Comparative profiling of microbial community of three economically important fishes reared in sea cages under tropical offshore environment.

The present study was undertaken to evaluate the microbial composition of farmed cobia pompano and milkfish, reared in sea-cages by culture-independent methods. This study would serve as a basis for assessing the general health of fish, identifying the dominant bacterial species present in the gut for future probiotic work and in early detection of potential pathogens. High-throughput sequencing of V3-V4 hyper variable regions of 16S rDNA on Illumina MiSeq platform facilitated unravelling of composite bacterial population. Analysis of 1.3 million quality-filtered sequences revealed high microbial diversity. Characteristic marine fish gut microbes: Vibrio and Photobacterium spp. showed prevalence in cobia and pompano whereas Pelomonas and Fusobacterium spp. dominated the gut of milkfish. Pompano hindgut with 10,537 operational taxonomy units (OTUs) exhibited the highest alpha-diversity index followed by cobia (10,435) and milkfish (2799). Additionally unique and shared OTUs in each gut type were identified. Gammaproteobacteria dominated in cobia and pompano while Betaproteobacteria showed prevalence in milkfish. We obtained 96 shared OTUs among the three species though the numbers of reads were highly variable. These differences in microbiota of farmed fish reared in same environment were presumably due to differences in the gut morphology, physiological behavior and host specificity.

Expression and immunolocalization of 20ß-hydroxysteroid dehydrogenase during testicular cycle and after hCG induction, in vivo in the catfish, Clarias gariepinus.

The maturation inducing hormone, 17α,20ß-dihydroxy-4-pregnen-3-one (17α,20ß-DP) is required for the meiotic maturation and is produced from the precursor 17α-hydroxyprogesterone by the enzyme 20ß-hydroxysteroid dehydrogenase (20ß-HSD) in several teleosts. Central role of 20ß-HSD in ovarian cycle and final oocyte maturation is well studied when compared to spermatogenesis. In the present study, we investigated the localization and expression of 20ß-HSD in testicular cycle and gonadotropin induced sperm maturation. During testicular ontogeny, 20ß-HSD expression was detectable at 50 and 100 days post-hatch (dph), while the expression was high at 150 dph. In testicular cycle, highest levels of mRNA and protein of 20ß-HSD were observed during spawning phase. Intraperitoneal injection of human chorionic gonadotropin (hCG) to prespawning catfish elevated both 20ß-HSD transcripts and protein levels when compared to saline treated controls in a time-dependent manner. Serum 17α,20ß-DP levels, measured during different phases of testicular cycle as well as following the treatment of hCG, showed a positive correlation with the expression of 20ß-HSD. Immunolocalization revealed the presence of 20ß-HSD protein predominantly in interstitial cells and spermatogonia/spermatocytes while 20ß-HSD was undetectable in haploid cells (spermatids/sperm). These results together with high expression during spawning phase of testicular cycle and after hCG treatment in the prespawning catfish suggests a pivotal role for 20ß-HSD during testicular recrudescence leading to sperm maturation. Further studies using various fish models on testicular 20ß-HSD may provide interesting details to understand its importance in teleostean spermatogenesis.

Cytochrome P450 aromatases: Impact on gonadal development, recrudescence and effect of hCG in the catfish, Clarias gariepinus.

Present study analyzed the importance of two forms of aromatases during ovarian development and recrudescence of north African/air-breathing catfish. We cloned both CYP19A1 (1941bp; ovarian form) and CYP19A2 (1786bp; brain form), which showed 47% homology between the two forms. Characterization of encoded proteins in non-steroidogenic COS-7 cells illustrated that both isoforms efficiently catalyzed the aromatization reaction by producing estradiol-17beta (E(2)) from testosterone. Tissue distribution pattern revealed preferential expression of CYP19A2 in brain while CYP19A1 predominated in ovary with trace amounts detected in other tissues including brain. Relative real-time PCR analysis revealed high transcript levels of both isoforms in the prespawning phase of ovarian cycle, which is in accordance with serum E(2) level. Aromatase activity in brain was comparatively lower than ovary, indicating the predominant requirement of aromatase in ovary. Ontogeny studies displayed sexual dimorphism, with early expression of CYP19A1 and CYP19A2 in ovary and brain, respectively. Phase-dependent rise of expression and enzyme activity of aromatase after hCG treatment revealed the stimulatory role of gonadotropin during preparatory and prespawning phases, preferentially to promote vitellogenesis. Lack of influence of hCG treatment during spawning phase endorses it further. A good correlation of expression, enzyme activity and serum E(2) levels suggests a crucial role of CYP19A1 during ovarian differentiation and ovarian cycle of catfish. Likewise, CYP19A2 might also be involved in these processes either indirectly or directly.

Cloning, expression and enzyme activity analysis of testicular 11beta-hydroxysteroid dehydrogenase during seasonal cycle and after hCG induction in air-breathing catfish Clarias gariepinus.

A full-length cDNA encoding 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) was cloned from testis of air-breathing catfish, Clarias gariepinus which showed high sequence homology to zebrafish and eel. The open reading frame of 11beta-HSD2 was then transfected to COS-7 cells, which converted 11beta-hydroxytestosterone (11-OHT) to 11-ketotestosterone (11-KT). Using NAD(+), 11beta-HSD2 from testicular microsomes oxidized 11-OHT with apparent K(m) 56+/-4nM and V(max) 55+/-6pmol/h/mgprotein values. Tissue distribution analysis revealed prominent expression in testis, anterior kidney, liver and gills. Expression of 11beta-HSD2 in testis and serum levels of 11-KT were high in the prespawning phase. Administration of human chorionic gonadotropin (hCG) during prespawning and resting phases revealed initial rise in 11beta-HSD2 transcript at 4h followed by gradual increase at 8h, 12h and peaking at 24h, only in testis of prespawning phase. Rate of conversion of 11-OHT to 11-KT by testicular microsomes during different testicular phases and after hCG administration corroborated well with the expression of 11beta-HSD2. Ontogeny study indicated that this enzyme is expressed during testicular development. Thus the spatio-temporal expression supported with putative dehydrogenase activity and circulating 11-KT levels clearly suggest a major role for 11beta-HSD2 during testicular differentiation and seasonal testicular cycle in catfish.

Thiourea-induced thyroid hormone depletion impairs testicular recrudescence in the air-breathing catfish, Clarias gariepinus.

We used thiourea-induced thyroid hormone depletion as a strategy to understand the influence of thyroid hormones on testicular recrudescence of the air-breathing catfish, Clarias gariepinus. Treatment with 0.03% thiourea via immersion for 21 days induced hypothyroidism (thyroid hormone depletion) as evidenced by significantly reduced serum T(3) levels. Thiourea-treated males had narrowed seminiferous lobules with fewer spermatozoa in testis, very little or no secretory fluid, reduced protein and sialic acid levels in seminal vesicles when compared to controls. The histological changes were accompanied by reduction in serum and tissue levels of testosterone (T) and 11-ketotestosterone (11-KT), a potent male specific androgen in fish. Qualitative changes in the localization of catfish gonadotropin-releasing hormone (cfGnRH) and luteinizing hormone (LH, heterologous system) revealed a reduction in the distribution of immunoreactive neuronal cells and fibers in thyroid depleted fish. Interestingly, thiourea-withdrawal group showed physiological and histological signs of recovery after 21 days such as reappearance of spermatozoa and partial restoration of 11-KT and T levels. These data demonstrate that thyroid hormones play a significant role in testicular function of catfish. The mechanism of action includes modulating sex steroids either directly or through the hypothalamo (GnRH)-hypophyseal (LH) axis.

Effect of methyl testosterone- and ethynyl estradiol-induced sex differentiation on catfish, Clarias gariepinus: expression profiles of DMRT1, Cytochrome P450aromatases and 3 beta-hydroxysteroid dehydrogenase.

The objective of the present study is to observe the effect of exogenous steroids, methyl testosterone (MT) and ethynyl estradiol (EEL) on gonadal differentiation and analyze its effect on the expression of several genes during testicular and ovarian differentiation in juvenile catfish. Exogenous hormone treatments (MT and EEL) were given by immersion at different days of hatching. The histological analysis revealed that the EEL- and MT-treatments resulted in the initiation of ovarian and testicular differentiation, respectively. This is further supported by specific expression of two forms of DMRT1 in the MT-treated group but not in the EEL-treated group at 47 days after hatching (dah). The reverse is true for the expression of ovarian aromatase. Results of the semi-quantitative RT-PCR show that brain aromatase transcript levels are high in 47 dah control (histologically female) and 47 dah EEL-treated fish, as compared to 47 dah MT-treated fish. At 60 dah, brain aromatase showed elevation in its expression. Interestingly, the expression pattern of 3 beta-HSD did not show any change in EEL- and MT-treated fish. The present study also provides a strategy to study sex differentiation, for those species where genetic sex population is unavailable.

Seabream GnRH: partial cDNA cloning, localization and stage-dependent expression in the ovary of snake head murrel, Channa striatus.

Vertebrate reproduction is under the neuroendocrine control of the hypothalamic decapeptide GnRH which synchronizes various reproductive events and influences other reproduction related aspects like spawning behavior and pheromonal action in fish. Multiple forms of GnRH peptides have been reported across diverse vertebrate and invertebrate classes. Here we report the partial seabream GnRH (sbGnRH) cDNA sequence cloned from the brain of Channa striatus (snake head murrel) a fresh water perciform with immense economic and medicinal value across Asiatic countries. sbGnRH mRNA was found in brain, gill and ovary of mature murrel with possible implications to the effect of GnRH on pheromonal phenomena and on reinitiation of oocyte meiosis. In keeping with the earlier reported role of GnRH in initiation of oocyte meiosis we here present evidence from RT-PCR, ICC demonstrating an increase in the level of sbGnRH mRNA in ovary from pre-vitellogenic to post-vitellogenic follicles.

Expression of 20beta-hydroxysteroid dehydrogenase and P450 17alpha-hydroxylase/c17-20 lyase during hCG-induced in vitro oocyte maturation in snake head murrel Channa striatus.

Partial cDNAs encoding carbonyl reductase like 20beta-hydroxysteroid dehydrogenase (20beta-HSD) and P450 17alpha-hydroxylase/c17-20 lyase (CYP17) were isolated from the ovary of snake head murrel and they exhibited high sequence identity to the Nile tilapia and rainbow trout, respectively. A low transcript level of both 20beta-HSD and CYP17 were detected in pre-vitellogenic follicles, while the transcript level was high in full-grown immature follicles. In hCG-induced in vitro oocyte maturation, we found a significant increase in 20beta-HSD transcript level after 2 h. The CYP17 transcripts also showed a considerable increase following hCG-induction compared to saline-treated controls. On the other hand, Western blot analysis demonstrated no significant change in the CYP17 protein level during hCG-induced in vitro oocyte maturation. Taken together, we suggest that in addition to 20beta-HSD, the CYP17 might have a role in the shift in steroidogenesis during meiotic maturation of snake head murrel.

Thyroid hormone modulation of ovarian recrudescence of air-breathing catfish Clarias gariepinus.

In the present study, thiourea-induced thyroid hormone depletion and thyroxine (T(4)) 'overdose' were used as a strategy to understand the influence of thyroid hormones on ovarian recrudescence of juvenile (3-months-old), immature (8-months-old) and adult (1-year-old) air-breathing catfish, Clarias gariepinus. Thiourea-induced thyroid hormone depletion in juvenile catfish impaired ovarian development, but no significant effect was observed in immature catfish and during late stage of ovarian recrudescence of mature catfish. T(4) treatment in females undergoing late stages of ovarian recrudescence induced rapid oocyte growth by promoting its early entry into maturational phase as evident from the presence of more number of vitellogenic and post-vitellogenic follicles, decrease in aromatse immunoreactivity and reduced estradiol-17beta levels. Hence, thyroid hormones have an important role to play during early stages of ovarian development and vitellogenesis of catfish and also indicating that thyroid has a stage dependent effect on ovary.

Thiourea-induced alteration in the expression patterns of some steroidogenic enzymes in the air-breathing catfish Clarias gariepinus.

Previous study from our laboratory on thiourea-induced thyroid hormone depletion in mature male catfish demonstrated that thyroid hormones play a significant role in testicular function. In the present study, we aimed to analyze the changes in the expression pattern of several steroidogenic enzyme genes after thyroid hormone depletion using semi quantitative RT-PCR in both adult male and female catfish. There was a marked decrease in the 11beta-hydroxylase expression in the testis and liver while no change was observed in case of kidney. A significant decrease in 11beta-hydroxysteroid dehydrogenase transcript level in testis, liver and kidney were observed in the thiourea treated males. The results obtained corroborated with our earlier findings of testicular regression after thyroid hormone depletion. In females, expression of aromatase transcript increased in experimental group compared to control. There was no considerable change observed in the transcript level of 3beta-hydroxysteroid dehydrogenase, P450 17alpha-hydroxylase/C17-20-lyase, and 20beta-hydroxysteroid dehydrogenase in both males and females. Thus, thyroid hormones might exert modulating effect on steroidogenic enzyme genes at the transcription level.