Effects of trophic 2,2', 4,4'-tetrabromodiphenyl ether (BDE-47) exposure in Oreochromis niloticus: A multiple biomarkers analysis.

Polybrominated diphenyl esters are emerging environmental contaminants with few toxicological data, being a concern for the scientific community. This study evaluated the effects of BDE-47 on the health of Oreochromis niloticus fish. The animals were exposed to three doses of BDE-47 (0, 0.253, 2.53, 25.3 ng g-1) every 10 days, for 80 days. The BDE-47 affected the hepatosomatic and gonadosomatic index in female and the condition factor by intermediate dose in both sexes. The levels of estradiol decreased and the T4 are increased, but the vitellogenin production was not modulated in male individuals. Changes in AChE, GST, LPO and histopathology were observed while the integrated biomarker response index suggests that the lowest dose of BDE-47 compromised the activity of antioxidant enzymes. The oral exposure to BDE-47 in environmental concentrations is toxic to O. niloticus and the use of multiple biomarkers is an attribution in ecotoxicology studies and biomonitoring programs.

Bisphenol A and Bisphenol S Induce Endocrine and Chromosomal Alterations in Brown Trout.

Bisphenol A is a widely used compound found in large amount of consumer products. As concerns have been raised about its toxicological and public health effect, the use of alternatives to bisphenol A are now increasing. Bisphenol S is one of the analogues being used as a replacement for bisphenol A despite the fact that little is known about the effects of bisphenol S on living organisms. In this study, we investigated the potential endocrine and genotoxic effects of bisphenol A and bisphenol S in juvenile brown trout (Salmo trutta). The fish were exposed to the compounds for either 2 weeks or 8 weeks via sustained-release cholesterol implants containing doses of 2 mg/kg fish or 20 mg/kg fish of the substances. The effects on the thyroid hormone levels and the estrogenic disrupting marker vitellogenin were evaluated, along with the genotoxic markers micronucleated cells and erythrocyte nuclear abnormalities. An increase in plasma vitellogenin was observed in fish exposed to the high dose of bisphenol A for 2 weeks. At this experimental time the level of the thyroid hormone triiodothyronine (T3) in plasma was elevated after bisphenol S exposure at the high concentration, and paralleled by an increase of micronucleated cells. Moreover, bisphenol A induced an increase of micronuclei frequency in fish erythrocytes after the exposure at the lowest dose tested. Taken together the results indicate that both bisphenol A and its alternative bisphenol S cause endocrine disrupting and genotoxic effects in brown trout, although suggesting two different mechanisms of damage underlying bisphenol A and bisphenol S activity.

The effects of bisphenol A on liver proteome and mucus vitellogenin in comparison to plasma as a non-invasive biomarker in immature Siberian sturgeons (Acipenser baerii).

This study was done to evaluate the effects of Bisphenol A (BPA) on Siberian sturgeon (Acipenser baerii). As liver is the main organ in the homeostatic adjustments to stress, we used a proteomics method to address molecular response in this tissue. Also, we compared the levels of vitellogenin in plasma and mucus to propose that the last one be a non-invasive method to analyze this biomarker. The fish received 1, 10, and 100 µg g-1 week-1 BPA intraperitoneally for two weeks. The samples were taken on days 0, 7, and 14. Plasma vitellogenin level increased as the highest value was recorded in the group with 100 µg g-1 week-1 of BPA. Changes in the mucus and blood vitellogenin showed a similar pattern, suggesting that mucus could be used for evaluating the changes in blood vitellogenin. Comparative proteomics was used to determine the proteome of the liver of A. baerii in the highest dose of BPA in comparison with the control. Sixteen proteins were identified that their expression changed at least twice between the studied groups. The proteomic results showed that BPA increased the expression of proteins involved in the detoxification and metabolism, activated glycolysis, and produced necrosis in the liver.

Micropollutants impair the survival of Oreochromis niloticus and threat local species from Iguaçu River, Southern of Brazil.

The wastewater contamination of urban rivers is a concern for biodiversity and a consequence from poor urban conservation policies. In the current study, the impact of urban and industrial activities was investigated in Iguaçu river (Southern Brazil) using juvenile Oreochromis niloticus, after trophic and chronic exposure (25, 50 and 100 %), over 81 days. After exposure liver, gills, gonads, brain, muscle, and blood were sampled for chemical, biochemical, histopathological, genotoxic and molecular analyses. Water levels of persistent organic pollutants such as polychlorinated biphenyls, organochlorine pesticides, polycyclic aromatics hydrocarbon (PAHs) and metals were investigated. The redox unbalance, histopathological and increase in vitellogenin expression in fish revealed both the bioavailability of micropollutants and their harmful effects. According to the results, the level of Iguaçu river pollution negatively impacts the health of O. niloticus revealing and highlighting the risk of this pollution exposure to biota and human populations.

Plasma Vitellogenin Reveals Potential Seasonal Estrogenicity in Fish from On-Site Wastewater Treatment Systems in Semi-Arid Streams Influenced by Snowmelt.

Effluents from on-site wastewater treatment systems can influence surface water quality, particularly when infrastructure is aging, malfunctioning, and improperly installed. Municipal wastewater often contains chemical compounds that can lead to adverse biological effects, such as reproductive impairment, in organisms that are chronically exposed. A significant number of these compounds are endocrine-disrupting chemicals. Water quality influences of on-site systems are poorly studied in semi-arid regions where instream flows are seasonally dependent on snowmelt, and when instream dilution of wastewater effluents is minimal during other times of the year. Here we examined surface water estrogenicity in low order tributaries of two unique semi-arid streams with on-site wastewater treatment systems, for which seasonal instream flow fluctuations occur in Park City, UT, USA. Water samples were collected from a total of five locations along two lotic systems downstream from active on-site treatment systems. Samples were extracted for targeted chemical analyses and to perform in vivo and in vitro bioassays with juvenile rainbow trout. Estrogenic activity was measured by quantifying the concentration and expression of vitellogenin (VTG) in plasma and liver, respectively. Plasma VTG presented elevated levels in fish exposed to water samples collected at the two sites in close proximity to on-site systems and during seasons with low stream discharge, though the levels observed did not suggest severe endocrine disruption. However, long-term exposure to these surface water could compromise the fish populations. While the sensitivity of in vitro bioassays was low and targeted chemical analyses did not identify causative compounds, the use of complementary lines of evidence (e.g., in vivo biological models) was advantageous in identifying estrogenic activity in waters influenced by effluents from on-site wastewater systems.

Impact of herbicide pretilachlor on reproductive physiology of walking catfish, Clarias batrachus (Linnaeus).

Herbicide pretilachlor is widely used in paddy fields to control annual weeds. The present study has been carried out in walking catfish, Clarias batrachus, to evaluate the impact of herbicide pretilachlor on reproductive physiology after chronic exposure. Based on the median lethal concentration value (96 h), fish were exposed to three nominal test concentrations of pretilachlor ((SL-I (1/20th LC50), SLII (1/15th LC50), and SL-III (1/10th LC50)) for 30, 45, and 60 days after which plasma sex steroid profile, plasma vitellogenin concentration, and gonadal aromatase activity were analyzed in both sexes. Plasma concentration of testosterone decreases in herbicide-exposed male fish. Significant increase in plasma 17ß-estradiol, plasma vitellogenin concentration, and gonadal aromatase activity were observed in herbicide-exposed male fish. All these alterations in reproductive parameters in male fish are dependent on concentration and exposure duration of herbicide. On the other hand, significant decrease in plasma concentration of testosterone was observed in female fish which was also dependent on concentration and exposure duration of herbicide. No significant changes in plasma 17ß-estradiol concentrations, plasma vitellogenin concentration, and gonadal aromatase activity were observed in female fish. Above findings clearly suggested that herbicide pretilachlor acts as endocrine disruptor in fish and affects overall reproductive physiology of fish, but its ability to induce reproductive toxicity in male and female differs considerably.

Tyramine and its receptor TYR1 linked behavior QTL to reproductive physiology in honey bee workers (Apis mellifera).

Honey bees (Apis mellifera) provide an excellent model for studying how complex social behavior evolves and is regulated. Social behavioral traits such as the division of labor have been mapped to specific genomic regions in quantitative trait locus (QTL) studies. However, relating genomic mapping to gene function and regulatory mechanism remains a big challenge for geneticists. In honey bee workers, division of labor is known to be regulated by reproductive physiology, but the genetic basis of this regulation remains unknown. In this case, QTL studies have identified tyramine receptor 1 (TYR1) as a candidate gene in region pln2, which is associated with multiple worker social traits and reproductive anatomy. Tyramine (TA), a neurotransmitter, regulates physiology and behavior in diverse insect species including honey bees. Here, we examine directly the effects of TYR1 and TA on worker reproductive physiology, including ovariole number, ovary function and the production of vitellogenin (VG, an egg yolk precursor). First, we used a pharmacology approach to demonstrate that TA affects ovariole number during worker larval development and increases ovary maturation during the adult stage. Second, we used a gene knockdown approach to show that TYR1 regulates vg transcription in adult workers. Finally, we estimated correlations in gene expression and propose that TYR1 may regulate vg transcription by coordinating hormonal and nutritional signals. Taken together, our results suggest TYR1 and TA play important roles in regulating worker reproductive physiology, which in turn regulates social behavior. Our study exemplifies a successful forward-genetic strategy going from QTL mapping to gene function.

Deleterious impacts of heat stress on steroidogenesis markers, immunity status and ovarian tissue of Nile tilapia (Oreochromis niloticus).

The water temperature of aquacultures is a primary factor of fish welfare, reproductive patterns, and immunity. To elucidate the molecular and biological processes of the temperature modulation of reproduction and immunity, female Nile tilapia (190 ± 10g) were allocated into five groups following acclimatization (150 females, three replicates, each n = 10). Each group was subjected to various temperatures (28 °C, 30 °C, 32 °C, 34 °C, and 37 °C), the group at 28 °C representing the control. Their serum levels of estradiol, cortisol, and vitellogenin were measured as well as serum triiodothyronine (T3) hormone, thyroxine (T4) hormone, and non-specific immunity (phagocytic and lysozyme activity). In addition, steroidogenic acute regulatory protein (STAR), vitellogenin gene receptor, and heat shock protein 70 (HSP70) gene expression were evaluated. The serum levels of estradiol, cortisol, and vitellogenin markedly declined (P < 0.05) in fish group at higher temperatures. In addition to T3, T4 was significantly affected (P < 0.05) in the control group. The expressions of the STAR gene (steroidogenesis) and vitellogenin receptors were also considerably down-regulated. The histopathological photomicrograph of fish subjected to high water temperature revealed injuries in ovary tissues, demonstrating its harmful effects. The experimental results verified the possible role of water temperature as a main stressor on Nile tilapia' physiology through modulation of steroidogenesis-related gene expression and immunity.

Identification and structural characterization of the factors involved in vitellogenesis and its regulation in the African Osteoglossiforme of aquacultural interest Heterotis niloticus (Cuvier, 1829).

The African bonytongue (Heterotis niloticus) is an excellent candidate for fish farming because it has outstanding biological characteristics and zootechnical performances. However, the absence of sexual dimorphism does not favor its reproduction in captivity or the understanding of its reproductive behavior. Moreover, no molecular data related to its reproduction is yet available. This study therefore focuses on the structural identification of the different molecular actors of vitellogenesis expressed in the pituitary gland, the liver and the ovary of H. niloticus. A transcriptomic approach based on de novo RNA sequencing of the pituitary gland, ovary and liver of females in vitellogenesis led to the creation of three transcriptomes. In silico analysis of these transcriptomes identified the sequences of pituitary hormones such as prolactin (PRL), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and their ovarian receptors (PRLR, FSHR, LHR). In the liver and ovary, estrogen receptors (ER) beta and gamma, liver vitellogenins (VtgB and VtgC) and their ovarian receptors (VLDLR) were identified. Finally, the partial transcript of an ovarian Vtg weakly expressed compared to hepatic Vtg was identified based on structural criteria. Moreover, a proteomic approach carried out from mucus revealed the presence of one Vtg exclusively in females in vitellogenesis. In this teleost fish that does not exhibit sexual dimorphism, mucus Vtg could be used as a sexing biomarker based on a non-invasive technique compatible with the implementation of experimental protocols in vivo.

Trade-off between aerobic performance and egg production in migratory macaroni penguins.

When successive stages of an organism's life-history overlap, conflicts and trade-offs can emerge due to competition among physiological pathways. For example, long periods of sustained locomotion in migrating birds are supported by the androgenic up-regulation of aerobic factors, such as new red blood cell production and hematocrit. However, towards the end of migration, many female birds begin up-regulating 17ß-estradiol (E2) to support vitellogenesis and egg production, but E2 secretion is known to have suppressive effects on red blood cell production (anti-erythropoiesis). We explored potential trade-offs between factors related to aerobic performance (hematocrit, reticulocyte index) and the expression of factors related to E2-mediated vitellogenesis (i.e. yolk precursor production) in female macaroni penguins (Eudyptes chrysolophus), a species in which the physiologies controlling egg production and migratory activity run simultaneously (e.g. females experience a migratory conflict). We collected blood samples from penguins immediately upon their return to the colony, prior to egg laying. Hematocrit was elevated when the penguins returned to the colony (50.05% ± 3.40 SD), which is similar to pre-laying values observed in other migratory bird species. Furthermore, mean reticulocyte levels were elevated (34.87% ± 2.34), which is the highest level yet recorded in birds. Similarly, both plasma vitellogenin and yolk-targeted very low density lipoprotein levels were upregulated (2.30 ± 0.06 µg Zn ml-1, and 9.70 ± 0.19 mmol l-1, respectively), indicating that penguins were reproductively active and producing eggs during migration and upon arrival on land. As predicted, a negative relationship between hematocrit and plasma vitellogenin was found, but we found no evidence to suggest that birds were experiencing reproductive anemia. Alternatively, we attribute the negative relationship to a hemodilution effect of yolk precursor secretion into circulation. It appears that female macaroni penguins are able to preserve hematocrit levels and new red blood cell production when migratory activity overlaps with reproductive processes.

Impacts of herbicide pendimethalin on sex steroid level, plasma vitellogenin concentration and aromatase activity in teleost Clarias batrachus (Linnaeus).

Pendimethalin (PM) is a selective herbicide, widely present in aquatic environment. It causes detrimental effects in fishes, but little is known regarding its reproductive toxicity. The present study was carried out in Clarias batrachus exposed to sub lethal concentrations of PM for 30, 45 and 60 days. Male fish showed a significant increase in plasma 17ß-estradiol (E2) however plasma E2 in females was not affected. Plasma testosterone levels were significantly decreased in both sexes. In male plasma vitellogenin (VTG) and gonadal aromatase activity was increased irrespective of herbicide concentration and exposure duration. In females concentration and time dependent reduction in plasma VTG but no significant change in the gonadal aromatase activity were observed. Results indicated that PM act as endocrine disruptor but act differentially in male and female fishes and plasma E2, T and VTG levels and aromatase activity can be considered as reliable biomarkers for PM toxicity in fishes.

Could the musk compound tonalide affect physiological functions and act as an endocrine disruptor in rainbow trout?

In the present study, the effect of polycyclic musk compound tonalide (AHTN) in two concentrations was studied in male rainbow trout (Oncorhynchus mykiss, Walbaum 1792). A feeding trial was conducted with AHTN incorporated into feed granules. One concentration was environmentally relevant (854 µg/kg); the second one was 10× higher (8699 µg/kg). The fish were fed twice a day with the amount of feed at 1 % of their body weight. After an acclimatization period, the experimental phase in duration of six weeks followed. At the end of the experiment, fish were sampled and the biometrical data were recorded. Subsequently, hematological and biochemical tests, histopathological examination, analysis of oxidative stress markers and evaluation of endocrine disruption using plasma vitellogenin were performed. In conclusion, an increase of hematocrit for both AHTN concentrations was found, but no significant changes were observed in biochemical profile. Moreover, AHTN caused lipid peroxidation in caudal kidney tissue, which was confirmed by histopathological images. The long-lasting AHTN exposure could thus be harmful for maintaining homeostasis in the rainbow trout organism. However, the vitellogenin concentration seemed not to be affected by AHTN.

Distribution of vitellogenin in Japanese flounder (Paralichthys olivaceus) for biomarker analysis of marine environmental estrogens.

Estrogen pollution in marine environments has become a research hotspot due to its adverse effects on the reproduction of wild organisms. To early detection of estrogen pollution, this study developed two methods for detecting Japanese flounder vitellogenin (Vtg), a sensitive biomarker for environmental estrogens. Firstly, monoclonal antibodies (mAb) specific to Vtg were prepared using purified lipovitellin (Lv), a main Vtg-derived yolk protein. Anti-Lv mAb (C1F1) had the highest titer (1:256,000) and was labeled with fluorescein isothiocyanate to establish a direct immunofluorescence (DIF) method for histological detection of Vtg in tissues. Additionally, using the purified Lv and mAb, an enzyme-linked immunosorbent assay (ELISA) was developed and this assay had a detection limit of 0.75 ng/mL and a working range of 1.95-250 ng/mL. Furthermore, Vtg induction in the plasma of Japanese flounder exposed to 17ß-estradiol (E2), 17α-ethinylestradiol (EE2), and bisphenol A (BPA) were quantified by ELISA, and Vtg induction in the liver of EE2-exposed Japanese flounder were measured by DIF. Finally, the distribution of Vtg in Japanese flounder was detected using these two methods. The results revealed that Vtg mainly appeared in the terminal tail fin, liver, kidney, intestine, and spleen. Considering the high concentration of Vtg and easy sample collection, the terminal tail fin could be a new alternative to plasma for Vtg quantification, while kidney and liver are suitable for histological detection of Vtg.

Reproductive cycle of northern largemouth bass Micropterus salmoides salmoides.

The seasonal dynamics of the northern largemouth bass (Micropterus salmoides salmoides) reproductive cycle was investigated in a Midwestern United States population. Mature largemouth bass (n = five males and five females) were collected each month over a one-year period. Environmental cues (water temperature and photoperiod) were recorded in situ using data loggers. Temporal changes in plasma sex steroid hormones (testosterone [T], 11-ketotestosterone [11-KT], and estradiol-17ß [E2 ]) and vitellogenin (Vtg), gamete development (histology), and organosomatic indices were highly associated with temporal water temperature and photoperiod dynamics. Female plasma concentrations of T and E2 and organosomatic indices exhibited similar temporal patterns, with peaks occurring in early summer. Gamete development in females was associated with increases in gonadosomatic index (GSI) and Vtg. In males, plasma concentrations of T and organosomatic indices exhibited bimodal peaks; high peaks occurred in the spring for T and GSI and in the fall for viscerosomatic and hepatosomatic indices. Male gamete development and GSI closely tracked temporal patterns in plasma T, with GSI lagging behind the T cycle by approximately one sampling period. Plasma concentrations of 11-KT were elevated in late winter and spring and were considerably higher than T throughout the reproductive cycle. Unlike the Florida largemouth bass (M. salmoides floridanus) advanced gamete development was observed in both sexes before winter, which may reduce the immediate energy required for gamete development in the spring. Results from this study also provide baseline information necessary for off-season spawning procedures and endocrine-disruption studies in northern largemouth bass.

Insights on the seasonal variations of reproductive features in the Eastern Atlantic Bluefin Tuna.

The Atlantic Bluefin Tuna (ABFT, Thunnus thynnus) is one of the most intensely exploited fisheries resources in the world. In spite of the years of studies on ABFT, basic aspects of its reproductive biology remain uncertain. To gain insight regarding the seasonal changes of the reproductive characteristics of the eastern stock of ABFT, blood and tissue samples were collected from mature specimens caught in the Mediterranean basin during the reproductive (May-June) and non-reproductive season (Oct-Nov). Histological analysis of the gonads of May-June samples indicated that there were females which were actively spawning (contained post-ovulatory follicles) and females that were not actively spawning that had previtellogenic and fully vitellogenic oocytes. In males, testis were at early or late stage of spermatogenesis during the reproductive season. In Oct-Nov, ovaries contained mostly previtellogenic oocytes as well as ß and α atretic follicles while the testis predominantly contained spermatogonia and few cysts with spermatocytes and spermatozoa. Gonadosomatic index (GSI) in females was highest among the actively spawning individuals while in males GSI was higher in early and late spermatogenic individuals compared to those that were spent. Plasma sex steroids levels varied with the reproductive season. In females, estradiol (E2), was higher in May-June while testosterone (T) and progesterone (P) did not vary. In males, E2 and T were higher in May-June while P levels were similar at the two sampling points. Circulating follicle stimulating hormone (FSH) was higher in Oct-Nov than in May-June both in males and females. Vitellogenin (VTG) was detected in plasma from both males and females during the reproductive season with levels in females significantly higher than in males. VTG was undetected in Oct-Nov samples. Since choriogenesis is an important event during follicle growth, the expression of three genes involved in vitelline envelope formation and hardening was measured and results showed significantly higher levels in ovaries in fish caught in May-June with respect to those sampled in Oct-Nov. In addition, a set of genes encoding for ion channels that are responsible for oocyte hydration and buoyancy, as well as sperm viability, were characterized at the two time points, and these were found to be more highly expressed in females during the reproductive season. Finally, the expression level of three mRNAs encoding for different lipid-binding proteins was analyzed with significantly higher levels detected in males, suggesting sex-specific expression. Our findings provide additional information on the reproductive biology of ABFT, particularly on biomarkers for the assessment of the state of maturation of the gonad, highlighting gender-specific signals and seasonal differences.

A Preliminary Survey of Estrogenic Effects in Cultured Adult Male Nile Tilapia (Oreochromis niloticus) in the Seven Lakes of San Pablo City, Philippines.

This study aimed to investigate the impact of estrogenic endocrine disruptors in adult male Nile Tilapia (Oreochromis niloticus) from the Seven Lakes (Bunot, Calibato, Mohicap, Palakpakin, Pandin, Sampaloc and Yambo) of San Pablo City in Laguna, Philippines. Fish were collected from aquaculture operations in each lake from July to September 2017 and examined for plasma vitellogenin (VTG) levels, hepatosomatic index (HSI) and gonadosomatic index (GSI). In comparison to the reference group (Pandin Lake), fish from Mohicap, Sampaloc and Yambo had higher levels of plasma VTG. HSI was increased in fish from Bunot Lake while it was markedly reduced in fish from Sampaloc Lake. Comparable GSI were obtained in all samples. While most examined fish are in active spermatogenesis, fish from Sampaloc Lake had immature gonads with no spermatozoa, and with minimal to moderate Leydig cell vacuolation and hyperplasia. The results suggest that cultured Nile tilapia from Sampaloc Lake is most adversely affected by endocrine-active compounds. The identification and quantification of these chemicals are recommended in future studies.

Chronic exposure to environmental concentrations of phenanthrene impairs zebrafish reproduction.

Phenanthrene (PHE) is a tricyclic polycyclic aromatic hydrocarbon which distributed extensively in the aquatic environment. However, the knowledge about its impact on fish reproduction is still limited, particularly under a chronic exposure regime. In this study, we exposed zebrafish (Danio rerio) embryos to environmentally relevant concentrations (0.2, 1.0, and 5.0 µg/L) of PHE for 4 months and assessed the impact on reproduction. The results demonstrated that egg production was decreased in fish exposed to PHE, with a significant reduction at 5.0 µg/L. The exposure significantly decreased the circulating concentrations of estradiol (E2) and testosterone (T) in female fish or E2 in male fish. In addition, plasma vitellogenin levels were significantly inhibited after PHE exposure in female fish. The transcription of hypothalamic-pituitary-gonadal (HPG) axis related genes (GnRH2, FSHß, LHß, 17ß-HSD, CYP11A1, and CYP19a) were significantly altered in a sex-specific manner. In addition, embryos derived from exposed parents exhibited increased malformation and decreased hatching success in the F1 generation. Taken together, these results demonstrate that chronic exposure to environmentally relevant concentration of PHE could cause adverse effects on reproduction and impair the development of offspring, ultimately leading to fish population decline in aquatic environment.

Development of specific enzyme-linked immunosorbent assays for multiple vitellogenins in marbled sole, Pleuronectes yokohamae.

Non-competitive, enzyme-linked immunosorbent assays (ELISAs) for three distinct sole vitellogenins (VtgAa, VtgAb and VtgC) were designed using their purified lipovitellin (Lv) products and corresponding digoxigenin-labeled, anti-Lv polyclonal antibodies, primarily for employment in monitoring estrogenic pollution of the environment. The working range of the ELISAs was from 0.97 to 1,000 ng/mL for all Vtg subtypes. Each ELISA appeared to be specific to the targeted Vtg subtype. Intra- and inter-assay coefficients of variation in the developed ELISAs were lower than 10%. Three Vtg subtypes were induced in serum of immature fish by estradiol-17ß (E2) injection (0.5 mg/kg body weight). All Vtg subtypes were induced one day after the injection, reaching peak levels (Lv equivalents) within three days, as follows: 39.1 ±â€¯28.9 µg/mL (VtgAa), 57.9 ±â€¯30.7 µg/mL (VtgAb) and 12.6 ±â€¯4.8 µg/mL (VtgC). In wild-caught males, VtgAa, VtgAb and VtgC were detected in ranges from 0.26 to 1.21, 0.19 to 8.69, and 0.17 to 53.50 µg/mL, respectively, over various sampling periods. In vitellogenic females sampled in January, the average level of VtgAb (8,744.43 ±â€¯733.93 µg/mL) was significantly higher than for VtgAa (150.33 ±â€¯22.35 µg/mL) or VtgC (57.08 ±â€¯6.00 µg/mL); thus VtgAb appeared to be the most dominant Vtg subtype. The present study entails the first report on development of subtype-specific Vtg ELISAs in marbled sole, which empowers us to detect and monitor estrogenic contamination in aquatic environments inhabited by this species.

Long-term exposure to waterborne nonylphenol alters reproductive physiological parameters in economically important marine fish.

Low concentrations of nonylphenol (NP) in aquatic environment can induce drastic effects on the endocrine system in animals. In this study, we examined the modulatory effects of NP on reproductive and physiological parameters in juveniles of the red seabream and black rockfish following waterborne NP exposure (0, 1, 10, and 50 µg L-1) for 60 days. In red seabream exposed to 50 µg L-1 NP, plasma levels of 17ß-estradiol (E2) and 11-ketotestosterone (11-KT) were significantly lower at 30 and 60 days, while E2 levels were slightly higher in 10 µg L-1-exposed individuals at day 30. Similarly, significantly lower levels of E2 and 11-KT were observed in 10 and 50 µg L-1-exposed black rockfish at 60 days, whereas the E2 level was higher in 1 µg L-1-exposed individuals at day 30. After exposure to NP, plasma and mRNA levels of vitellogenin (VTG) were significantly higher in both species at 30 and 60 days, similar to the inducible effects from synthetic estrogen. Plasma cortisol levels were significantly elevated by relatively higher concentrations of NP (10 and 50 µg L-1) at 30 and 60 days. Finally, 60 days of exposure of 50 µg L-1 NP significantly decreased the gonadosomatic index (GSI) and increased the hepatosomatic index (HSI) in both species. The results obtained from this study provide an evidence of the endocrine disrupting potential of waterborne NP on early stages of economically important marine fish. The NP-triggered endocrine modulation can induce effects on the development of reproductive and metabolic organs in fish species.

Development of specific chemiluminescent immunoassays for three subtypes of vitellogenin in grey mullet (Mugil cephalus).

Chemiluminescent immunoassays (CLIAs) were developed for each of three subtypes of vitellogenin (VtgAa, VtgAb and VtgC) in grey mullet, primarily for use in monitoring estrogenic pollution of the environment. The working range of VtgAa-CLIA and VtgAb-CLIA was from 0.975 to 1,000 ng/ml, while that of VtgC-CLIA was from 0.487 to 1,000 ng/ml. Each CLIA appeared to be specific to the targeted Vtg subtype. Intra- and inter-assay coefficients of variation in the developed CLIAs were lower than 10%. In male serum, VtgAa, VtgAb and VtgC were detected in ranges from 0.01 to 0.38, 0.02 to 1.01, and 0.01 to 3.12 µg/ml, respectively, during various sampling periods. In vitellogenic females (October), serum VtgAb levels (1,192.05 ±â€¯237.81 µg/ml) were significantly higher than levels of the other two Vtg subtypes (120.82 ±â€¯30.42 and 119.23 ±â€¯16.95 µg/ml for VtgAa and VtgC, respectively). When immature mullet were fed diets containing 17α-ethinylestradiol (EE2) at three different doses (0.4, 40 and 4,000 ng/g body weight), all Vtg subtypes were induced by 40 ng/g and 4,000 ng/g EE2. The VtgC (610.30 ±â€¯150.18 µg/ml) was most highly expressed among the three Vtgs in fish fed 40 ng/g EE2, while VtgAb (33.25 ±â€¯13.58 mg/ml) was highest in expression in fish fed 4,000 ng/g EE2. The present study provided practical subtype-specific Vtg assays for the first time in grey mullet, providing the necessary means to evaluate estrogenic activities in aquatic environments.