Comparative stable isotope analyses of green sturgeon (Acipenser medirostris) and white sturgeon (A. transmontanus) in the San Francisco estuary.

Green sturgeon (Acipenser medirostris) and white sturgeon (A. transmontanus) are closely related, sympatric species that inhabit the San Francisco estuary. Green sturgeon have a more marine life history but both species spawn in the Sacramento River and reside for some duration in San Francisco Bay. These sturgeons are of conservation concern, yet little is known about their dietary competition when they overlap in space and time. To examine evidence of dietary differentiation, we collected whole blood and blood plasma from 26 green sturgeon and 35 white sturgeon in San Francisco Bay. Using carbon and nitrogen stable isotope analyses, we compared their relative trophic levels and foraging locations along the freshwater to marine gradient. Sampling blood plasma and whole blood allowed comparison of dietary integration over shorter and longer time scales, respectively. Plasma and whole blood δ13 C values confirmed green sturgeon had more marine dietary sources than white sturgeon. Plasma δ15 N values revealed white sturgeon fed at lower trophic levels than green sturgeon recently, however, whole blood δ15 N values demonstrated the two species fed at the same trophic level over longer time scales. Larger individuals of both species had higher δ13 C values than smaller individuals, reflecting more marine food sources in adulthood. Length did not affect δ15 N values of either species. Isotope analyses supported the more marine life history of green than white sturgeon and potentially highlight a temporary trophic differentiation of diet between species during and preceding the overlapping life stage in San Francisco Bay.

Organochlorine and Metal Contaminants in the Blood Plasma of Green Sturgeon Caught in Washington Coastal Estuaries.

Non-invasive monitoring was used to evaluate the concentrations of 40 contaminants in the blood plasma of the North American Green Sturgeon Acipenser medirostris caught and released from three estuaries in Washington State. The highest contaminant concentrations were found in fish caught in the most urbanized sites. Few statistical differences were found when evaluating contaminant levels according to sex, maturation stage, or distinct population segments of Green Sturgeon. The results indicate that recent exposure to legacy contaminants was reflected in Green Sturgeon plasma. Aldrin, 4,4-DDE, α-HCH, copper, and selenium were the most frequently detected contaminants. This study also explored the challenges of assessing toxicity in threatened species using non-lethal approaches. There is currently a lack of environmental contaminant monitoring data in estuaries frequented by Green Sturgeon and limited plasma to tissue toxicity correlations.

Quantification of steroid hormones in low volume plasma and tissue homogenates of fish using LC-MS/MS.

Quantification of steroid hormones in fish is an important step for toxicology and endocrinology studies. Among the hormone analysis techniques, liquid chromatography tandem mass spectrometry (LC-MS/MS) has widely been used for measuring hormones in various biological samples. Despite all improvements in the technique, detection of several hormones in a low volume of serum or plasma is still challenging. We developed a robust method for simultaneous quantification of 14 steroid hormones including corticosterone, cortisol, 11-ketotestosterone, progesterone, testosterone, 17OH-progesterone, aldosterone, dihydrotestosterone, estrone, 17ß-estradiol, estriol, ethinylestradiol, levonorgestrel and equilin from volumes as low as 10 µL serum or plasma in a short run by LC-MS/MS. The lowest limit of detection in 10 µL serum was 0.012 ng/mL measured for cortisol, progesterone, testosterone, 17OH-progesterone and estrone. Use of high (25 times more) serum volume improved detection limit of hormones by 2-40 times. The method was compared with the radioimmunoassay technique in which testosterone and 17ß-estradiol were highly correlated with R2 of 0.95 and 0.96, respectively. We validated the method by measuring four selected hormones, in low and high plasma volumes of largemouth bass (Micropterus salmoides). In addition, we developed a method to quantify hormones in whole body fish homogenates of small fish and compared the values to plasma concentrations, using fathead minnow (Pimephales promelas). Calculated concentrations of the hormones in plasma were consistent with those in the homogenate and 11-ketotestosterone and 17ß-estradiol were significantly different in males and females. The ability to measure hormones from whole body homogenates was further evaluated in two model small fish species, zebrafish (Danio rerio) and juvenile silverside (Menidia beryllina). These results suggest that whole tissue homogenate is a reliable alternative for hormone quantification when sufficient plasma is not available.

Tissue Contaminant Burdens in San Francisco Estuary White Sturgeon (Acipenser transmontanus): Implications for Population Recovery.

The San Francisco Estuary (SFE) is heavily influenced by anthropogenic activities, including historic and chronic contaminant inputs. These contaminants can adversely affect SFE fish populations, particularly white sturgeon, because they are a benthic dwelling, long-lived species. We measured a suite of metals and organic contaminants in liver and gonad tissues of 25 male and 32 female white sturgeon as well as several physiological indicators of sturgeon health. Most sturgeon (68% of males and 83% of females) were estimated to be between 13 and 17 years of age. Sturgeon tissues had elevated concentrations of several metals, including As, Ba, Cd, Cu, Cr, Pb, Hg, Ni, Se, and Zn. The most frequently detected organic contaminants in sturgeon livers and gonads were DDE, PCBs, PBDEs, and galaxolide. Selenium was detected at levels similar to those shown to cause impaired liver physiology and reproductive success in white sturgeon. Observed Hg levels were higher than those shown to result in lower condition factor and gonadosomatic indices in white sturgeon. Liver galaxolide levels correlated with decreased plasma estradiol levels in female sturgeon. The Cd, As, and Cu warrant further investigation, because they were detected at levels known to impair fish health. Our results suggest contaminants are negatively affecting SFE white sturgeon health and fitness. Future SFE white sturgeon contaminant research is suggested.

Confirmation of ovarian homogeneity in post-vitellogenic cultured white sturgeon, Acipenser transmontanus.

Assessing stage of oocyte maturity in female sturgeon by calculating oocyte polarization index (PI) is a necessary tool for both conservation propagation managers and caviar producers to know when to hormonally induce spawning. We tested the assumption that sampling ovarian follicles from one section of one ovary is sufficient for calculating an oocyte PI representative of oocyte maturity for an individual animal. Short-wavelength near-infrared spectroscopy (SW-NIR) scans were performed on three positions per ovary for five fish prior to caviar harvest. Samples of ovarian follicles were subsequently taken from the exact location of the SW-NIR scans for calculation of oocyte PI and follicle diameter. Oocyte PI was statistically different though not biologically relevant within an ovary and between ovaries in four of five fish. Follicle diameter was statistically different but not biologically relevant within an ovary in three of five fish. There were no differences in follicle diameter between ovaries. No statistical differences were observed between SW-NIR spectra collected at different locations within an ovary or between ovaries. These results emphasize the importance of utilizing both oocyte PI measurement and progesterone-induced oocyte maturation assays while deciding when to hormonally induce spawning in sturgeon females.

Research tools to investigate movements, migrations, and life history of sturgeons (Acipenseridae), with an emphasis on marine-oriented populations.

Worldwide, sturgeons (Acipenseridae) are among the most endangered fishes due to habitat degradation, overfishing, and inherent life history characteristics (long life span, late maturation, and infrequent spawning). As most sturgeons are anadromous, a considerable portion of their life history occurs in estuarine and marine environments where they may encounter unique threats (e.g., interception in non-target fisheries). Of the 16 marine-oriented species, 12 are designated as Critically Endangered by the IUCN, and these include species commercially harvested. We review important research tools and techniques (tagging, electronic tagging, genetics, microchemistry, observatory) and discuss the comparative utility of these techniques to investigate movements, migrations, and life-history characteristics of sturgeons. Examples are provided regarding what the applications have revealed regarding movement and migration and how this information can be used for conservation and management. Through studies that include Gulf (Acipenser oxyrinchus desotoi) and Green Sturgeon (A. medirostris), we illustrate what is known about well-studied species and then explore lesser-studied species. A more complete picture of migration is available for North American sturgeon species, while European and Asian species, which are among the most endangered sturgeons, are less understood. We put forth recommendations that encourage the support of stewardship initiatives to build awareness and provide key information for population assessment and monitoring.

Importance of environmental endocrinology in fisheries management and aquaculture of sturgeons.

Less is known about the reproductive endocrinology of sturgeons compared to modern teleosts. However, tools to assess the reproductive endocrinology and effects of environmental factors on reproduction do exist. This review utilizes case studies to describe the parameters involved in environmental endocrinology and the management and recovery efforts for the phylogenetically ancient sturgeon and paddlefish (Clade Chondrostei). Specifically, we discuss the use of environmental endocrinology to determine sex and stage of maturity and identify oviposition on spawning grounds, the importance of understanding endocrine disruption pathways, the challenges and benefits of assessing stress in wild populations of sturgeon, and three major physiological events in the reproductive development of farmed sturgeon understanding of which appears to be crucial for improving sturgeon aquaculture.

Distinguishing ovarian maturity of farmed white sturgeon (Acipenser transmontanus) by Fourier transform infrared spectroscopy: a potential tool for caviar production management.

Fourier transform infrared spectroscopy (FT-IR, 4000-400 cm(-1)) was applied to blood plasma of farmed white sturgeon (N = 40) to differentiate and predict the stages of ovarian maturity. Spectral features of sex steroids (approximately 3000 cm(-1)) and vitellogenin (approximately 1080 cm(-1)) were identified. Clear segregation of maturity stages (previtellogenesis, vitellogenesis, postvitellogenesis, and follicular atresia) was achieved using principal component analysis (PCA). Progression of oocyte development in the late phase of vitellogenesis was also monitored using PCA based on changes in plasma concentrations of sex steroid and lipid content. The observed oocyte polarization index (PI, a measure of nuclear migration) was correlated with changes in plasma sex steroid levels revealed by FT-IR PCA results. A partial least squares (PLS) model predicted PI values within the range 0.12-0.40 (R = 0.95, SEP = 2.18%) from differences in spectral features. These results suggest that FT-IR may be a good tool for assessing ovarian maturity in farmed sturgeon and will reduce the need for the invasive ovarian biopsy required for PI determination.

Calcium regulation in wild populations of a freshwater cartilaginous fish, the lake sturgeon Acipenser fulvescens.

Lake sturgeon, Acipenser fulvescens, are one of a few species of cartilaginous fishes that complete their life cycle entirely in freshwater. Sturgeons maintain very low concentrations of circulating calcium (Ca(2+)) compared with other vertebrates, and therefore, face unique challenges in regard to Ca(2+) regulation, which are likely to be magnified during vitellogenic stages of the reproductive cycle. In the present study, Ca(2+) concentrations and associated hormones of female and male lake sturgeon were examined in two wild populations, and were related to reproductive stage. In both populations, free, bound and total Ca(2+) were low, peaking in mid-late vitellogenic females. Internal Ca(2+) and phosphate (PO(4)(3-)) concentrations were inversely related to environmental concentrations, suggesting that these ions are preferentially retained and that mechanisms for mobilization are up-regulated under diminished environmental concentrations. Plasma 17beta-estradiol, 11-ketotestosterone and testosterone, peaked in mid-late vitellogenic females, while the androgens peaked in spawning males. Urine Ca(2+) was more tightly regulated than other divalent ions and decreased in spawning fish. Therefore, the increases in free plasma Ca(2+), the very low circulating concentrations of free and total Ca(2+), and the increase in PO(4)(3-) and bound Ca(2+) in low Ca(2+) environments indicate unique adaptations to Ca(2+) regulation in the lake sturgeon.

Identification of plasma glucocorticoids in pallid sturgeon in response to stress.

Compared to teleosts, little is known about the stress response in chondrosteans, and the glucocorticoid(s) most responsive to stress have never been definitively determined in sturgeon. In terms of cortisol production, pallid sturgeon (Scaphirhynchus albus) have a low physiological response to stress compared to other sturgeons (Acipenser s.p.). Because of this, our null hypothesis was that cortisol is not the predominant glucocorticoid secreted in response to stress in pallid sturgeon. Our objective was to identify the putative glucocorticoids present in the plasma of pallid sturgeon during the stress response. Pallid sturgeon were subjected to a severe confinement stress (12 h) with an additional handling stressor for the first 6 h. Control fish were not subjected to confinement but were handled only to collect blood. Blood plasma was collected at time 0, 6, and 12 h. Gas chromatography/mass spectrometry was used to screen the plasma for the spectrum of glucocorticoids and determine the putative steroid secreted during the stress response. Cortisol was the primary glucocorticoid detected in stressed pallid sturgeon. In addition, the cortisol metabolites cortisone, alloTHE (5alpha-pregnane-3alpha,17alpha,21-triol-11,20-dione), allo-alpha-cortolone (3alpha,17alpha,20alpha,21-tetrahydro-5alpha-pregnan-11-one), and allo-beta-cortolone (3alpha,17alpha,20beta,21-tetrahydro-5alpha-pregnan-11-one) were detected. Plasma cortisol increased from a resting concentration of 0.67 ng/ml to 10.66 ng/ml at 6h followed by a decrease to 6.78 ng/ml by 12 h. Plasma glucose increased significantly by time 6 and 12 h in both stressed and unstressed groups and remained elevated at time 12h, while resting lactate concentrations were low to non-detectable and did not increase significantly with the stressor over time. Cortisol was the primary glucocorticoid synthesized and secreted in response to a stressor in pallid sturgeon. Though the proportional increase in plasma cortisol in stressed pallid sturgeon was lower than many other species of sturgeon, the concentration was high enough to elicit a secondary stress response as seen by changes in plasma glucose.

Evidence of detrimental effects of environmental contaminants on growth and reproductive physiology of white sturgeon in impounded areas of the Columbia River.

This study sought to determine whether wild white sturgeon from the Columbia River (Oregon) were exhibiting signs of reproductive endocrine disruption. Fish were sampled in the free-flowing portion of the river (where the population is experiencing reproductive success) and from three reservoirs behind hydroelectric dams (where fish have reduced reproductive success). All of the 18 pesticides and almost all of the 28 polychlorinated biphenyls (PCBs) that were analyzed in livers and gonads were detected in at least some of the tissue samples. Metabolites of p,p -dichlorodiphenyltrichloroethane (DDT) [p,p -dichlorodiphenyldichloroethylene (DDE) and p,p -1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD)]were consistently found at relatively high levels in fish. Some males and immature females showed elevated plasma vitellogenin; however, concentrations were not correlated with any of the pesticides or PCBs analyzed. Negative correlations were found between a number of physiologic parameters and tissue burdens of toxicants. Plasma triglycerides and condition factor were negatively correlated with total DDT (DDD + DDE + DDT), total pesticides (all pesticides detected - total DDT), and PCBs. In males, plasma androgens and gonad size were negatively correlated with total DDT, total pesticides, and PCBs. Fish residing in the reservoir behind the oldest dam had the highest contaminant loads and incidence of gonadal abnormalities, and the lowest triglycerides, condition factor, gonad size, and plasma androgens. These data suggest that endocrine-disrupting chemicals may be accumulating behind dams over time. Overall, results of this study indicate that exposure to environmental contaminants may be affecting both growth and reproductive physiology of sturgeon in some areas of the Columbia River.

Ovarian steroidogenesis in white sturgeon (Acipenser transmontanus) during oocyte maturation and induced ovulation.

Ovarian follicles and plasma were collected from two female white sturgeon, Acipenser transmontanus, injected with sturgeon pituitary homogenate followed 12h later with GnRHa to induce ovulation. The oocytes of one female underwent germinal vesicle breakdown (GVBD) but ovulation did not occur in response to hormonal stimulation (Female 1), while the oocytes of the other female underwent GVBD and ovulation (Female 2). Follicles collected 12h after the first injection to induce ovulation were incubated with radioinert pregnenolone (P5) or tritiated-P5 ([3H]P5) plus radioinert P5. Steroids were extracted from media and intact follicles, and the extracts were fractionated by high performance liquid chromatography (HPLC). Fractions from the incubation with radioinert precursor were used in a bioassay to determine the potency of the steroid products to induce GVBD. Plasma levels of testosterone (T), estradiol, and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) were measured by radioimmunoassay during induced ovulation, and plasma collected at the time of ovulation (actual or expected) was analyzed by HPLC. A peak in plasma 17,20beta-P was detected at the time of the second injection to induce ovulation in Female 2 (the time at which follicles were collected for incubation with [3H]P5). The HPLC analysis revealed several progestins in the plasma of Female 2 at ovulation that were not present in Female 1 at the time of expected ovulation. A variety of C19 and C21 steroids were produced in vitro by ovarian follicles from both females. The "suggestive" identities of the major metabolites were 11-deoxycortisol, androstenedione, 17-hydroxyprogesterone (17OHP), and 17,20beta-P in Female 1 and cortisol, 17,20beta, 21-trihydroxyprogesterone (20beta-S), 11-deoxycortisol, T, 17OHP, and 17,20beta-P in Female 2. Several of the steroids were active in a GVBD bioassay, but the fractions that contained the steroid coeluting the authentic 11-deoxycortisol on the HPLC and 17,20beta-P (positively identified by gas chromatography-mass spectrometry) were found to be the most potent. The results from this study combined with the results of Webb et al. (2001b) suggest the potential roles of 11-deoxycortisol, 17,20beta-P, 20beta-S, and P4 as maturation-inducing steroids in sturgeon.