Sex-biased regulation of respiratory burst, phagocytic activity and plasma immune factors in lined seahorse (Hippocampus erectus) after subchronic benzo[a]pyrene exposure.

Both wild and aquacultured seahorses are currently under great threat from marine pollution, notably from the potent contaminant and carcinogen benzo[a]pyrene (BaP). However, very little data are available regarding the immunomodulating effects of BaP in seahorses. Therefore, in this study, we exposed lined seahorses (Hippocampus erectus) for 7 d to BaP at three dosages (0.5, 5, and 50 µg/L) to evaluate sexual dimorphism in immune response. We measured eight immune parameters in the blood, including respiratory burst (RB), phagocytic activity (PA), monocytes/leucocytes, immunoglobulin M, complement 3, complement, interferon-a, and interleukin-2. Male seahorses had significantly higher parameters than females, except in terms of monocytes/leucocytes (P < 0.05). Although flow cytometry showed that RB and PA variation per BaP dose were roughly similar across sexes, RB and PA exhibited distinct patterns. Additionally, fluorescence intensity and leucocyte percentage were positively correlated in PA but not RB for all treatment and sex combinations. Through ELISA, we showed that the other six parameters had complex responses that nevertheless varied in a BaP-dosage and sex-dependent manner. Overall, adult male seahorses had higher immunocompetence than females before BaP exposure, and sexual dimorphism continued to be apparent during BaP exposure. Furthermore, all eight parameters were sensitive to BaP. Based on these results, we highly recommend H. erectus as a sentinel species for crude contamination, whereas PA and RB are valuable bioindicators of marine contaminants such as BaP.

Hormonal changes over the spawning cycle in the female three-spined stickleback, Gasterosteus aculeatus.

Female three-spined sticklebacks are batch spawners laying eggs in a nest built by the male. We sampled female sticklebacks at different time points, when they were ready to spawn and 6, 24, 48 and 72h post-spawning (hps) with a male. Following spawning, almost all females (15 out of 19) had ovulated eggs again at Day 3 post-spawning (72hps). At sampling, plasma, brain and pituitaries were collected, and the ovary and liver were weighed. Testosterone (T) and estradiol (E2) were measured by radioimmunoassay. Moreover, the mRNA levels of follicle-stimulating hormone (fsh-ß) and luteinizing hormone (lh-ß) in the pituitary, and of the gonadotropin-releasing hormones (GnRHs: gnrh2, gnrh3) and kisspeptin (kiss2) and its G protein-coupled receptor (gpr54) in the brain were measured by real-time qPCR. Ovarian weights peaked in "ready to spawn" females, dropped after spawning, before again progressively increasing from 6 to 72hps. Plasma T levels showed peaks at 24 and 48hps and decreased at 72hps, while E2 levels increased already at 6hps and remained at high levels up to 48hps. There was a strong positive correlation between T and E2 levels over the spawning cycle. Pituitary lh-ß mRNA levels showed a peak at 48hps, while fsh-ß did not change. The neuropeptides and gpr54 did not show any changes. The changes in T and E2 over the stickleback spawning cycle were largely consistent with those found in other multiple-spawning fishes whereas the marked correlation between T and E2 does not support T having other major roles over the cycle than being a precursor for E2.

Plasma levels of immune factors and sex steroids in the male seahorse Hippocampus erectus during a breeding cycle.

To better understand the endocrine- and immune-response pattern during reproduction in a fish species having parental care behaviors and also to accumulate the endocrine- and immune-related data for future explanations of the low reproductive efficiency in seahorse species, the variations of immune factors and sex steroids in the plasma of the male lined seahorse Hippocampus erectus at different breeding stages, i.e., pre-pregnancy, pregnancy (early, middle, and late periods), and post-pregnancy, were investigated in the present study. The immune factors included monocytes/leucocytes (M/L), leucocyte phagocytic rate (LPR), immunoglobulin M (Ig M), interleukin-2 (IL-2), interferon-α (IFN-α), and lysozyme (LZM). The sex steroids included testosterone (T), 11-ketotestosterone (11-KT), 11ß-hydroxytestosterone (11ß-OHT), 17α-methyltestosterone (17α-MT), 17ß-estradiol (E2), and 17α-hydroxy-20ß-dihydroprogesterone (17α-20ß-P). Moreover, the immune metabolic activity of epithelium cells in the brood pouch at different breeding stages was also analyzed through ultrastructural observations of the abundance of cytoplasmic granules, mitochondria, endoplasmic reticulum, lysosomes, and exocytosis. The results show that a higher immune level was observed during pregnancy, particularly in the early and middle periods, and a lower immune level was noted during pre-pregnancy. Correspondingly, the epithelium cells in the brood pouch also showed a stronger immune metabolic activity during pregnancy and weaker activity during pre-pregnancy. Four sex steroids of T, 11ß-OHT, 17α-MT, and E2 were higher during pre-pregnancy and lower during post-pregnancy, whereas 11-KT and 17α-20ß-P, which were positively correlated with part immune factors, were higher during pregnancy. No negative correlations between sex steroids and immune factors were observed. In conclusion, the higher immune competence during pregnancy may indicate that parental care could improve immunity, which may be the major factor for no immunosuppressive effect of sex steroids during reproduction in the seahorse H. erectus, unlike noncaregiving fishes in which inhibitions of sex steroids on immunity are frequently observed. Moreover, higher 11-KT and 17α-20ß-P during pregnancy than during pre-pregnancy and post-pregnancy may suggest that these two steroids are also involved in parental care regulation.

Biomarker responses to estrogen and androgen exposure in the brook stickleback (Culaea inconstans): A new bioindicator species for endocrine disrupting compounds.

Small-bodied freshwater fish are commonly used in regulatory testing for endocrine disrupting compounds (EDCs) but most lack a sensitive and quantifiable androgen-specific biomarker. Brook stickleback (Culaea inconstans) are a North American freshwater fish whose males produce an androgen-regulated glycoprotein in the kidney called spiggin. Although spiggin induction in females has been used as an androgen-specific biomarker of exposure in other stickleback species it has not been characterized in brook stickleback. Therefore, our objective was to develop a bioassay using brook stickleback to measure estrogenic and androgenic responses and establish the sensitivity of traditional and novel biomarkers of exposure. We first developed and optimized a qPCR assay to measure spiggin and vitellogenin transcript levels in kidney and liver tissue, respectively. Basal levels were differentially expressed in mature wild-caught male and female brook stickleback. To determine their sensitivity to EDCs, fish were exposed to nominal concentrations of 1, 10 and 100ng/L of 17α-methyltestosterone (MT) or 17α-ethinylestradiol (EE2) for 21days (sampled at 7 and 21days) under semi-static renewal conditions. MT and EE2 exposure induced spiggin and vitellogenin transcripts in female kidneys and male livers, respectively. Exposure to EE2 also increased hepatosomatic index in both sexes and decreased gonadosomatic index in females. Histopathological alterations were observed in the kidney of EE2-exposed fish and an increase in kidney epithelium cell height occurred in MT-exposed females. Given the sensitivity of these endpoints, the brook stickleback is a promising new freshwater fish model for EDC evaluation and a potential bioindicator for EDCs in North American freshwater environments.

Di-n-butyl phthalate causes antiestrogenic effects in female Murray rainbowfish (Melanotaenia fluviatilis).

Di-n-butyl phthalate (DnBP) is an industrial pollutant with antiandrogenic effects reported in male mammals and fish. Little research has been done on the endocrine effects of DnBP in female fish. The present study investigated the changes in ovarian histology and serum vitellogenin concentrations in adult Murray rainbowfish (Melanotaenia fluviatilis) after exposure to 125 µg/L, 250 µg/L, 500 µg/L, and 1000 µg/L DnBP for 7 d. Treatment at 125 µg/L to 1000 µg/L DnBP for 7 d had no significant effect on the survival, condition factor, gonadosomatic index, hepatosomatic index, and developmental stage of the fish. Based on the histological investigation, the sizes of the previtellogenic oocytes in the fish treated at 250 µg/L to 1000 µg/L were found to be significantly higher than in the corresponding control fish (p ≤ 0.05). The early vitellogenic oocytes in the fish treated at 1000 µg/L were significantly smaller relative to those in the unexposed fish (p ≤ 0.05). Histological changes like chorion folding, shrunken ooplasm, impaired yolk production, granulomatous inflammation, and interstitial fibrosis were observed in the ovaries of the fish treated with DnBP. The circulating levels of plasma vitellogenin were significantly lower in the fish exposed to 500 µg/L and 1000 µg/L DnBP (p ≤ 0.05). These data show that a continuous exposure to subacute concentrations of DnBP for 7 d can cause antiestrogenicity in female adult Murray rainbowfish.

Oxidative stress and band 3 protein function in Liza aurata and Salmo irideus erythrocytes: effect of different aquatic conditions.

The aim of this study was to assess the effect of the different aquatic conditions on anion transport of fish erythrocytes through the measurement of the sulphate uptake operating from band 3, the determination of reduced glutathione (GSH) and oxidized glutathione (GSSG). To this purpose, blood samples of 30 Liza aurata and 30 Salmo irideus fishes were collected via caudal vein and washed with physiological buffer. Successively, erythrocytes suspended at 3% haematocrit were used to measure the SO(4) (=) influx by atomic absorption spectrophotometry at 425 nm wavelength and the GSH concentration using an immunodiagnostic assay intended for the quantitative determination of glutathione in ethylenediaminetetraacetic acid in blood. All results were analyzed using unpaired Student's t-test and a P < 0.05 was considered statistically significant. The results of this study showed statistically significant differences about the sulphate uptake and the glutathione levels in S. irideus with respect to L. aurata (P < 0.0001). In conclusion, the different aquatic conditions play an important role on anion transport in fish erythrocytes, showing that environmental differences induce perturbations in erythrocyte membrane and should be evaluated to prevent physiological damages in fishes.

Osmoregulation of the resident estuarine fish Atherinella brasiliensis was still affected by an oil spill (Vicuña tanker, Paranaguá Bay, Brazil), 7 months after the accident.

An oil tanker loaded with methanol and bunker oil has exploded in November 2004 in Paranaguá Bay, in front of Paranaguá Harbor, southern Brazil. In order to investigate the chronic effects of an oil spill on a resident estuarine fish, the Brazilian silverside Atherinella brasiliensis was sampled 1, 4, and 7 months after the spill, from 2 sites inside Paranaguá Bay, and also from a reference site inside nearby Guaratuba Bay, non-affected by the spill. Increases in plasma osmolality (reaching ~525 mOsm/kg H2O, or ~70% above values in reference fish) and chloride (reaching 214 mM in site C, or ~51% above values in reference fish) were detected 4 months after the spill, in parallel with branchial carbonic anhydrase inhibition (to 56% of the activity measured in reference fish) in silversides obtained from the contaminated sites. Plasma cortisol concentration increased progressively in samples from fish obtained 4 (462 ng/mL) and 7 (564-650 ng/mL) months after the spill, when compared to values in reference fish (192 ng/mL). Osmoregulation of a resident estuarine fish is still affected by an oil spill, months after the accident. It is, thus, a sensitive tool for the evaluation of the chronic effects of oil spills inside tropical estuarine systems, and A. brasiliensis is proposed as an adequate sentinel species for monitoring protocols.

[Contents of monovalent cations and ATP in erythrocytes of marine fishes in experimental hypoxia].

Coupling of membrane and metabolic functions in nuclear erythrocytes was investigated under experimental hypoxia conditions in fishes (Liza aurata, Scorpaena porcus) with different tolerance to oxygen deficiency. It was shown, that resistant to hypoxia Scorpaena porcus keeps in erythrocytes transmembrane gradients of K+ and Na+ and cellular concentration ATP under 15% of oxygen saturation of sea water. It was connected with the decrease in Na+, K+ -ATPase and hexokinase activity. The reaction to oxygen deficiency was opposite in sensitive to hypoxia Liza aurata erythrocytes. A decrease in ionic gradients and concentration of ATP in red blood cells was observed while the activity of Na+, K+ -ATPase and hexokinase was high. The reasons of the differences obtained are discussed.

LDL oxidation, antioxidant capacity and growth of cultured grey mullet ( Mugil cephalus ) fed dietary sorghum distillery residue pretreated with polyethylene glycol.

Dietary sorghum distillery residue (SDR) showed antioxidant and blood thinning effects on grey mullet during winter, but inhibited their growth. The objective of this study was to establish a preliminary treatment of the dietary SDR with polyethylene glycol (PEG), a tannin-binding agent, to enhance growth and blood antioxidant capacity of grey mullet ( Mugil cephalus ) feed. The feeding trial was carried out from June to November. The water temperature was between 25 and 30 degrees C; the specific growth rate of mullet was reduced significantly by feeding diet containing 20% SDR in comparison to fish fed the control diet or diet containing 20% SDR and PEG. In the period of October-November, the water temperature decreased to 19-25 degrees C; the specific growth rates of the 20% SDR-PEG group and the 20% SDR group were 0.13 and 0.19% day(-1), respectively, significantly higher than those fed the control diet (0.07% day(-1)). Feeding with 20% SDR or 20% SDR-PEG diets resulted in prolonged lag phase of low-density lipoprotein (LDL) oxidation compared to fish fed the control diet. The total antioxidant capacity of the plasma of the grey mullet fed 20% SDR-PEG was 1.24 mmol/L, significantly higher than those in the fish fed 20% SDR diet (0.84 mmol/L) or the control (0.72 mmol/L). In vivo observations found that preliminary treatment of SDR with PEG eliminated the endogenous undesirable growth inhibitory factors but maintained its protective effects against LDL oxidation in blood and improved the total antioxidant capacity and cold adaptation of grey mullet. The ethanol extract of SDR contained 31.9 +/- 7.8 mg/g gallic acids equivalent. The concentration needed to scavenge 50% of the DPPH radicals (IC(50)) was 0.86 mg/mL. Increased gallic acid equivalent and decreased IC(50) of DPPH scavenging activity of SDR fed to fish increased the total antioxidant capacity in blood plasma of grey mullet significantly.

Choriogenin and vitellogenin in red lip mullet (Chelon haematocheilus): purification, characterization, and evaluation as potential biomarkers for detecting estrogenic activity.

Two vitelline envelope precursors (choriogenin H: Chg H; choriogenin L: Chg L) and an egg yolk precursor (vitellogenin B: VgB) were purified from red lip mullet. The mass of intact Chg H and Chg L were estimated to be approximately 215 kDa and approximately 69 kDa, respectively. In SDS-PAGE, Chg H and Chg L separated to positions corresponding to approximately 51 kDa and approximately 44 kDa, respectively. The mass of intact VgB was approximately 530 kDa and resolved into a polypeptide of approximately 185 kDa in SDS-PAGE. Specific antisera were raised against each purified protein and specific immunoassays were developed. When Chg H, Chg L and VgB were induced in the serum of immature mullet by injection with various doses of estradiol-17beta (E(2)), VgB exhibited the most sensitive response exhibiting high variation in its induced levels. The variation in induced levels of Chg H and L was relatively minimal although induction required higher doses of E(2) than with VgB. Serum samples obtained from immature mullet populations collected from their natural habitat exhibited similar profiles in the levels of these proteins. The present study suggests that the utilization of multiple biomarkers holds great importance for the reliable and accurate evaluation of estrogenic activity in aquatic environments.

Simultaneous determination of oxidized and reduced glutathione in eel's (Monopterus albus) plasma by transient pseudoisotachophoresis coupled with capillary zone electrophoresis.

Both the reduced form of glutathione (GSH) and the oxidized form of glutathione (GSSG) in eel's ( Monopterus albus) plasma were for the first time determined by transient pseudoisotachophoresis coupled with capillary zone electrophoresis. The method of transient pseudoisotachophoresis coupled with capillary zone electrophoresis has been thoroughly optimized and adequately evaluated for the simultaneous determination of GSH and GSSG in eel's plasma. The detection limits (S/N = 3) of the method developed were 0.2 and 0.05 micromol/L for GSH and GSSG, respectively. The linearity of the calibration curves was valid in the range of 0-10 micromol/L GSH and 0-0.70 micromol/L GSSG. The method was simple, fast, and reproducible. It was found that the respective concentrations of GSH and GSSG were in the range of 9.1-14.5 and 0.31-0.58 micromol/L in the adult eel's plasma, and 10.8-17.9 and 0.49 - 0.68 micromol/L in the juvenile eel's plasma of the three populations determined. Each blood sample was a composite of five eels. For each of the three populations, the concentrations of GSH and GSSG in the adult eel's plasma were lower than those in the juvenile eel's plasma, and the concentrations of GSH and GSSG in the plasma of population 1 (deep yellow finless eels) were higher than those in populations 2 (light yellow finless eels) and 3 (green finless eels) for either the adult or the juvenile eels.