Diethylstilbestrol arrested spermatogenesis and somatic growth in the juveniles of yellow catfish (<Emphasis Type="Italic">Pelteobagrus fulvidraco</Emphasis>), a fish with sexual dimorphic growth

In fish, spermatogenesis and somatic growth are mainly regulated by hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-somatic (HPS) axes, respectively. Xenoestrogens have been reported to impair spermatogenesis in some fishes, and arrest somatic growth in some others, whereas, whether xenoestrogens are capable of disrupting spermatogenesis and somatic growth simultaneously in fish that exhibits sexual dimorphic growth is little known, and the underlying mechanisms remain poorly understood. In this study, male juveniles of yellow catfish (Pelteobagrus fulvidraco), which exhibits a sexual dimorphic growth that favors males, were exposed to diethylstilbestrol (DES) for 28 days. After exposure, DES significantly disrupted the spermatogenesis (decreased gonadal-somatic index (GSI) and germ cell number) and arrested the somatic growth (declined body weight) of the catfish juveniles. Gene expression and plasma steroid analyses demonstrated the suppressed mRNA levels of genes in HPG axis (gnrh-II, fshβ, and lhβ in the brain and dmrt1, sf1, fshr, cyp17a1, cyp19a1a, and cyp11b2 in the testis) and decreased 17β-estrodial (E2) and 11-ketotestosterone (11-KT) levels in plasma. Further analysis revealed the arrested germ cell proliferation (cyclin d1), meiosis (dmc1, sycp3), and enhanced apoptosis (decreased bcl-2 and elevated bax/bcl-2 ratio) in the testis. Besides, DES also suppressed the mRNA levels of genes in HPS axis (ghrh, gh, and prl in the brain and ghr, igf1, igf2a, and igf2b in the liver). The suppressed HPG and HPS axes were thus supposed to disturb spermatogenesis and arrest somatic growth in yellow catfish. The present study greatly extended our understanding on the mechanisms underlying the toxicity of DES on spermatogenesis and somatic growth of fish.     

The effects of starvation on fast-start escape and constant acceleration swimming performance in rose bitterling (<Emphasis Type="Italic">Rhodeus ocellatus</Emphasis>) at two acclimation temperatures

To investigate the effects of starvation and acclimation temperature on the escape ability of juvenile rose bitterling (Rhodeus ocellatus), we measured the fast-start escape and constant acceleration swimming performance of fish fasted for 0 (control), 1 and 2 weeks and half-lethal periods (6 or 4 weeks) at two temperatures (15 and 25 °C). Fish acclimated at a high temperature exhibited shorter response latency (R), higher maximum linear velocity (V _max) and longer escape distance during escape movement (D _120ms) than those at the low temperature. Starvation resulted in a significant decrease in V _max and D _120ms at either low or high temperature and a significant increase in R at only the high temperature in the half-lethal period groups (P < 0.05). The relationship between V _max (Y, m s^?1) and starvation time (X, week) was Y ₁₅ = ?0.062X + 1.568 (r = ?0.665, n = 36, P < 0.001) at low temperature and Y ₂₅ = ?0.091X + 1.755 (r = ?0.391, n = 40, P = 0.013) at high temperature. The relationship between U _cat (Y, cm s^?1) and starvation time (X, week) was Y ₁₅ = ?1.649X + 55.418 (r = ?0.398, n = 34, P = 0.020) at low temperature and Y ₂₅ = ?4.917X + 62.916 (r = ?0.793, n = 33, P < 0.001) at high temperature. The slopes of equations showed a significant difference between low and high temperature (F _1,63 = 9.688, P = 0.003), which may be due to the different energy substrate utilization when faced with food deprivation at different temperatures.     

Demographic and competition studies on <Emphasis Type="Italic">Brachionus ibericus</Emphasis> and <Emphasis Type="Italic">Proales similis</Emphasis> in relation to salinity and algal (<Emphasis Type="Italic">Nannochloropsis oculata</Emphasis>) density

We isolated the rotifers Brachionus ibericus and Proales similis from the sediment of shrimp tanks and studied their individual demographic characters and competition between them at two food levels (0.25?×?10⁶, 1.00?×?10⁶ cells ml^?1 of Nannochloropsis oculata at 25 °C) and salinities ranging from 10 to 30‰. Our hypothesis was that growth rates would be higher with increasing food levels and salinities. Observations were taken twice a day for life table studies and daily once for population growth experiments. Using survivorship and fecundity data, we derived various life history variables. Although the average life span (7.6?±?0.4 days) and gross reproductive rate (33.8?±?2.9 neonate female^?1 day^?1) of B. ibericus were higher than those of P. similis (average life span 5.4?±?0.6 days and gross reproductive rate 13.0?±?0.6 neonate female^?1 day^?1), the population growth experiments showed that P. similis had higher r values (0.32?±?0.005 day^?1) than B. ibericus (0.23?±?0.002 day^?1) at 1.0?×?10⁶ cells ml^?1 of N. oculata. The rotifer P. similis was more adversely affected due to the presence of B. ibericus than vice versa. The data are important for developing techniques for a large-scale culture of these rotifers as food in aquaculture.     

Gene expression of thyrotropin- and corticotrophin-releasing hormones is regulated by environmental salinity in the euryhaline teleost <Emphasis Type="Italic">Sparus aurata</Emphasis>

In euryhaline teleosts, the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-interrenal axes (HPT and HPI, respectively) are regulated in response to environmental stimuli such as salinity changes. However, the molecular players participating in this physiological process in the gilthead seabream (Sparus aurata), a species of high value for aquaculture, are still not identified and/or fully characterized in terms of gene expression regulation. In this sense, this study identifies and isolates the thyrotropin-releasing hormone (trh) mRNA sequence from S. aurata, encoding prepro-Trh, the putative factor initiating the HPT cascade. In addition, the regulation of trh expression and of key brain genes in the HPI axis, i.e., corticotrophin-releasing hormone (crh) and corticotrophin-releasing hormone-binding protein (crhbp), was studied when the osmoregulatory status of S. aurata was challenged by exposure to different salinities. The deduced amino acid structure of trh showed 65–81% identity with its teleostean orthologs. Analysis of the tissue distribution of gene expression showed that trh mRNA is, though ubiquitously expressed, mainly found in brain. Subsequently, regulation of gene expression of trh, crh, and crhbp was characterized in fish acclimated to 5-, 15-, 40-, and 55-ppt salinities. In this regard, the brain gene expression pattern of trh mRNA was similar to that found for the crh gene, showing an upregulation of gene expression in seabream acclimated to the highest salinity tested. Conversely, crhbp did not change in any of the groups tested. Our results suggest that Trh and Crh play an important role in the acclimation of S. aurata to hypersaline environments.     

Effects of dietary microencapsulated <Emphasis Type="Italic">Cymbopogon flexuosus</Emphasis> essential oil on reproductive-related parameters in male <Emphasis Type="Italic">Rhamdia quelen</Emphasis>

In aquaculture, nutrition and supplemented diets have been shown to affect broodstock reproductive performance. In this study, we investigated the effects of dietary supplementation with Cymbopogon flexuosus essential oil (CFEO) microcapsules on reproductive-related parameters in silver catfish (Rhamdia quelen) male broodfish. Adult male broodstocks were separated into three groups according to the concentrations of supplemented CFEO (0.0?=?control; 1.0 or 3.0 mL per kg of diet). After 20 days under experimental conditions, the animals were euthanized and the gonads were harvested for gonadosomatic index, sperm analysis, oxidative stress, and histopathology; testosterone levels were measured in the plasma; gene expression of prl, smtl, pomca, and pomcb was assessed in the pituitary gland by real-time PCR. The results showed no alterations on reproductive parameters in R. quelen males treated with Cymbopogon flexuosus essential oil compared to the control-diet animals. In conclusion, CFEO microcapsules supplied for 20 days in the concentrations of 1.00 or 3.00 mL per kilogram of diet did not affect the reproduction criteria evaluated in this study in male silver catfish.     

Immunomodulatory effects of <Emphasis Type="Italic">Rhodomyrtus tomentosa</Emphasis> leaf extract and its derivative compound,rhodomyrtone, on head kidney macrophages of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

Rhodomyrtus tomentosa is a medicinal plant that shows biological effects including immunomodulatory activity on human and other mammals but not in fish. In this study, we evaluated the in vitro immunomodulatory effects of R. tomentosa leaf extract and its active compound, rhodomyrtone, on the immune responses, using rainbow trout (Oncorhynchus mykiss) head kidney (HK) macrophages as a model. The tested immune functions included the expression of genes involved in innate immune and inflammatory responses and the production of reactive oxygen species (ROS). Gene expression was evaluated after exposure to 10 μg mL^?1 of R. tomentosa and 1 μg mL^?1 of rhodomyrtone for 4 and 24 h. R. tomentosa and rhodomyrtone induced changes in the expression of pro-inflammatory cytokines (il1β, il8, and tnfα), anti-inflammatory cytokines (il10 and tgfβ), inducible enzymes (inos, cox2, and arginase), and an antioxidant enzyme (gpx1). Co-exposure of R. tomentosa with LPS resulted in a prominent reduction in the expression of genes related to an inflammatory process (il1β, il8, tnfα, inos, saa, hepcidin, and gpx1), suggesting anti-inflammatory effects. Similarly, co-exposure of rhodomyrtone with LPS led to a downregulation of inflammation-related genes (il1β, inos, saa, and hepcidin). In addition, exposure to both natural plant products caused a reduction in cellular ROS levels by HK macrophages. The present results indicate that R. tomentosa and rhodomyrtone exerted immunostimulatory and anti-inflammatory effects on fish macrophages, thus opening up the possibility of using these natural products to further develop immunostimulants for health management in aquaculture.     

Gene structure and expression analyses of multiple vitellogenesis-inhibiting hormones in the whiteleg shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

In the whiteleg shrimp Litopenaeus vannamei, the existence of six sinus gland peptides (SGPs) possessing vitellogenesis-inhibiting hormone (VIH) function has been previously demonstrated. Genomic and cDNA sequence information for two of these is already known. Here, we cloned cDNAs for the remaining three, i.e., those encoding Liv-SGP-A, -B, and -F. The deduced amino acid sequences were comprised of a signal peptide, a CHH precursor-related peptide, a mature peptide, and a C-terminal amidation signal. The genomic DNA for Liv-SGP-A, -B, -C, -F, and -G, was found to be organized as three exons and two introns. The insertion of the two introns were conserved in all VIH genes, with intron I being inserted six residues following the signal peptide, and intron II after the 40th residue of the mature peptide. We also quantified the relative simultaneous expression of Liv-SGP-A, -B, -C, -F, and -G in relation to molting and unilateral eyestalk ablation. The expression levels of each gene in the eyestalks of adults did not change significantly during molting or following unilateral eyestalk ablation. However, unilateral eyestalk ablation significantly decreased expression levels at 10 and 20 days for Liv-SGP-A and -C, and at 20 days for Liv-SGP-G in the eyestalks of subadults. This study has therefore clarified the genomic structure of the five major subtype I VIHs in L. vannamei, and elucidated their expression levels in the eyestalks in relation to molting and unilateral eyestalk ablation.     

Alternative microalgal diets for cultivation of the fairy shrimp <Emphasis Type="Italic">Branchinella thailandensis</Emphasis> (Branchiopoda: Anostraca)

Fairy shrimp is known as a nutritional food for fish and crustaceans in aquaculture. In most hatcheries, the microalga Chlorella sp. appears to be the most common, suitable, and nutritious food to feed fairy shrimp. In this study, we attempted to determine other alternative algal diets for cultivation of fairy shrimp Branchinella thailandensis. Seven experimental diets including three treatments of dried Spirulina sp. at 0.75 (S1), 1.5 (S2), and 3.0 mg dry weight individual^?1 (S3); three treatments of Chlorococcum humicola at 5 × 10⁵ (Ch1), 1 × 10⁶ (Ch2), and 2 × 10⁶ cells mL^?1 (Ch3); and a control diet (Chlorella vulgaris at 1 × 10⁶ cells mL^?1) were fed to 5-day-old shrimp for 15 days. Evaluation of growth performance, egg production, survival percentage, and nutritional and carotenoid content of the experimental fairy shrimp revealed that Ch3 is the most suitable algal diet. Our results suggest that C. humicola is the best alternative food source for the cultivation of B. thailandensis. In addition, dried Spirulina powder is also a good choice when live algae are not available and can be used as an alternative feed in fairy shrimp cultures.     

Inhibitory neurotransmitter serotonin and excitatory neurotransmitter dopamine both decrease food intake in Chinese perch (<Emphasis Type="Italic">Siniperca chuatsi</Emphasis>)

Aminergic neurotransmitters play important roles in the regulation of food intake. However, their effects on feeding in fish have been less explored and still unclear. In the present study, the effects of serotonin (5-HT) and dopamine (DA) on food intake were evaluated through intraventricular (ICV) administration in Chinese perch (Siniperca chuatsi) and the mRNA expression levels of neuropeptide Y (NPY), agouti gene-related protein (AgRP), and pro-opiomelanocortin (POMC) were detected. At 1 h post-injection, 5-HT significantly decreased food intake in a dose-dependent manner. The mRNA expression of NPY and AgRP were significantly decreased (p < 0.05), whereas the mRNA expression of POMC was significantly increased (p < 0.05), suggesting the involvement of NPY, AgRP, and POMC in inhibitory action of 5-HT on food intake in Chinese perch. DA significantly decreased (p < 0.05) food intake and AgRP mRNA expression at 1 h post-injection, indicating the inhibitory effect of DA on food intake might be mediated through AgRP. This might shed new light on the regulation of food intake in Chinese perch.     

Dietary supplementation of curcumin augments heat stress tolerance through upregulation of <Emphasis Type="Italic">nrf-2</Emphasis>-mediated antioxidative enzymes and <Emphasis Type="Italic">hsps</Emphasis> in <Emphasis Type="Italic">Puntius sophore</Emphasis>

Heat stress is one of the major environmental concerns in global warming regime and rising temperature has resulted in mass mortalities of animals including fishes. Therefore, strategies for high temperature stress tolerance and ameliorating the effects of heat stress are being looked for. In an earlier study, we reported that Nrf-2 (nuclear factor E2-related factor 2) mediated upregulation of antioxidative enzymes and heat shock proteins (Hsps) provide survivability to fish under heat stress. In this study, we have evaluated the ameliorative potential of dietary curcumin, a potential Nrf-2 inducer in heat stressed cyprinid Puntius sophore. Fishes were fed with diet supplemented with 0.5, 1.0, and 1.5% curcumin at the rate 2% of body weight daily in three separate groups (n = 40 in each group) for 60 days. Fishes fed with basal diet (without curcumin) served as the control (n = 40). Critical thermal maxima (CTmax) was determined for all the groups (n = 10, in duplicates) after the feeding trial. Significant increase in the CTmax was observed in the group fed with 1.5% curcumin- supplemented fishes whereas it remained similar in groups fed with 0.5%, and 1% curcumin-supplemented diet, as compared to control. To understand the molecular mechanism of elevated thermotolerance in the 1.5% curcumin supplemented group, fishes were given a sub-lethal heat shock treatment (36 °C) for 6 h and expression analysis of nrf-2, keap-1, sod, catalase, gpx, and hsp27, hsp60, hsp70, hsp90, and hsp110 was carried out using RT-PCR. In the gill, expression of nrf-2, sod, catalase, gpx, and hsp60, hsp70, hsp90, and hsp110 was found to be elevated in the 1.5% curcumin-fed heat-shocked group compared to control and the basal diet-fed, heat-shocked fishes. Similarly, in the liver, upregulation in expression of nrf-2, sod, catalase, and hsp70 and hsp110 was observed in 1.5% curcumin supplemented and heat shocked group. Thus, this study showed that supplementation of curcumin augments tolerance to high temperature stress in P. sophore that could be attributed to nrf-2-induced upregulation of antioxidative enzymes sod, catalase, gpx, and the hsps.     

Anti-oxidative functions of <Emphasis Type="Italic">mt2</Emphasis> and <Emphasis Type="Italic">smtB</Emphasis> mRNA expression in the gills and brain of zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) upon cadmium exposure

There were not any past studies about metallothionein isoforms (smtB and mt2) having anti-oxidative functions on zebrafish after Cd²⁺ exposure. On the other hand, the anti-oxidative enzymatic factors such as superoxide dismutase (sod), glutathione peroxidase (gpx1a), and catalase (cat) are used as references to investigate whether the smtB and mt2 have anti-oxidative responses on the gills and brain of zebrafish after 1–6 h of 0 and 1.78 μM Cd²⁺ exposure. The anti-oxidative system such as sod, cat, and gpx1a mRNA expressions demonstrated a cascade response upon Cd²⁺-induced oxidative stress in the present study. Interestingly, the smtB mRNA expression levels increased by 3.2- to 6.1-fold, and mt2 raised by 4.1- to 11.3-fold in gills at 1 and 3 h after exposure to Cd²⁺, respectively. On the other hand, the smtB mRNA levels increased by 10.6- to 58.6-fold, but mt2 mRNA levels increased by 2.3- to 11.1-fold in brain at 1 and 3 h after exposure to Cd²⁺, respectively. In addition, both tissues showed increased apoptosis levels at 3 h, and recovery after 6 h of Cd²⁺ exposure. From the results, we suggest that both mt2 and smtB play a role in anti-oxidation responses within 6 h after exposure to Cd²⁺. In conclusion, the smtB mRNA levels have a higher response than mt2 in the brain, but both mRNA expressions appear to have a similar pattern in the gill. We suggest that smtB plays an important role to defend oxidative stress in the brain of adult zebrafish upon acute Cd²⁺ exposure.     

Effects of temperature and fatigue on the metabolism and swimming capacity of juvenile Chinese sturgeon (<Emphasis Type="Italic">Acipenser sinensis</Emphasis>)

Chinese sturgeon (Acipenser sinensis) is a critically endangered species. A flume-type respirometer, with video, was used to conduct two consecutive stepped velocity tests at 10, 15, 20, and 25 °C. Extent of recovery was measured after the 60-min recovery period between trials, and the recovery ratio for critical swimming speed (U _crit) averaged 91.88% across temperatures. Temperature (T) effects were determined by comparing U _crit, oxygen consumption rate (MO ₂), and tail beat frequency (TBF) for each temperature. Results from the two trials were compared to determine the effect of exercise. The U _crit occurring at 15 °C in both trials was significantly higher than that at 10 and 25 °C (p < 0.05). The U _crit was plotted as a function of T and curve-fitting allowed calculation of the optimal swimming temperature 3.28 BL/s at 15.96 °C (trial 1) and 2.98 BL/s at 15.85 °C (trial 2). In trial 1, MO ₂ increased rapidly with U, but then declined sharply as swimming speed approached U _crit. In trial 2, MO ₂ increased more slowly, but continuously, to U _crit. TBF was directly proportional to U and the slope (dTBF/dU) for trial 2 was significantly lower than that for trial 1. The inverse slope (tail beats per body length, TB/BL) is a measure of swimming efficiency and the significant difference in slopes implies that the exercise training provided by trial 1 led to a significant increase in swimming efficiency in trial 2.     

Transcripts of genes encoding reproductive neuroendocrine hormones and androgen receptor in the brain and testis of goldfish exposed to vinclozolin,flutamide, testosterone,and their combinations

Vinclozolin (VZ) is a pesticide that acts as an anti-androgen to impair reproduction in mammals. However, VZ-induced disruption of reproduction is largely unknown in fish. In the present study, we have established a combination exposure in which adult goldfish were exposed to VZ (30 and 100 μg/L), anti-androgen flutamide (Flu, 300 μg/L), and androgen testosterone (T, 1 μg/L) to better understand effects of VZ on reproductive endocrine system. mRNA levels of kisspeptin (kiss-1 and kiss-2) and its receptor (gpr54), salmon gonadotropin-releasing hormone (gnrh3) and androgen receptor (ar) in the mid-brain, and luteinizing hormone receptor (lhr) in the testis were analyzed and compared with those of control following 10 days of exposure. kiss-1 mRNA level was increased in goldfish exposed to 100 µg/L VZ and to Flu, while kiss-2 mRNA level was increased following exposure to Flu and to combinations of 30 µg/L VZ with Flu, 100 µg/L VZ with T, and Flu with T. gpr54 mRNA level was increased in goldfish exposed to Flu and to combination of 30 µg/L VZ with Flu and 100 µg/L VZ with T. gnrh3 mRNA level was increased in goldfish exposed to 100 µg/L VZ, to Flu, and to combinations of 30 µg/L VZ with Flu, 100 µg/L VZ with T, and Flu with T. The mid-brain ar mRNA level was increased in goldfish exposed to Flu and to combinations of 30 µg/L VZ with Flu, 100 µg/L VZ with T, and Flu with T. Testicular lhr mRNA level was increased in goldfish exposed to Flu and to combination of 30 µg/L VZ with Flu. These results suggest that VZ and Flu are capable of interfering with kisspeptin and GnRH systems to alter pituitary and testicular horonal functions in adult goldfish and the brain ar mediates VZ-induced disruption of androgen production.     

Effects of low-voltage constant direct current on plasma biochemical profiles and gene expression levels in crucian carp <Emphasis Type="Italic">Carassius carassius</Emphasis>

This study investigated the plasma biochemical profiles and gene expression levels of crucian carp Carassius carassius subjected to anesthetization with low-voltage constant direct current (DC) and tricaine methanesulfonate (MS222). Fish in the MS222 treatments and the DC treatments were exposed to either two concentrations of MS222 (100 and 200 mg/l) or constant DC with a voltage of 26 V, at a voltage gradient of 0.68 V/cm, respectively. The results showed that low-voltage constant DC immobilized fish more quickly, resulting in significant elevations of plasma cortisol levels compared to exposure of high or low doses of MS222. Electronarcosis led to an increased expression of heat shock proteins 70 (HSP70) as well as HSP90 compared to the treatment group with MS222. Also, electronarcosis enhanced mitochondrial respiratory chain gene (atp6, cox1 and nd5) and antioxidant enzyme gene (CAT, SOD and GSH-px1) expression levels. In conclusion, low-voltage constant DC can disturb fish plasma biochemical profiles and gene expression levels.     

Characterization of <Emphasis Type="Italic">pax3a</Emphasis> and <Emphasis Type="Italic">pax3b</Emphasis> genes in artificially induced polyploid and gynogenetic olive flounder (<Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>) during embryogenesis

Although chromosome set manipulation techniques including polyploidy induction and gynogentic induction in flatfish are becoming increasingly mature, there exists a poor understanding of their effects on embryonic development. PAX3 plays crucial roles during embryonic myogenesis and neurogenesis. In olive flounder (Paralichthys olivaceus), there are two duplicated pax3 genes (pax3a, pax3b), and both of them are expressed in the brain and muscle regions with some subtle regional differences. We utilized pax3a and pax3b as indicators to preliminarily investigate whether chromosome set manipulation affects embryonic neurogenesis and myogenesis using whole-mount in situ hybridization. In the polyploid induction groups, 94 % of embryos in the triploid induction group had normal pax3a/3b expression patterns; however, 45 % of embryos in the tetraploid induction group showed abnormal pax3a/3b expression patterns from the tailbud formation stage to the hatching stage. Therefore, the artificial induction of triploidy and tetraploidy had a small or a moderate effect on flounder embryonic myogenesis and neurogenesis, respectively. In the gynogenetic induction groups, 87 % of embryos in the meiogynogenetic diploid induction group showed normal pax3a/3b expression patterns. However, almost 100 % of embryos in the gynogenetic haploid induction group and 63 % of embryos in the mitogynogenetic diploid induction group showed abnormal pax3a/3b expression patterns. Therefore, the induction of gynogenetic haploidy and mitogynogenetic diploidy had large effects on flounder embryonic myogenesis and neurogenesis. In conclusion, the differential expression of pax3a and pax3b may provide new insights for consideration of fish chromosome set manipulation.