Expression dynamics of solute carrier family 15 member 4 (SLC15A4) and its potential regulatory role in ovarian development of the Indian white shrimp, Penaeus indicus.

Solute carrier proteins (SLC) are essential membrane transport proteins responsible for transporting lipids, amino acids, sugars, neurotransmitters, and drugs across the biological membranes. Dysfunction of these carrier proteins may lead to an imbalance of biological mechanisms and also in the failure of the transporting pathways of several signaling neurotransmitters. In the present study, a 646 bp of a solute carrier protein (SLC15A4) was cloned and sequenced from the Indian white shrimp, Penaeus indicus. Multiple sequence alignment using ClustalW and phylogenetic analysis of putative SLC15A4 fragment from P. indicus (PiSLC15A4) was performed using Mega X tool. Tissue distribution analysis was carried out using real-time PCR. The differential expressions of PiSLC15A4 were also analyzed in the ovaries and brain tissues of wild-caught female shrimps at different maturation stages and in the brain tissues of captive females subjected to induce maturation by eyestalk ablation. Significant diversity in SLC15A4 sequence obtained from P. indicus was observed when compared to the other species. Tissue distribution analysis confirmed the ubiquitous expression of PiSLC15A4 in all the tissues examined. The differential expressions of PiSLC15A4 indicated higher expression of the gene in brain tissue of females at the vitellogenic stage, while the expressions in ovaries were significantly higher in the immature stage. The differential expressions of PiSLC15A4 in the brain tissues were substantially higher in eyestalk ablated shrimps compared to the eyestalk intact females. The study suggests a role for SLC15A4 in the endocrine signaling pathways stimulating ovarian maturation in P. indicus.

Mitochondrial DNA based diversity studies reveal distinct and substructured populations of pearlspot, Etroplus suratensis (Bloch, 1790) in Indian waters.

Pearlspot (Etroplus suratensis) is one of the most commercially important brackish water fish species widely found along the coastal regions of peninsular India and Sri Lanka. Pearlspot is known for its tender flesh, delectable taste, culinary tourism and highyielding market value. Information on the genetic makeup of stocks/populations is extremely vital as it forms the basis for future genetic studies. For this, we utilized ATPase6/8 genes of mtDNA of pearlspot populations collected from nine different locations ranging from Ratnagiri in Maharashtra state on the west coast to Chilika in Odisha on the east coast. Sequence analyses of these genes revealed 33 polymorphic sites, which include 17 singleton and 16 parsimony informative sites. Pair-wise genetic differentiation study (FST = 0.75) indicated significant (P<0.001) differences among all the pairs of stocks except those from Chilika and Nagayalanka. The spatial analysis of molecular variance (SAMOVA) significantly delineated the population into four groups (FCT = 0.69, P = 0.0001), namely northwest (Ratnagiri and Goa); southwest (Mangalore and lakes at Vembanad, Ashtamudi and Vellayani in Kerala); southeast (Pulicat in Tamil Nadu) and northeast (Chilika in Odisha and Nagayalanka in Andhra Pradesh). The above delineation is supported by clades of the phylogenetic tree and also the clusters of median joining haplotype network. The high haplotype diversity (0.84), low nucleotide diversity (0.003), and negative values of Tajima's D (-1.47) and Fu's Fs statistic (-14.89) are characteristic of populations having recently undergone demographic expansion. Mantel test revealed significant isolation by distance. The study identifies highly delineated structured populations with restricted gene flow. If such a stock is overfished, it is highly unlikely that it would recover through migration. For any future breeding programme in this species, it would be desirable to form a base population which incorporates the genetic material from all the locations so that we get a wide gene pool to select from.

Sex differentiation and sex change in the protandrous black porgy, Acanthopagrus schlegeli.

Protandrous black porgy fish, Acanthopagrus schlegeli, have a striking life cycle with a male sex differentiation at the juvenile stage and male-to-female sex change at 3 years of age. We had characterized the sex differentiation and sex change in this species by the integrative approaches of histology, endocrine and molecular genetics. The fish differentiated in gonad at the age around 4-months and the gonad further developed with a bisexual gonad for almost for 3 years and sex change at 3 year of age. An antagonistic relationship in the testicular and ovarian tissues was found during the development of the gonadal tissue. Male- (such as sf-1, dmrt1, dax-1 and amh) and female- (such as wnt4, foxl2 and cyp19a1a) promoting genes were associated with testicular and ovarian development, respectively. During gonadal sex differentiation, steroidogenic pathway and estrogen signaling were also highly expressed in the brain. The increased expression of sf-1 and wnt4, cyp19a1a in ovarian tissue and decreased expression of dax-1 in the ovarian tissue may play important roles in sex change from a male-to-female. Endocrine factors such as estradiol and luteinizing hormone may also involve in the natural sex change. Estradiol induced the expression of female-promoting genes and resulted in the precocious sex change in black porgy. Our series of studies shed light on the sex differentiation and sex change in protandrous black porgy and other animals.

Insights on Molecular Mechanisms of Ovarian Development in Decapod Crustacea: Focus on Vitellogenesis-Stimulating Factors and Pathways.

Vitellogenesis in crustaceans is an energy-consuming process. Though the underlying mechanisms of ovarian maturation in decapod Crustacea are still unclear, evidence indicates the process to be regulated by antagonistically-acting inhibitory and stimulating factors specifically originating from X-organ/sinus gland (XO/SG) complex. Among the reported neuromediators, neuropeptides belonging to the crustacean hyperglycemic hormone (CHH)-family have been studied extensively. The structure and dynamics of inhibitory action of vitellogenesis-inhibiting hormone (VIH) on vitellogenesis have been demonstrated in several species. Similarly, the stimulatory effects of other neuropeptides of the CHH-family on crustacean vitellogenesis have also been validated. Advancement in transcriptomic sequencing and comparative genome analysis has led to the discovery of a large number of neuromediators, peptides, and putative peptide receptors having pleiotropic and novel functions in decapod reproduction. Furthermore, differing research strategies have indicated that neurotransmitters and steroid hormones play an integrative role by stimulating neuropeptide secretion, thus demonstrating the complex intertwining of regulatory factors in reproduction. However, the molecular mechanisms by which the combinatorial effect of eyestalk hormones, neuromediators and other factors coordinate to regulate ovarian maturation remain elusive. These multifunctional substances are speculated to control ovarian maturation possibly via the autocrine/paracrine pathway by acting directly on the gonads or by indirectly exerting their stimulatory effects by triggering the release of a putative gonad stimulating factor from the thoracic ganglion. Acting through receptors, they possibly affect levels of cyclic nucleotides (cAMP and cGMP) and Ca2+ in target tissues leading to the regulation of vitellogenesis. The "stimulatory paradox" effect of eyestalk ablation on ovarian maturation continues to be exploited in commercial aquaculture operations, and is outweighed by the detrimental physiological effects of this procedure. In this regard, the development of efficient alternatives to eyestalk ablation based on scientific knowledge is a necessity. In this article, we focus principally on the signaling pathways of positive neuromediators and other factors regulating crustacean reproduction, providing an overview of their proposed receptor-mediated stimulatory mechanisms, intracellular signaling, and probable interaction with other hormonal signals. Finally, we provide insight into future research directions on crustacean reproduction as well as potential applications of such research to aquaculture technology development.

Salinity effects on the expression of osmoregulatory genes in the euryhaline black porgy Acanthopagrus schlegeli.

Black porgy is a marine euryhaline species with a capacity to cope with demands in a wide range of salinities and thus is a perfect model-fish to study osmoregulatory responses to salinity-acclimated processes and their hormonal control. The present study was performed to understand the regulatory changes in hormone, hormone receptors and important osmoregulatory genes in pituitary, gill, intestine and kidney in response to acute salinity stress. Transcript levels were analyzed by quantitative real-time PCR following acute salinity challenge by direct transfer of seawater (SW) acclimatized fish to fresh water (FWBP) and vice versa (SWBP). SW acclimation significantly increased plasma osmolality and intestine Na+/K+-ATPase (NKA) activity while FW acclimation increased plasma cortisol and branchial NKA activity. Plasma osmolality and chloride concentration decreased in FWBP whereas GH levels remained unchanged in both FWBP and SWBP. Comparative analysis of gene profiles between FWBP and SWBP showed that pituitary prolactin transcript increased significantly in FWBP. Prolactin receptor (PRLR) transcripts increased in gill of FWBP while it decreased in gill and kidney of SWBP. NKA transcripts increased in gill of both FWBP and SWBP, while it decreased in intestine of FWBP and increased in intestine and kidney of SWBP. Glucocorticoid receptor (GR) transcripts decreased in intestine and kidney of FWBP while it increased in gill and intestine of SWBP. No significant changes were observed in growth hormone receptor (GHR) transcripts of both FWBP and SWBP in pituitary, gill, intestine and kidney. Our current data demonstrated the correlation between PRLR gene expression in relation to FW adaptation, and GR gene expression in relation to SW adaptation in euryhaline black porgy. The results indicate that black porgy has an excellent osmoregulatory capacity and is capable of withstanding large variations in salinity.

Characterization and expression profile of farnesoic acid O-methyltransferase gene from Indian white shrimp, Penaeus indicus.

Methyl farnesoate (MF), a sesquiterpenoid synthesized in the mandibular organ, regulates many physiological processes in crustaceans including growth and reproduction. In the present study, farnesoic acid O-methyltransferase (FAMeT), the key enzyme responsible for final step conversion of farnesoic acid (FA) to methyl farnesoate (MF), was cloned and characterized from the nervous tissues of Penaeus indicus. Multiple sequence alignment, prediction of conserved domain regions, phosphorylation sites identification and phylogenetic analysis indicated that putative FAMeT fragment from P. indicus (PiFAMeT), shares a high degree of sequence identity to FAMeT proteins isolated from other crustaceans species. Quantitative real-time PCR analysis revealed ubiquitous expression of PiFAMeT in all the tissues examined, with comparative higher mRNA levels in nervous tissue and ovary. Additionally, the levels of PiFAMeT also showed gradual increase of expression correlating with the advancement in ovarian maturation. Further to support their role in promoting ovarian development, serotonin treatment (5HT, 50 µg/g body weight) was given to eyestalk intact and unilaterally eyestalk ablated females which resulted in significant increase in PiFAMeT transcript levels at day 7 and day 14. The relatively higher levels of PiFAMeT, reflecting higher levels of MF, suggest a role during secondary vitellogenesis thereby regulating ovarian development in P. indicus. Further research is required to understand the synergistic interaction of MF pathways with serotonergic and other regulatory pathways in regulating ovarian maturation in penaeid shrimps.

Age-dependent differential expression of genes involved in steroid signalling pathway in the brain of protandrous black porgy, Acanthopagrus schlegeli.

The mechanisms underlying brain sex differentiation in animals are poorly understood. In the present study, using black porgy, Acanthopagrus schlegeli, as primary experimental model, we investigated the temporal expression patterns of receptors for androgen (ar) and estrogen (esr1 and esr2a) in the brain during posthatching ages and analyzed them against the timing of gonadal germ cell development. We hypothesized that endogenous estrogens naturally masculinize the brain of black porgy. The expression of sex steroid receptors was studied in relation to a wider suite of other related genes (nr5a2, nr0b1, star, and cyp19a1b) to provide some insight into the monomale sex differentiation pattern observed in this species. Our results revealed a highly significant increase in esr1 together with the increase in esr2a at 120 dph (days posthatching), suggesting a significant role for esr in sex differentiation in this species. Temporal expression patterns of nr5a2, nr0b1, star, sex steroid receptors, and cyp19a1b in the brain provided evidence for their physiological roles in the monomale sex differentiation in this species. The expression of nr5a2, star, ar, esr1, esr2a, and cyp19a1b increased at 120 dph, a period when brain sex differentiation probably occurs in this species. The study also suggests that neurosteroidogenesis in black porgy may be regulated by both nr5a2-dependent and nr5a2-independent mechanisms. The results demonstrated striking differences in the abundance of the gene transcripts in discrete brain region throughout ontogeny. In addition, the sex steroid hormone levels and aromatase activity in brain at different developmental states and the changes in the gene expression patterns in response to aromatase inhibitor treatment are also discussed.

The expression of nr0b1 and nr5a4 during gonad development and sex change in protandrous black porgy fish, Acanthopagrus schlegeli.

Protandrous black porgy fish, Acanthopagrus schlegeli, have a striking life cycle, with a mono-male sex differentiation at the juvenile stage and male-to-female sex change at 3 yr of age. We report for the first time integrative molecular data on these interesting phenomena. Sex differentiation occurred between 4 and 5 mo of age. Testicular nr5a4 transcripts increased to high levels during sex differentiation (5 mo old), whereas nr0b1 (Dax-1) did not increase until the age of 8 mo. High nr5a4 and nr0b1 expression in testicular tissue, in contrast to low nr5a4 and high nr0b1 expression in ovarian tissue, were found in the male phase of 0(+)- to 2-yr-old fish (before sex change). Increased nr5a4, decreased nr0b1, and increased cyp19a1a were found in the ovarian tissues undergoing development from primary oocytes to vitellogenic oocytes during the natural sex change in 2(+)-yr-old fish. Removal of testicular tissue in 1(+)-yr-old fish resulted in both increased ovarian nr5a4 and genes in the steroidogenic pathway and decreased nr0b1 together with the appearance of vitellogenic oocytes. Ovary developed into the active stage with the increased expression of star and steroidogenic enzymes, including aromatase, in concordance with the decreased expression of nr0b1 in the testis-excised fish. Long-term estradiol (E2) administration resulted in early sex change, but the ovaries were mainly with primary oocytes. Low nr5a4, high nr0b1, and low steroidogenic enzymes, including cyp19a1a expression, were also observed in these E2-fed ovarian tissues. Thus, nr5a4 but not nr0b1 was associated with male sex differentiation. Testicular development required cooperative functions of both nr5a4 and nr0b1. The present study suggests that nr5a4 and nr0b1 have an antagonistic interaction for the oocyte development. Testicular tissue exerted inhibitory effects on ovarian development. It is probable that nr0b1 regulates the timing of vitellogenic development and sex change in black porgy.

Dual roles of cyp19a1a in gonadal sex differentiation and development in the protandrous black porgy, Acanthopagrus schlegeli.

Protandrous black porgy fish, Acanthopagrus schlegeli, have a striking life cycle, with male sex differentiation at the juvenile stage, a bisexual gonad during first 2 yr of life, and a male-to-female sex change (with vitellogenic oocytes) at 3 yr of age. The present study investigated the role of aromatase (cyp19a1a/Cyp19a1a) in gonadal development in this species, especially in relation to sexual differentiation and sex change. Fish of various ages were treated with estradiol (E2) or aromatase inhibitor (AI) to determine whether manipulation of the hormonal environment has an impact on these processes. We report an integrative immunohistochemical, cellular, and molecular data set describing these interesting phenomena. During male sex differentiation, high levels of cyp19a1a/Cyp19a1a expression were observed in the undifferentiated gonad (4 mo of age), in marked contrast to the low cyp19a1a/Cyp19a1a levels detected in the differentiated testis at the age of 5-6 mo. A low dose of E2 (0.25 mg/kg feed) stimulated testicular growth and function in sexually differentiated fish, whereas a high dose of E2 (6 mg/kg feed) induced female development. Furthermore, administration of AI suppressed male development and promoted female sexual differentiation. An increased number of figla transcripts (an oocyte-specific gene) were observed prior to cyp19a1a expression, concomitant with the development of oogonia and early primary oocytes in the ovaries of both E2- and AI-treated groups. Immunohistochemical Pcna staining showed that the regression of testicular tissue occurred prior to the development of ovarian tissue in both E2- and AI-induced females. The importance of cyp19a1a in female development was further demonstrated by the increase in cyp19a1a transcripts during the naturally occurring sex change. Transcripts of foxl2 increased in the gonads of 2- to 3-yr-old black porgy during the early stages of the natural sex change, followed by a gradual elevation of cyp19a1a levels. The levels of both genes peaked in the resulting ovarian tissue. Thus, cyp19a1a/Cyp19a1a plays dual roles in the gonadal development, namely, in testicular development during the initial period of sexual differentiation and later in ovarian development during the natural sex change.