig et al., 2009) and ‘the bester’ [F1 hybrid beluga and sterlet sturgeon (Huso huso L. female × Acipenser ruthenus L. male); Amiri et al., 1996a], where T peaked at 5.023 and 28 ng ml−1, respectively in each species. The present study found that adult females had the highest mean estradiol levels of all categories assessed, which has also been observed in Atlantic sturgeon in the Hudson River (Van Eenennaam et al., 1996). The mean value of E2 in SRE Atlantic sturgeon (3.7 ± 0.3 ng ml−1) was similar to that of ovulating (1.99 ± 1.32 ng ml−1) and gravid female (5.10 ± 4.05 ng ml−1) means of Atlantic sturgeon in the Hudson River (Van Eenennaam et al., 1996), indicating that some SRE sturgeon may be undergoing vitellogenesis. This trend also corresponds to findings in maturing female white sturgeon (Webb et al., 2002), showing the utility of a sex-determining equation between species, as used in the present study. The mean E2 concentration in the present study (3.7 ± 0.3 ng ml−1) fell within the range of female adult ‘the bester’ hybrid (2–4 ng ml−1; Amiri et al., 1996b) and female lake sturgeon (0.025–9.536 ng ml−1; Craig et al., 2009) but was lower than adult female Gulf sturgeon (mean, 9.97 ng ml−1; Heise et al., 2009). Additionally, in the present study, the immature female E2 mean was an intermediate value (2.9 ± 0.2 ng ml−1) between mature females and all male sturgeon. This finding was unique to the present study, whereas Webb et al. (2002) found that immature female white sturgeon had E2 concentrations with no significant difference from male immature and maturing white sturgeon. The innate differences in T and E2 levels between the sexes illustrates the utility of circulating steroid hormones as a non-lethal method for sex determination of Atlantic sturgeon. However, the lack of significant differences of hormone values between juveniles and adults of each sex was unexpected. Juvenile individuals would be expected to have lower basal levels of T and E2, and conversely, adults would have significantly higher levels (e.g. Webb et al., 2002; Craig et al., 2009; Petochi et al., 2011). These high levels of T and E2 in the SRE could be attributed to individuals that are considered late-stage juvenile by FL that were undergoing sexual maturation at the time of sampling. The gonadal changes associated with maturation could manifest as higher circulating levels of reproductive hormones in the blood. The logistic model created in the present study provides the first species-specific method of sex determination with hormones for both juvenile and adult Atlantic sturgeon, providing a new tool for future studies using RIA. The classification used in this study from Webb et al. (2002) has been successfully used in lake sturgeon (Acipenser fulvescens), showing the utility of these types of classification equations even across sturgeon species (Craig et al., 2009; Shaw et al., 2012; Thiem et al., 2013). However, the model created herein needs further investigation with spawning populations, in which mean hormone concentrations are generally higher than the mean values of T and E2 in the SRE (Van Eenennaam et al., 1996). It is important to note that in the present study we could not validate sex by dissection or endoscopy because of the endangered status of the species. However, the combination of sex-determining methods from Webb et al. (2002) and Van Eenennaam et al. (1996) allows for a high degree of accuracy in the findings. A unique and unexpected finding of the present study was a shift to a female-dominated sex ratio that was observed in 2014, as non-spawning populations usually maintain a near 1:1 (male:female) sex ratio (Trencia et al., 2002). Therefore, a female-biased sex ratio of 1:3 (male:female) observed in a non-spawning system, such as the SRE, is uncommon. For example, in the St Lawrence River, Atlantic sturgeon have been found in varying sex ratios depending on the area, but all were relatively balanced, ranging from 1:1.13 to 1:1.32 (male:female) during the non-spawning seasons (Trencia et al., 2002). Likewise, in a shovelnose sturgeon (Scaphirhynchus platorynchus) population, the total sex ratio was 1.2:1 (male:female), but shifted to 2.3:1 when only spawning individuals were considered (Moos, 1978). Although sex ratios by season had a small sample size (n = 9), precluding a statistical test, more male Atlantic sturgeon were observed during April and May. Atlantic sturgeon in the SRE enter the system in mid-spring (Little 2013), with results from the present study probably substantiating that a male cohort is the first to aggregate (Table 3). This finding is not uncommon as male-dominated sex ratios have been documented in other sturgeon species. For example, in shortnose sturgeon (Acipenser brevirostrum), during spawning aggregations sex ratios have been documented ranging from 2.5:1 to 7:1 (male:female) in various systems (Pekovitch, 1979; Taubert, 1980a, b; Buckley and Kynard, 1985b; Collins and Smith, 1997). Likewise, in lake sturgeon (Acipenser fulvescens) a ratio of 2.1:1 (male:female) was observed in a spawning aggregation (Thiem et al., 2013). However, unlike male sturgeon in other systems, the male-skewed sex ratio is probably not a product of a spawning aggregation, and further investigation of this observation on a large temporal scale would help to elucidate whether the observed skewed sex ratio has ecological or biological significance. Although the SRE cannot be used as a spawning ground owing to lack of assess by unpassable dams, preliminary research suggests it may serve as a foraging site (Little, 2013). Sand lance (Ammodytes americanus) comprise the majority (Percent Index of Relative Importance = 83.4%) of the Atlantic sturgeon diet in the SRE, which has not been documented elsewhere. The high nutrient content of Atlantic sand lance could be beneficial energy for growth of juveniles and future reproduction in adults, in which nutrient-rich food sources can increase fecundity and egg viability (Ouellet et al., 2001; Cerdá et al., 2004). However, more research is needed in this area to determine whether foraging in the SRE affects energy stores (i.e. fatty acids) that in turn could affect Atlantic sturgeon population health outside of this small estuarine system. In summary, the present study used a non-lethal approach to determine the sex, reproductive status and the overall sex ratio of Atlantic sturgeon in the SRE. By doing so, the present investigation illustrates the need to gain a better understanding of the importance of small estuaries to the GOM DPS unit and the Atlantic sturgeon population as a whole. For future conservation of this species, non-lethal methods should be applied to other understudied river systems to complete a larger data set of Atlantic sturgeon throughout their range. Acknowledgements We thank the undergraduate and graduate students of the Sulikowski laboratory at the University of New England; in particular, Bianca Prohaska. Furthermore, the authors thank Drs Gayle Zydlewski and Michael Kinnison for their helpful comments and guidance during the writing process of this work. Finally, we thank the Marine Science Center staff for logistical support. This research was approved by the University of New England's Animal Care and Use Committee (institutional animal care and use protocol no. 20101221SUL and UNE-20140401SULIJ) and was part of a research requirement for the undergraduate honours programme at the University of New England. This manuscript represents Marine Science Center contribution number 94. Funding This work was supported by National Oceanic and Atmospheric Administration (grant no. NA10NMF4720023; special permit 45-04); Summer Undergraduate Research Experience (SURE) at the University of New England; and the Maine Space Grant Consortium. References Amiri BM , Maebayashi M, Adachi S, Yamauchi K ( 1996 a) Testicular development and serum sex steroid profiles during the annual sexual cycle of the male sturgeon hybrid, the bester . J Fish Biol 48 : 1039 – 1050 . OpenURL Placeholder Text WorldCat Amiri BM , Maebayashi M, Adachi , S, Yamauchi K ( 1996 b) Ovarian development and serum sex steroid and vitellogenin profiles in the female cultured sturgeon hybrid, the bester . J Fish Biol 48 : 1164 – 1178 . Google Scholar Crossref Search ADS WorldCat Atlantic States Marine Fisheries Commission ( 1990 ) Interstate fishery management plan for Atlantic sturgeon. Fisheries Management Report No. 17. Atlantic States Marine Fisheries Commission, Washington, DC, 73 pp. Atlantic States Marine Fisheries Commission ( 1998 ) Amendment 1 to the interstate fishery management plan for Atlantic sturgeon. Management Report No. 31. Atlantic States Marine Fisheries Commission, Washington, DC, 43 pp. Atlantic sturgeon Status Review Team (ASSRT) ( 2007 ) Status Review of Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus). Report to National Marine Fisheries Service, Northeast Regional Office. 23 February 2007, 174 pp. Bain MB ( 1997 ) Atlantic and shortnose sturgeons of the Hudson River: common and divergent life history attributes . Environ Biol Fishes 48 : 347– 358 . Google Scholar Crossref Search ADS WorldCat Barannikova IA , Bayunova LV, Semenkova TB ( 2004 ) Serum levels of testosterone, 11- ketotestosterone and oestradiol-17β in three species of sturgeon during gonadal development and final maturation induced by hormonal treatment . J Fish Biol 64 : 1330– 1338 . Google Scholar Crossref Search ADS WorldCat Boreman J ( 1997 ) Sensitivity of North American sturgeons and paddlefish to fishing mortality . Environ Biol Fishes 48 : 399– 405 . Google Scholar Crossref Search ADS WorldCat Brothers LL , Belknap DF, Kelley JT, Janzen CD ( 2008 ) Sediment transport and dispersion in a cool-temperate estuary and embayment, Saco River estuary, Maine, USA . Mar Geol 251 : 183 – 194 . Google Scholar Crossref Search ADS WorldCat Buckley J , Kynard B ( 1985 b) Yearly movements of shortnose sturgeons in the Connecticut River . Trans Am Fish Soc 114 : 813 – 820 . Google Scholar Crossref Search ADS WorldCat Ceapa C , Williot P, Le Menn F, Davail-Cuisset B ( 2002 ) Plasma sex steroids and vitellogenin levels in stellate sturgeon (Acipenser stellatus Pallas) during spawning migration in the Danube River . J Appl Ichthyol 18 : 391 – 396 . Google Scholar Crossref Search ADS WorldCat Cerdá J , Carrillo M, Zanuy S, Ramos J, Higuera M ( 2004 ) Influence of nutritional composition of diet on sea bass, Dicentrarchus labrax L., reproductive performance and egg and larval quality . Aquaculture 128 : 345 – 361 . Google Scholar Crossref Search ADS WorldCat Collins MR , Smith TIJ ( 1997 ) Distribution of shortnose and Atlantic sturgeons in South Carolina . N Am J Fish Manage 17 : 995 – 1000 . Google Scholar Crossref Search ADS WorldCat Colombo RE , Wills PS, Garvey JE ( 2004 ) Use of ultrasound imaging to determine sex of shovelnose sturgeon . N Am J Fish Manage 24 : 322 – 326 . Google Scholar Crossref Search ADS WorldCat Craig JM , Papoulias DM, Thomas MV, Annis ML, Boase J ( 2009 ) Sex assignment of lake sturgeon (Acipenser fluvescens) based on plasma sex hormone and vitellogenin level . J Appl Ichthyol 25 : 60 – 67 . Google Scholar Crossref Search ADS WorldCat Cuisset B , Fostier A, Williot P, Bennetau-Pelissero , C, Le Menn , F ( 1995 ) Occurrence and in vitro biosynthesis of 11-ketotestosterone in Siberian sturgeon, Acipenser baeri Brandt maturing females . Fish Physiol Biochem 14 : 313 – 322 . Google Scholar Crossref Search ADS PubMed WorldCat Davail-Cuisset B , Rouault T, Williot P ( 2011 ) Estradiol, testosterone, 11-ketotestosterone, 17,20β-dihydroxy-4-pregnen-3-one and vitellogenin plasma levels in females of captive European sturgeon, Acipenser sturio . J Appl Ichthyol 27 : 666 – 672 . Google Scholar Crossref Search ADS WorldCat Doroshov SI , Moberg GP, Van Eenennaam JP ( 1997 ) Observations on the reproductive cycle of cultured white sturgeon, Acipenser transmontanus . Environ Biol Fishes 48 : 265 – 278 . Google Scholar Crossref Search ADS WorldCat Furey NB , Sulikowski JA ( 2011 ) The fish assemblage structure of the Saco River estuary . Northeast Nat 18 : 37 – 44 . Google Scholar Crossref Search ADS WorldCat Heise RJ , Bringolf RB, Patterson R, Cope WG, Ross ST ( 2009 ) Plasma vitellogenin and estradiol concentrations in Adult Gulf Sturgeon from the Pascagoula River Drainage, Mississippi . Trans Am Fish Soc 138 : 1028 – 1035 . Google Scholar Crossref Search ADS WorldCat Kahn J , Mohead M ( 2010 ) A Protocol for Use of Shortnose, Atlantic, Gulf, and Green Sturgeons. US Department of Commerce, NOAA Technical Memo. NMFS-OPR-45, 62 pp. Kahnle AW , Hattala KA, McKown KA, Shirey CA, Collins MR, Squiers TS Jr, Savoy T ( 1998 ) Stock status of Atlantic sturgeon of Atlantic Coast estuaries. Report for the Atlantic States Marine Fisheries Commission. Draft III. Kahnle AW , Laney RW, Spear BJ ( 2005 ) Proceedings of the workshop on status and management of Atlantic sturgeon. Raleigh, NC 3–4 November 2003. Special Report No. 84 of the Atlantic States Marine Fisheries Commission. Keyvanshokooh S , Gharaei A ( 2010 ) A review of sex determination and searches for sex-specific markers in sturgeon . Aquac Res 41 : e1 – e7 . Google Scholar Crossref Search ADS WorldCat Kynard B , Kieffer M ( 2002 ) Use of a borescope to determine the sex and egg maturity stage of sturgeons and the effect of borescope use on reproductive structures . J Appl Ichthyol 18 : 505 – 508 . Google Scholar Crossref Search ADS WorldCat Little CE ( 2013 ) Movement and habitat use of Atlantic (Acipenser oxyrinchus oxyrinchus) and shortnose sturgeon (Acipenser brevirostrum) in the Saco River estuary system. Master's Thesis, University of New England, Biddeford, ME, USA, 75 pp. Moberg GP , Watson JD, Doroshov SI, Papkoff H, Pavlick RJ Jr ( 1995 ) Physiological evidence for two sturgeon gonadotropins in Acipenser transmontanus . Aquaculture 135 : 27 – 39 . Google Scholar Crossref Search ADS WorldCat Moos RE ( 1978 ) Movement and reproduction of shovelnose sturgeon, Scaphirhynchus platorynchus (Rafinesque), in the Missouri River South Dakota. PhD Dissertation, University of South Dakota, Vermillion, SD, USA, 213 pp. Niklitschek EJ , Secor DH ( 2005 ) Modeling spatial and temporal variation of suitable nursery habitats for Atlantic sturgeon in the Chesapeake Bay . Estuar Coast Shelf Sci 64 : 135 – 148 . Google Scholar Crossref Search ADS WorldCat Ouellet P , Lambert Y, Berube I ( 2001 ) Cod egg characteristics and viability in relation to low temperature and maternal nutritional condition . ICES J Mar Sci 58 : 672 – 686 . Google Scholar Crossref Search ADS WorldCat Pekovitch AW ( 1979 ) Distribution and some life history aspects of shortnose sturgeon (Acipenser brevirostrum) in the upper Hudson River Estuary . Hazleton Environmental Sciences Corporation, Illinois . 67 pp. Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Petochi BT , Di Marco P, Donadelli V, Longobardi A, Corsalini I, Bertotto D, Finoia MG, Marino G ( 2011 ) Sex and reproductive stage identification of sturgeon hybrids (Acipenser naccarii × Acipenser baerii) using different tools: ultrasounds, histology and sex steroids . J Appl Ichthyol 27 : 637 – 642 . Google Scholar Crossref Search ADS WorldCat Prohaska BK , Tsang PCW, Driggers WB, Hoffmayer ER, Sulikowski JA ( 2013 ) Development of a non-lethal and minimally invasive protocol to study elasmobranch reproduction . Mar Coast Fish 5 : 181 – 188 . Google Scholar Crossref Search ADS WorldCat Reynolds W , Casterlin M ( 1985 ) Vagile macrofauna and the hydrographic environment of the Saco River estuary and adjacent waters of the Gulf of Maine . Hydrobiologia 128 : 207 – 215 . Google Scholar Crossref Search ADS WorldCat Scott WB , Crossman EJ ( 1973 ) Freshwater fishes of Canada. Bulletin 184. Fisheries Research Board of Canada, Ottawa, ON, Canada, 966 pp. Secor DH ( 2002 ) Atlantic sturgeon fisheries and stock abundances during the late nineteenth century. AFS Symposium 28 : 89 – 98 . Secor DH , Waldman JR ( 1999 ) Historical abundance of Delaware Bay Atlantic sturgeon and potential rate of recovery. AFS Symposium 23 : 203 – 216 . Semenkova T , Barannikova I, Kime DE, McAllister BG, Bayunova L, Dyubin V, Kolmakov N ( 2002 ) Sex steroid profiles in female and male stellate sturgeon (Acipenser stellatus Pallas) during final maturation induced by hormonal treatment . J Appl Ichthyol 18 : 375 – 381 . Google Scholar Crossref Search ADS WorldCat Shaw SL , Chipps SR, Windels SK, Webb MAH, McLeod DT, Willis DW ( 2012 ) Lake sturgeon population attributes and reproductive structure in the Namakan Reservoir, Minnesota and Ontario . J Appl Ichthyol 28 : 168 – 175 . Google Scholar Crossref Search ADS WorldCat Smith TIJ , Clungston JP ( 1997 ) Status and management of Atlantic sturgeon, Acipenser oxyrinchus, in North America . Environ Biol Fish 48 : 335 – 346 . Google Scholar Crossref Search ADS WorldCat Sulikowski JA , Tsang PCW, Howell HW ( 2004 ) An annual cycle of steroid hormone concentrations and gonad development in the winter skate, Leucoraja ocellata, from the western Gulf of Maine . Mar Biol 144 : 845 – 853 . Google Scholar Crossref Search ADS WorldCat Sulikowski , JA , Wheeler CR, Gallagher AJ, Prohaska BK, Langan JA, Hammerschlag N ( 2016 ) Variation in the reproductive ecology of a marine apex predator, the tiger shark (Galeocerdo cuvier), at a protected female aggregation site . Aquatic Biol 24 : 175 – 184 . Google Scholar Crossref Search ADS WorldCat Taubert BD ( 1980 a) Biology of shortnose sturgeon, Acipenser brevirostrum, in the Holyoke Pool, Connecticut River, Massachusetts. Doctoral dissertation, University of Massachusetts, Amherst, MA, USA. Taubert BD ( 1980 b) Reproduction of shortnose sturgeon, Acipenser brevirostrum, in the Holyoke Pool, Connecticut River, Massachusetts . Copeia 1980 : 114 – 117 . Google Scholar Crossref Search ADS WorldCat Thiem JD , Hatin D, Dumont P, Van Der Kraak G, Cooke SJ ( 2013 ) Biology of lake sturgeon (Acipenser fulvescens) spawning below a dam on the Richelieu River, Quebec: behaviour, egg deposition, and endocrinology. Can J Zool 91 : 175 – 186 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Trencia G , Verreault G, Georges S, Pettigrew P ( 2002 ) Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus) fishery management in Quebec, Canada, between 1994 and 2000 . J Appl Ichthyol 18 : 455 – 462 . Google Scholar Crossref Search ADS WorldCat Tsang PCW , Callard IP ( 1987 ) Morphological and endocrine correlates of the reproductive cycle of the aplacental viviparous dogfish, Squalus acanthias . Gen Comp Endocrinol 66 : 182 – 189 . Google Scholar Crossref Search ADS PubMed WorldCat US Army Corps of Engineers ( 2013 ) Section 111 Shore Damage Mitigation Project Draft Decision Document and Environmental Assessment Including Finding of No Significant Impact and Section 404(b)(1). New England District, U.S. Army Corps of Engineers, Concord, MA, pp 11. Van Eenennaam JP , Doroshov SI, Moberg GP, Watson JG, Moore DS, Linares J ( 1996 ) Reproductive conditions of the Atlantic sturgeon (Acipenser oxyrhynchus) in the Hudson River . Estuaries 19 : 769 – 777 . Google Scholar Crossref Search ADS WorldCat Walker TI ( 2005 ) Reproduction in fisheries science. In WC Hamlett, BGM Jamieson,eds, Reproductive Biology and Phylogeny of Chondrichthyes (Sharks, Batoids and Chimaeras) . Science Publishers , Enfield , pp 81 – 126 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Webb MAH , Feist GW, Foster EP, Schreck CB, Fitzpatrick MS ( 2002 ) Potential classification of sex and stage of gonadal maturity of wild white sturgeon using blood plasma indicators . Trans Am Fish Soc 131 : 132 – 142 . Google Scholar Crossref Search ADS WorldCat Author notes " Editor: Steven Cooke © The Author 2016. Published by Oxford University Press and the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
TI - Using circulating reproductive hormones for sex determination of Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus) in the Saco River estuary, Maine
JF - Conservation Physiology
DO - 10.1093/conphys/cow059
DA - 2016-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/using-circulating-reproductive-hormones-for-sex-determination-of-kNJjz2fJ5E
VL - 4
IS - 1
DP - DeepDyve
ER -