Corticosteroid plasma kinetics and gonadal receptor gene expression during the reproductive cycle in female Eurasian Perch: Investigation of the roles of corticosteroids in vitellogenesis.

To improve the quality of reproduction in Eurasian perch, Perca fluviatilis L., which is a promising candidate for Eurasian freshwater aquaculture that is currently cultivated in recirculating aquaculture systems (RAS), investigating the hormones that mediate and affect reproduction in this species is indispensable. The literature defines a group of four major corticosteroids (11-deoxycorticosterone, 11-deoxycortisol, corticosterone and cortisol) that might mediate critical stages of reproduction in female perch. Unfortunately, neither the basic roles nor the kinetics of these four corticosteroids throughout the reproductive cycle of female perch have been well defined to date. In this study, we therefore elucidated the plasma kinetics of these four corticosteroids during the reproductive cycle of domesticated female perch while monitoring the expression of the different receptors and enzymes that mediate their production and possible functions. Additionally, we performed an in vitro experiment during late vitellogenesis to investigate the possible direct roles of these steroids during that stage. Our results revealed that these four corticosteroids were detectable throughout the reproductive cycle, and the levels of most of them (11-deoxycorticosterone, 11-deoxycortisol, and cortisol) fluctuated significantly depending on the stage of reproduction. 11-Deoxycorticosterone and 11-deoxycortisol exhibited their highest levels, 1.8 ng/ml and 58 ng/ml, respectively, at the beginning of the reproductive cycle. By the end of the reproductive cycle, 11-deoxycortisol and cortisol plasma levels exhibited a surge, reaching 58 ng/ml and 150 ng/ml, respectively. During the perch reproductive cycle, the corticosteroid receptor complex is not regulated only at the hormone level, as the expression levels of all corticosteroid receptor genes showed a progressive and similar decline. In vitro exposure of vitellogenic oocytes to some of these corticosteroids (11-deoxycorticosterone and 11-deoxycortisol) induced an increase in yolk globule diameter and a decrease in the density of yolk globules, which indicates the involvement of both of these hormones in yolk globule coalescence. Taken together, these results implicate corticosteroids in the reproductive cycle, although the related cellular mechanisms remain to be investigated.

The effect of two different experimental rearing temperatures on the quality and the large-scale cryopreservation of Eurasian perch (Perca fluviatilis) sperm.

Eurasian perch is an important fish species for European aquaculture diversification, but the quality of reproduction still remains one of the main limitations for further industry development. In particular, the optimal condition to obtain the best quality of sperm is poorly understood. The aim of our study was to measure the possible effects of two experimental rearing temperatures (6 °C and the conventionally used 12 °C) and of hormonal stimulation, on the motility parameters (pMOT, VCL, VSL, LIN, ALH, BCF), osmolality and fertilizing capacity of Eurasian perch sperm at the end of the reproductive cycle. A prior untested, large-scale (5 mL cryotube and Polystyrene box) cryopreservation method was implemented using fresh sperm obtained from the two above mentioned temperature groups. Males were injected with 100 µg body weight kg-1 sGnRHa. No significant difference was recorded between the two rearing temperatures and between the saline control and sGnRHa treated groups on the different features of sperm quality. A similar fertilization rate was monitored in all sGnRHa treated (6 °C: 69 ± 13%, 12 °C: 81 ± 11%) and saline control groups (6 °C: 79 ± 10%, 12 °C: 87 ± 4%). Correspondingly, no significant difference in hatching rate was observed in the sGnRHa injected (6 °C: 27 ± 9%, 12 °C: 40 ± 20%) and saline control (6 °C: 35 ± 18%, 12 °C: 36 ± 7%) males. However, a notable negative effect of freezing process on sperm movement was observed following thawing in both temperature groups. No significant difference in the motility parameters was measured between the two temperature groups following large-scale cryopreservation. Furthermore, a similar result was observed in the fertilizing capacity (6 °C: 79 ± 10%, 12 °C: 75 ± 8) of thawed sperm as well as in the hatching rate (6 °C: 52 ± 13%, 12 °C: 46 ± 19%). Our results indicate that fresh Eurasian perch sperm can tolerate a reduced rearing temperature following hormonal treatment. The adopted large-scale cryopreservation method could be used efficiently in the future for the fertilization of large amounts of Eurasian perch eggs following a precise standardization process.

Spawning Performance and Sex Steroid Levels in Female Pikeperch Sander lucioperca Treated with Poly(lactic-co-glycolic acid) Microparticles.

Pikeperch Sander lucioperca is a piscivorous species considered a promising candidate for the diversification of intensive aquaculture. This study aimed to determine the effect of a sustained-release delivery system incorporating mammalian gonadotropin-releasing hormone agonist (mGnRHa) into poly(lactic-co-glycolic acid) (PLGA) microparticles on the sex steroid levels and aspects of artificial reproduction of pikeperch. Fish were divided into four groups and injected with 20 µg mGnRHa/kg, 5-day release microparticles encapsulated with 5 µg GnRHa/kg BW (PLGA 5), 20 µg GnRHa/kg (PLGA 20), or 1 mL/kg 0.9% NaCl (control). Cumulative percentage ovulation was 100% in the PLGA 5 group, significantly higher than in other tested groups. No differences among groups were observed in latency or fecundity. The level of 11-ketotestosterone (11-KT) peaked at 40 h post-injection, and was sustained during ovulation, in all treated groups. The 17ß-estradiol (E2) concentration increased in the mGnRHa-only group immediately after hormone injection, while both PLGA groups showed a reduction in E2 after injection, continuing to decrease until ovulation. A low dose of mGnRHa in PLGA microparticles significantly improves induction of ovulation and results in acceptable reproductive performance, which may positively affect pikeperch production under controlled conditions.

Split it up and see: using proxies to highlight divergent inter-populational performances in aquaculture standardised conditions.

BACKGROUND: Considering wild inter-populational phenotypic differentiation can facilitate domestication and subsequent production of new species. However, comparing all populations across a species range to identify those exhibiting suitable key traits for aquaculture (KTA; i.e. important for domestication and subsequent production) expressions is not feasible. Therefore, proxies highlighting inter-populational divergences in KTA are needed. The use of such proxies would allow to identify, prior to bioassays, the wild population pairs which are likely to present differentiations in KTA expressions in aquaculture conditions. Here, we assessed the relevance of three alternative proxies: (i) genetic distance, (ii) habitat divergence, and (iii) geographic/hydrologic distances. We performed this evaluation on seven allopatric populations of Perca fluviatilis for which divergences in KTA had already been shown. RESULTS: We showed differences in the correlation degree between the alternative proxy-based and KTA-based distance matrices, with the genetic proxy being correlated to the highest number of KTA. However, no proxy was correlated to all inter-populational divergences in KTA. CONCLUSION: For future domestication trials, we suggest using a multi-proxy assessment along with a prioritisation strategy to identify population pairs which are of interest for further evaluation in bioassays.

Is the use of recombinant cGnRH may be a future alternative to control the fish spawning? Let us go with the goldfish example.

The use of recombinant gonadotropin-releasing hormone (rGnRH) has very rarely been tested in fish to promote spawning. This study evaluated the impact of recombinant chicken gonadotropin-releasing hormone (rcGnRH) with metoclopramide on the release of sex steroids and final maturation induction in goldfish (Carassius auratus) broodstock. For this purpose, goldfish broodstock was divided into four groups and treated with 0.9% NaCl with 20 mg/kg metoclopramide (Met) (C); 10 µg/kg body weight (BW) rcGnRH with 20 mg/kg metoclopramide (rcGn10); 15 µg/kg BW rcGnRH with 20 mg/kg metoclopramide (rcGn15); and 20 µg/kg BW rcGnRH with 20 mg/kg metoclopramide (rcGn20). The capability of the rcGnRH for eliciting biological response was tested in vivo by evaluating the changes of 17ß estradiol (E2), testosterone (T), and 17α, 20ß-dihydroxy-4-pregnen-3-one (DHP) and the induced spawning. Blood samples were obtained at 0 h, 12 h, and 24 h after injection. The rcGn10, rcGn15, and rcGn20 treatments induced lower E2 concentration, especially 24 h post-injection. T levels were significantly higher in rcGn10, rcGn15, and rcGn20 treatments 12 h post-injection than at 0 h and then decreased at 24 h post-injection. Furthermore, the rcGnRH tested significantly enhanced DHP secretion in rcGn10, rcGn15, and rcGn20 treatments 12 h post-injection before a decline at 24 h post-injection. No significant difference between the sampling times was found in the C treatment for the 3 sex steroids tested. The results also displayed that rcGnRH at 10-20 µg/kg of body weight can trigger spawning with the highest speed and efficiency of spawning at 20 µg/kg. The obtained results represent a possible strategy for enhancing the artificial reproduction and ovulation of broodstock fish by rGnRH and further support the use of recombinant hormones to promote reproduction in aquaculture.

In vitro follicle culture shows that progestagens are the maturation-inducing hormones (MIH) and possible regulators of the ovulation-mediating hormone PGE2 in female Eurasian perch Perca fluviatilis.

In European aquaculture, Eurasian perch, Perca fluviatilis L., is perceived as one of the most highly valuable freshwater fish species and a strong candidate for the development of freshwater aquaculture. In the pursuit of improving the quality of reproduction in this domesticated species, investigating the hormones mediating the final oocyte maturation (FOM) is therefore indispensable. But, the exact nature of the maturation-inducing hormone (MIH) in Eurasian perch is unknown. To further validate the existence of a maturation-inducing activity behind potential hormonal candidates in this species, we in vitro tested a group of nine hormones: cortisol (Co), 11-deoxycortisol (11-D), corticosterone (coS), 11-deoxycorticosterone (DOC), 17α,20ßdihydroxy-4-pregnen-3-one (DHP) and 17α,20ß,21 trihydroxy-4-pregnen-3-one (THP), prostaglandin E2 (PGE2), estradiol-17ß (E2) and testosterone (T), in their ability to trigger FOM advancement and the production of sex steroids potentially involved in FOM. Using mature female perch, two in vitro experiments were conducted with oocytes at the start of the FOM. The follicles were incubated for 62 h in Cortland media with and without human chorionic gonadotropin (hCG). By the end of the incubation, only DHP and THP triggered the full advancement in FOM even at low doses with the effect of DHP being in vivo validated. However, the de novo productions of E2 and DHP were not shown to be regulated by either of the MIH candidates. Progestagens are hence more credible candidates as MIH than corticosteroids in Eurasian perch. Our in vitro study also revealed that both PGE2 and DHP are strongly associated with ovulation and that PGE2 might have slightly contributed to such DHP activity.

Determination of annual reproductive cycle in male sterlet, Acipenser ruthenus using histology and ultrasound imaging.

The aim of this study was to evaluate seasonal testicular development in the cultured sterlet, Acipenser ruthenus. During annual sexual cycle of male sterlet, stages of gonad maturity were examined using histology and ultrasonography approaches. The histology identified males at different stages of maturity among fish sampled monthly. According to the seasonal changes in the testes, reproductive cycle was divided into four stages including resting, pre-spawning, spawning, and post-spawning. The histology examination revealed considerable variation in testicular developmental stages. These changes were identified based on persistent spermatogenesis and asynchronous gonad development in testes, showing that regulation of annual gonadal cycle is influenced by season. Also, the results obtained using ultrasound suggested that reproductive stages can be identified based on morphology and tissue echogenicity. At each phase of testicular development, gonadosomatic index (GSI) and number of spermatogenic cysts were variable. The present study focused on determination of annual reproductive development in cultured male sterlet which clearly identifies reproductive stage in each season as valuable guide for future researches on reproductive biology of sterlet. This study presents basic knowledge about reproductive biology in sterlet contributing to optimal broodstocks management that allows comparison of reproductive development among sturgeon species.

First identification of dopamine receptors in pikeperch, Sander lucioperca, during the pre-ovulatory period.

Dopamine (DA) is a ubiquitous neurotransmitter exerting a range of pleiotropic actions through two DA receptor families, the D1 and the D2. To date in vertebrates, a maximum of four receptor subtypes have been identified within the D1 family, D1 (former D1A), D5 (former D1B), D6 (former D1C and D1D) and D7 (former D1E), while the D2 family encloses five subtypes, D2, D3, D4, D8 (former D2like or D2l) and D9 (former D4-related sequence or D4-rs). In teleosts, no study has investigated in parallel all the DA receptors to identify and localize the whole receptor repertoire from both families. In pikeperch, Sander lucioperca, a species of interest for aquaculture development, the existence, number and location of the DA receptors are totally unknown. To address these questions, RNA-seq with de novo transcriptome reconstruction, functional annotation and phylogenetic analysis were performed to characterize the transcript repertoire of DA receptors in the brain of female pikeperch at the pre-ovulatory period. Ten different cDNA were identified and showed to belong to the D1 family: two D1, one D5a, one D6a and one D6b and to the D2 family: two spliced variants of D2, one D3, one D8 and one D9. Unlike zebrafish, the subtypes D4 and D7 have not yet been isolated in pikeperch. As expected D1, D3, D8 and D9 are mostly expressed in brain parts except for the cerebellum (D1 and D3). The inter-species differences in the number of DA receptors and the inter-organ differences in the gene expression of all receptors support the complexity of the dopaminergic actions in vertebrate.

Seasonal simulated photoperiods influence melatonin release and immune markers of pike perch Sander lucioperca.

Melatonin is considered as the time-keeping hormone acting on important physiological functions of teleosts. While the influence of melatonin on reproduction and development is well described, its potential role on immune functions has little been considered. In order to better define an immune modulation by the melatonin hormone, we hypothesized that natural variations of photoperiod and subsequent changes in melatonin release profile may act on immune status of pikeperch. Therefore, we investigated during 70 days the effects of two photoperiod regimes simulating the fall and spring in western Europe, on pikeperch physiological and immune responses. Samples were collected at 04:00 and 15:00 at days 1, 37 and 70. Growth, plasma melatonin levels, innate immune markers and expression of immune-relevant genes in head kidney tissue were assessed. While growth and stress level were not affected by the seasonal simulated photoperiods, nocturnal levels of plasma melatonin were photoperiod-dependent. Innate immune markers, including lysozyme, complement, peroxidase and phagocytic activities, were stimulated by the fall-simulated photoperiod and a significant correlation was made with plasma melatonin. In addition to bring the first evidence of changes in fish immunocompetence related to photoperiod, our results provide an additional indication supporting the immunomodulatory action of melatonin in teleosts.

Constant long photoperiod inhibits the onset of the reproductive cycle in roach females and males.

Photoperiod and temperature are commonly accepted as the determinant factors for the control of the reproductive cycle in freshwater fishes. However, this determining effect is dependent on fish species. While applying a constant long photoperiod has an inhibitory effect in some species, the same photoperiodic manipulation has a stimulating effect in others. In cyprinids, a decrease in temperature or photoperiod can induce the gonad recrudescence. However, in roach Rutilus rutilus an early spring spawner cyprinid, there is little knowledge about the cueing role of each environmental factor. The aim of this work was to study the effect of a constant long photoperiod on the gametogenesis in roach. Fish were kept under either naturally simulated photoperiod or artificial constant long photoperiod and sampled at three times: at the beginning of photoperiod decrease, at the beginning of temperature decrease, and at the end of temperature decrease. Morphological parameters (gonado-somatic, hepato-somatic, and viscera-somatic indexes), plasma sexual steroids, and proportion of gametogenesis stages were estimated at each sampling time. The results showed that a constant, long photoperiod exerted inhibitory effects on gametogenesis advancement in both females and males that could stem from decrease of sex steroid production. Roach displayed a similar response to photoperiodic manipulations to other early spring spawners like percids, such as European perch, yellow perch and pikeperch. These results clearly showed the cueing role of the photoperiod in the induction of the reproductive cycle in roach.

Design, production and purification of a novel recombinant gonadotropin-releasing hormone associated peptide as a spawning inducing agent for fish.

GnRH is a neuropeptide known to regulate reproduction in vertebrates. The purpose of this study was to design and produce recombinant gonadotropin-releasing hormone associated peptide (rGnRH/GAP) as an alternative of the previous GnRHs and native extracted hormone from tissue, to induce final maturation in fish. Decapeptide as well as GAP area sequences were compared between GnRH1, GnRH2, and mGnRH from Acipenser sp and Huso huso, respectively. Considering the conserved amino acids and the replacement of un-stable amino acids with those that were more stable against proteolytic digestion as well as had a longer half-life, the sequence was designed. The sequences of decapeptide and GAP region were synthesized and then cloned on pET28a expression vector and transformed into expression host Escherichia coli BL21(DE3). The supernatant of cultured recombinant bacteria was used for purification using TALON Metal affinity resin. The purity of the GnRH/GAP was confirmed by single 8 kDa band on SDS-PAGE and Western blot. Bioinformatics studies were performed for evaluation of homology between GnRH protein sequences and prediction of 3D protein structure using Swiss Model. The result showed that the structure prediction of the recombinant GnRH decapeptide was relatively similar to decapeptide of GnRH2 from Beluga (Huso huso). The GAP structure was similar to GAP1 of Nile tilapia (Oreochromis niloticus) and sturgeon and GnRH2 of Chinese sturgeon (Acipenser sinensis). The mass analysis showed that the sequence was exactly the same as designated sequence. Biology activity of rGnRH/GAP was tested in mature goldfish (Carassius auratus) and results showed that rGnRH/GAP had a positive effect in final maturation. Indeed 17α, 20ß-dihydroxy-4-pregnen-3-one (DHP) was increased 17 h and 24 h after injection with rGnRH/GAP and spawning stemmed from that injection. These novel findings introduce the potential of utilizing rGnRH/GAP in aquaculture.

Physiological and proteomic responses to corticosteroid treatments in Eurasian perch, Perca fluviatilis: Investigation of immune-related parameters.

The comparative effects of cortisol and 11-deoxycorticosterone (DOC), two major corticosteroids in fish, have yet received little attention in teleosts. We evaluated the proteomic and immune responses of Eurasian perch to chronic corticosteroid treatments. We implanted immature perch with cortisol (80mg/kg) or DOC (4mg/kg) and measured the proportions of blood leucocytes, immune indices in the plasma, spleen and liver (complement and lysozyme activity, total immunoglobulin and immune gene expression in the tissues) and differential proteome expression (corticosteroid versus control) in the liver and the spleen on days 2, 4 and 14 post-treatment. Implantation of cortisol decreased the ratio of blood leucocytes and depressed Ig levels in both organs while DOC modulated the proportion of leucocyte sub-populations (increase in lymphocytes and decrease in granulocytes). In contrast, the innate humoral immunity was not strongly influenced by any of corticosteroid implants. The only immune parameter that was significantly affected was lysozyme, after DOC treatment. A number of proteins were differentially regulated by these hormones and some were identified in the liver (21 for cortisol and 8 for DOC) and in the spleen (10 for cortisol and 10 for DOC). None of the proteins was directly linked to immunity, except the natural killer enhancing factor, which was repressed by cortisol in the spleen. Our results also confirm that the proteins involved in energetic and glucose metabolism are affected by corticosteroids. Furthermore, these corticosteroids differently regulate immune status in Eurasian perch and they primarily impact leucocytes, as opposed to innate immune function.

Effects of hCG and salmon gonadoliberine analogue on spermiation in the Eurasian perch (Perca fluviatilis).

This study analysed (i) the effect of human chorionic gonadotropin (hCG) and salmon gonadoliberine analogue (sGnRHa) on the effectiveness of induction of spermiation and (ii) the effect of latency time following the application of those spawning agents on the quantity and quality of the sperm of Eurasian perch, Perca fluviatilis, obtained during out-of-season spawning. For this study, pond-reared fish were used which had been acclimated to the controlled conditions. Three groups were distinguished which were treated with either saline (0.9% NaCl; control group), hCG (500 IU kg-1) or sGnRHa (100 µg kg-1). The fish were kept in a recirculating system at 12 °C throughout the study, during which sperm was collected every two days between the 2nd and 10th day following hormonal treatment. During the study, quantitative (e.g. sperm volume, total sperm production) and qualitative (measured with a computer-assisted sperm analysis system - i.e. CASA) parameters were monitored. The results of the study indicate that the hormonal treatment had a highly beneficial effect on the spermiation rate (100% in experimental groups from day 6 following injection) as well as quantity, which increased 50% in experimental groups (over 2200 × 109 of spermatozoa per kg of body weight) by day 4 following injection. For the sperm quality, both spawning agents tested had a rather positive effect, although sperm motility rate (MOT) was seen to be significantly reduced on day 10 following the application of hCG (MOT = 72.8% ± 8.1), which was not observed after the application of sGnRHa (minimum mean MOT 81.7% ± 6.1). The results clearly indicate that hormonal treatment had a positive effect on spermiation in Eurasian perch, most apparent from day 6 following injection, regardless of the hormonal agent used. Though application of sGnRHa allowed a high volume of high quality sperm to be stripped for two days longer (up to day 10 post-injection) compared to the application of hCG.

Sublethal effect assessment of a low-power and dual-frequency anti-cyanobacterial ultrasound device on the common carp (Cyprinus carpio): a field study.

The use of ultrasonication for cyanobacterial control in freshwater bodies has become increasingly popular during the last decades despite controversial efficiency on large scale application. Apart from that, little information is currently available regarding ultrasound toxicity potential towards non-target species. This work was designed to address this issue in the common carp using a low-power (7-9 W output) and dual-frequency (23 and 46 kHz) anti-cyanobacterial ultrasound device. Results showed that carps were unaffected by ultrasound exposure when exposed in floating cages in fish ponds over a 30-day period. The experiment duration was the main factor influencing all measured biological parameters in exposed and non-exposed organisms. Indeed, it was positively associated with an increase in fish condition factor. Cortisol level also tended to slightly increase over the number of days of experiment but its variation did not enable to sort out any ultrasound exposure-related stress. Moreover, an overall diminution along the experimental period of the expression level of a set of biomarkers could be reported, encompassing cellular antioxidant enzyme activities such as superoxide dismutase (SOD), glutathione peroxydase (GPx), catalase and glutathione S-transferase (GST), and lactate dehydrogenase activity. Subtle changes in these biomarkers were dependent of the type of enzyme activity and especially of the origin of fish (i.e., sampled pond) regardless of the presence of ultrasound equipment, reflecting thereby fish adaptation to local environmental conditions in each pond. In conclusion, this study does not provide indication that ultrasonication in the aforementioned conditions affects the welfare and physiological homeostasis of carps.

Influence of waterborne gallic and pelargonic acid exposures on biochemical and reproductive parameters in the zebrafish (Danio rerio).

Gallic and pelargonic acids are biologically derived substances receiving a growing interest as eco-friendly biocides with potential applications in freshwater system management. However, some data gaps remain to address their chronic ecotoxicity issue, particularly for fish. This work aimed at investigating the sublethal effects of a long-term waterborne exposure of zebrafish to these compounds. Mature fish were exposed to gallic or pelargonic acid at the concentrations of 0, 0.05, 0.5 and 5 mg/L during one month under semi-static conditions. Fecundity, hatching rate and median hatching time were regularly evaluated. Circulating sex hormone levels (11 ketotestosterone -11 KT, 17 ßestradiol -E2-), plasma vitellogenin (Vtg), and gonad histology were monitored in males and females after exposure. Lactate dehydrogenase (LDH), total glutathione peroxydase (GPx) and glutathione-S transferase (GST) activities were assessed as enzymatic biomarkers of exposure in fish liver. Significant increases of GPx activity were reported in females exposed to both type of chemicals regardless the contamination level. Moreover, 5 mg/L gallic acid induced a decrease in 11-KT levels for males. For fish exposed to pelargonic acid, decreases in circulating hormone levels were reported respectively at 0.05 and 5 mg/L for 11-KT in males, and at 0.5 mg/L for E2 in females. However, no histological alteration in gonads neither significant variation in reproductive performances were detected following zebrafish exposure to gallic or pelargonic acid. Additional investigations concerning the mode of application and the environmental fate of these substances may warrant their further use in freshwater systems at concentrations compatible with biocidal/allelochemical effects. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 227-240, 2017.

Di-(2-ethylhexyl)-phthalate disrupts pituitary and testicular hormonal functions to reduce sperm quality in mature goldfish.

Di-(2-ethylhexyl) phthalate (DEHP) interferes with male reproductive endocrine system in mammals, however its effects on fish reproduction are largely unknown. We evaluated sperm quality and investigated reproductive endocrine system in mature goldfish (Carassius auratus) exposed to nominal 1, 10, and 100µg/L DEHP. To examine DEHP estrogenic activity, one group of goldfish was exposed to 17ß-estradiol (5µg/L E2) for comparison. Following 30d of exposure, sperm production was decreased and suppressed in DEHP and E2 treated goldfish, respectively. Sperm motility and velocity were decreased in goldfish exposed to 100 and 10µg/L DEHP at 15s post-sperm activation, respectively. Compared to control, 11-ketotestosterone (11-KT) levels were decreased at 10 and 1µg/L DEHP at day 15 and 30, respectively. In E2 treated goldfish, 11-KT levels were decreased compared to control during the period of exposure. E2 levels were increased in goldfish exposed to E2, but remained unchanged in DEHP treated goldfish during the period of exposure. StAR mRNA levels encoding regulator of cholesterol transfer to steroidogenesis were decreased in DEHP and E2 treated goldfish following 15 and 30d of exposure, respectively. Luteinizing hormone (LH) levels were decreased in DEHP and E2 treated goldfish following 15 and 30d of exposure, respectively. In DEHP treated goldfish, gnrh3, kiss1 and its receptor (gpr54) mRNA levels did not change during the experimental period. In E2 treated goldfish, gnrh3 mRNA levels were decreased at day 7, but kiss1 and gpr54 mRNA levels were increased at day 30 of exposure. The mRNA levels of genes encoding testicular LH and androgen receptors remained unchanged in DEHP and E2 treated goldfish. In contrast to E2 treated goldfish, vitellogenin production was not induced in DEHP treated goldfish and mRNA levels of genes with products mediating estrogenic effects remained unchanged or decreased. In conclusion, DEHP interferes with testis and pituitary hormonal functions to reduce sperm quality in goldfish and does not exhibit estrogenic activity.

The trenbolone acetate affects the immune system in rainbow trout, Oncorhynchus mykiss.

In aquatic systems, the presence of endocrine-disrupting chemicals (EDC) can disrupt the reproductive function but also the immune system of wildlife. Some studies have investigated the effects of androgens on the fish immune parameters but the mechanisms by which the xenoandrogens alter the immunity are not well characterized. In order to test the effects of trenbolone acetate (TbA) on fish immune system, we exposed rainbow trout male juveniles during three weeks to TbA levels at 0.1 and 1µg/L. The present results suggest that TbA impacts, in a tissue-dependent manner, the rainbow trout immunity by affecting primarily the humoral immunity. Indeed, TbA inhibited lysozyme activity in plasma and liver and enhanced the alternative complement pathway activity (ACH50) in kidney. In plasma, the modulation of the complement system was time-dependent. The mRNA expression of genes encoding some cytokines such as renal TGF-ß1, TNF-α in skin and hepatic IL-1ß was also altered in fish exposed to TbA. Regarding the cellular immunity, no effect was observed on the leucocyte population. However, the expression of genes involved in the development and maturation of lymphoid cells (RAG-1 and RAG-2) was decreased in TbA-treated fish. Among those effects, we suggest that the modulation of RAG-1 and mucus apolipoprotein-A1 gene expression as well as plasma and hepatic lysozyme activities are mediated through the action of the androgen receptor. All combined, we conclude that trenbolone affects the rainbow trout immunity.

Acute toxicity and sublethal effects of gallic and pelargonic acids on the zebrafish Danio rerio.

Gallic and pelargonic acids are naturally found in a variety of plants and food products. Despite their extensive use in man-made applications, little is known regarding their potential risks to aquatic vertebrates. The aim of this work was to assess the acute toxicity of these polyphenolic and fatty acid compounds to the zebrafish. In order to get insights into sublethal effects, the enzyme activity of usual biomarkers related to oxidative stress and biotransformation were also assessed in fish. These latter included total superoxide dismutase, catalase as well as total glutathione peroxidase for antioxidant defence mechanisms and glutathione S-transferase for biotransformation related enzyme. Gallic acid was practically non-toxic (96-h lethal concentration (LC50) > 100 mg/L) whereas pelargonic acid was slightly toxic (96-h LC50 of 81.2 mg/L). Moreover, biomarker analyses indicated enhanced superoxide dismutase activity in fish exposed to 20, 40 and 100 mg/L of gallic acid compared to control. A dose-dependent induction of glutathione peroxidase and glutathione S-transferase was reported following gallic acid exposure at the tested concentrations of 10, 20 and 40 mg/L, with the exception of 100 mg/L of substance where basal activity levels were reported. In the case of pelargonic acid, there was no change in antioxidant enzyme activity while an inhibition of glutathione S-transferase was observed from organisms exposed to 45, 58 and 76 mg/L of test solution. The results concerning sublethal effects on biological parameters of zebrafish highlighted thereby the need for further investigations following chronic exposure to both organic acids.

Effects of low dose endosulfan exposure on brain neurotransmitter levels in the African clawed frog Xenopus laevis.

Understanding the impact of pesticides in amphibians is of growing concern to assess the causes of their decline. Among pesticides, endosulfan belongs to one of the potential sources of danger because of its wide use and known effects, particularly neurotoxic, on a variety of organisms. However, the effect of endosulfan was not yet evaluated on amphibians at levels encompassing simultaneously brain neurotransmitters and behavioural endpoints. In this context, tadpoles of the African clawed frog Xenopus laevis were submitted to four treatments during 27 d: one control, one ethanol control, and two low environmental concentrations of endosulfan (0.1 and 1 µg L(-1)). Endosulfan induced a significant increase of brain serotonin level at both concentrations and a significant increase of brain dopamine and GABA levels at the lower exposure but acetylcholinesterase activity was not modified by the treatment. The gene coding for the GABA transporter 1 was up-regulated in endosulfan contaminated tadpoles while the expression of other genes coding for the neurotransmitter receptors or for the enzymes involved in their metabolic pathways was not significantly modified by endosulfan exposure. Endosulfan also affected foraging, and locomotion in links with the results of the physiological assays, but no effects were seen on growth. These results show that low environmental concentrations of endosulfan can induce adverse responses in X. laevis tadpoles. At a broader perspective, this suggests that more research using and linking multiple markers should be used to understand the complex mode of action of pollutants.

Influence of short-term exposure to low levels of 17α-ethynylestradiol on expression of genes involved in immunity and on immune parameters in rainbow trout, Oncorhynchus mykiss.

Fish are exposed to endocrine-disrupting chemicals (EDC), which are well known to disturb not only the reproductive system but also the immune system in vertebrates. However, the mechanisms by which these compounds are able to modify fish immunity are not well understood. In order to test the EE2 effects on immunity in selected organs, we exposed rainbow trout male juveniles for 3 weeks to EE2 concentrations ranging from 0.01 to 1 µg/L. The results of this study suggest that EE2 affects the immunity of rainbow trout in a tissue dependent manner. This molecule affects both cellular and humoral immune systems. Indeed, blood leukocyte populations, as well as hepatic and plasma lysozyme, plasma MPO and renal complement activities, are modulated by EE2. Moreover, EE2 alters the gene expression of some mucus compounds, hepatic expression of complement sub-unit and lysozyme, or genes involved in the hepatic phagocytosis and transport of immunoglobulin across the liver.