The role of steroid/thyroid hormones in the regulation of endocrine cells at the level of the pituitary has remained unclear. Therefore, using single-cell quantitative real-time PCR, we examined absolute amounts of transcripts for nuclear receptors [estrogen receptors (ERs) alpha, beta, and gamma; androgen receptors (ARs) a and b; glucocorticoid receptors (GRs) 1, 2a, and 2b; and thyroid hormone receptors (TRs) alpha1, alpha2, and beta] in pituitary cells of immature (IM) and mature (M) male tilapia, Oreochromis niloticus. In the two reproductive stages, ACTH cells expressed only ERbeta, whereas all other pituitary cell types expressed ERalpha + beta, and a subpopulation coexpressed ARa, ARb, GR1, GR2b, and TRbeta but lacked ERgamma, GR2a, TRalpha1, and TRalpha2. IM males had high percentages of LH cells (IM 46.0% vs. M 10.0%), GH cells (IM 23.3% vs. M 7.9%), and prolactin cells (IM 68.8% vs. M 6.0%) with ERbeta, and TSH cells (IM 19.2% vs. M 0.0%) and MSH cells (IM 25.6% vs. M 0.0%) with ERalpha + TRbeta. A high percentage of FSH cells in IM males expressed ERbeta (IM 46.9% vs. M 18.8%), and FSH cells in M males showed significantly high GR1 transcripts (IM 76.0 +/- 5.0 vs. M 195.0 +/- 10.7 copies per cell; P < 0.05), suggesting that FSH cells are regulated differently in the two reproductive stages. Coexpression of ERalpha + beta in high percentages of cells of the GH family (GH, IM 43.8% vs. M 14.3%; prolactin, IM 8.3% vs. M 59.7%; somatolactin, IM 22.2% vs. M 42.2%) suggests that the expression of both ERs is important for functionality. Thus, differential coexpression of genes for nuclear receptors in subpopulations of pituitary cell types suggests multiple steroid/thyroid hormone regulatory pathways at the level of the pituitary during the two reproductive stages.
Copper is one of the major heavy metal pollutants found in the aquatic environment. Therefore, it is important for determining the genes that play a key role in copper metabolism in aquatic organisms. This study, thus, aimed to identify a new copper-inducible gene in swordtail fish, Xiphophorus helleri. Using ACP-based RT-PCR coupled with RLM-RACE, we cloned Wap65, a mammalian homologue of hemopexin gene. The gene exhibits high identity at amino acid levels with the Wap65 gene of other fish species (42-68%) and mammalian hemopexin gene (35-37%). In addition, ten cysteine and two histidine residues are conserved in the swordtail fish Wap65 gene. These cysteine residues are vital for structural integrity, and histidine residues provide high binding affinity towards heme. As revealed by RT-PCR, the gene was upregulated in swordtail fish that were exposed to copper in a dose- and time-dependent manner. Therefore, the identification of Wap65, a mammalian homologue of hemopexin, as a new copper-inducible gene will provide greater insight into the role of this gene in copper metabolism.
Newly discovered kisspeptin (metastin), encoded by the Kiss1/KISS1 gene, is considered as a major gatekeeper of puberty through the regulation of GnRH. In the present study, we cloned a novel kisspeptin gene (kiss2) in the zebrafish Danio rerio and the medaka Oryzias latipes, which encodes a sequence of 125 and 115 amino acids, respectively, and its core sequence (FNLNPFGLRF, F-F form) is different from the previously characterized kiss1 (YNLNSFGLRY, Y-Y form). Our in silico data mining shows kiss1 and kiss2 are highly conserved across nonmammalian vertebrate species, and we have identified two putative kisspeptins in the platypus and three forms in Xenopus. In the brain of zebrafish and medaka, in situ hybridization and laser capture microdissection coupled with real-time PCR showed kiss1 mRNA expression in the ventromedial habenula and the periventricular hypothalamic nucleus. The kiss2 mRNA expression was observed in the posterior tuberal nucleus and the periventricular hypothalamic nucleus. Quantitative real-time PCR analysis during zebrafish development showed a significant increase in zebrafish kiss1, kiss2 (P < 0.002), gnrh2, and gnrh3 (P < 0.001) mRNA levels at the start of the pubertal phase and remained high in adulthood. In sexually mature female zebrafish, Kiss2 but not Kiss1 administration significantly increased FSH-beta (2.7-fold, P < 0.05) and LH-beta (8-fold, P < 0.01) mRNA levels in the pituitary. These results suggest that the habenular Kiss1 and the hypothalamic Kiss2 are potential regulators of reproduction including puberty and that Kiss2 is the predominant regulator of gonadotropin synthesis in fish.
Matched MeSH terms: Fish Proteins/genetics*; Zebrafish Proteins/genetics
There is very little information on the capacity of freshwater carnivorous fish to biosynthesize highly unsaturated fatty acids (HUFA). The striped snakehead fish (Channa striata) is a carnivorous species cultured inland of several Southeast Asian countries due to its pharmaceutical properties in wound healing enhancement. We described here the full-length cDNA cloning of a striped snakehead fatty acid desaturase (fads), which is responsible for desaturation of unsaturated fatty acids in the HUFA biosynthesis. Bioinformatics analysis reveals a protein coding region with length of 445 amino acids containing all characteristic features of desaturase enzyme, including a cytochrome b5-domain with the heme-binding motif, two transmembrane domains and three histidine-rich regions. The striped snakehead fads amino acid sequence shares high similarity with known fads of other teleosts. The mRNA expression of striped snakehead fads also showed an ontogenic-related increase in expression in 0-20 days after hatch larva. Using ISH, we localized the presence of fads in larva brain, liver and intestinal tissues.
Transferrin is a protein super-family involved in iron transport, a central process in cellular homeostasis. Throughout the evolution of vertebrates, transferrin members have diversified into distinct subfamilies including serotransferrin, ovotransferrin, lactoferrin, melanotransferrin, the inhibitor of carbonic anhydrase, pacifastin, and the major yolk protein in sea urchin. Previous phylogenetic analyses have established the branching order of the diverse transferrin subfamilies but were mostly focused on the transferrin repertoire present in mammals. Here, we conduct a comprehensive phylogenetic analysis of transferrin protein sequences in sequenced vertebrates, placing a special focus on the less-studied nonmammalian vertebrates. Our analyses uncover a novel transferrin clade present across fish, sauropsid, and amphibian genomes but strikingly absent from mammals. Our reconstructed scenario implies that this novel class emerged through a duplication event at the vertebrate ancestor, and that it was subsequently lost in the lineage leading to mammals. We detect footprints of accelerated evolution following the duplication event, which suggest positive selection and early functional divergence of this novel clade. Interestingly, the loss of this novel class of transferrin in mammals coincided with the divergence by duplication of lactoferrin and serotransferrin in this lineage. Altogether, our results provide novel insights on the evolution of iron-binding proteins in the various vertebrate groups.
In this study, we reported a molecular characterization of a novel proto-type galectin-1 from the striped murrel Channa striatus (named as CsGal-1). The full length CsGal-1 was identified from an established striped murrel cDNA library and further we confirmed the sequence by cloning. The complete cDNA sequence of CsGal-1 is 590 base pairs (bp) in length and its coding region encoded a poly peptide of 135 amino acids. The polypeptide contains a galactoside binding lectin domain at 4-135. The domain carries a sugar binding site at 45-74 along with its signatures (H(45)-X-Asn(47)-X-Arg(49) and Trp(69)-X-X-Glu(72)-X-Arg(74)). CsGal-1 shares a highly conserved carbohydrate recognition domain (CRD) with galectin-1 from other proto-type galectin of teleosts. The mRNA expressions of CsGal-1 in healthy and various immune stimulants including Aphanomyces invadans, Aeromonas hydrophila, Escherchia coli lipopolysaccharide and poly I:C injected tissues of C. striatus were examined using qRT-PCR. CsGal-1 mRNA is highly expressed in kidney and is up-regulated with different immune stimulants at various time points. To understand its biological activity, the coding region of CsGal-1 gene was expressed in an E. coli BL21 (DE3) cloning system and its recombinant protein was purified. The recombinant CsGal-1 protein was agglutinated with mouse erythrocytes at a concentration of 4μg/mL in a calcium independent manner. CsGal-1 activity was inhibited by d-galactose at 25mM(-1) and d-glucose and d-fructose at 100mM(-1). The results of microbial binding assay showed that the recombinant CsGal-1 protein agglutinated only with the Gram-negative bacteria. Interestingly, we observed no agglutination against Gram-positive bacteria. Overall, the study showed that CsGal-1 is an important immune gene involved in the recognition and elimination of pathogens in C. striatus.
Neurokinin B (NKB) was recently identified as a key regulator of reproduction in mammals and fish. Fish were found to possess a specific novel neurokinin termed NKF. To study the role of NKB/NKF in the regulation of fish reproduction and to investigate the role of NKB/NKF and their receptors in the piscine pituitary, we have identified the NKB/tachikinin 3 receptor (tac3r) system in tilapia. Bioinformatics and phylogenetic analyses have demonstrated that the tilapia holds 1 putative tac3 gene and 2 NKB receptor genes (tac3ra and tac3rb) that clustered with other piscine Tac3 and NKB receptor lineages. Furthermore, we found that in African cichlids, NKB peptides differ from other vertebrate NKBs in their C-terminal sequence, possessing isoleucine instead of valine as the X in the NKB FXGLM-NH2-terminal consensus sequence. Signal transduction analysis demonstrated that tilapia NKB (tiNKB), tiNKF, and human NKB activated both CRE-luc and SRE-luc transcriptional activity of both tilapia and human NKB receptors. Two hours after ip injection of tiNKB, the plasma levels of both FSH and LH were increased, whereas tiNKF was more effective in increasing LH levels. However, tiNKB was more effective than tiNKF in increasing both FSH and LH from tilapia pituitary dispersed cells. Using in situ hybridization and fluorescent immunohistochemistry, we have shown that LH cells possess tac3, tac3ra, and tac3rb mRNAs, whereas FSH cells possess mainly tac3rb and tac3ra and tac3 to a much lesser extent. These results suggest that the members of the NKB/tac3r system may serve as paracrine/autocrine regulators of gonadotropin release in fish pituitary.
Ferritin is a highly-conserved iron-storage protein that has also been identified as an acute phase protein within the innate immune system. The iron-storage function is mediated through complementary roles played by heavy (H)-chain subunit as well as the light (L) in mammals or middle (M)-chain in teleosts, respectively. In this study, we report the identification of five ferritin subunits (H1, H2, M1, M2, M3) in the Atlantic salmon that were supported by the presence of iron-regulatory regions, gene structure, conserved domains and phylogenetic analysis. Tissue distribution analysis across eight different tissues showed that each of these isoforms is differentially expressed. We also examined the expression of the ferritin isoforms in the liver and kidney of juvenile Atlantic salmon that was challenged with Aeromonas salmonicida as well as in muscle cell culture stimulated with interleukin-1β. We found that each isoform displayed unique expression profiles, and in certain conditions the expressions between the isoforms were completely diametrical to each other. Our study is the first report of multiple ferritin isoforms from both the H- and M-chains in a vertebrate species, as well as ferritin isoforms that showed decreased expression in response to infection. Taken together, the results of our study suggest the possibility of functional differences between the H- and M-chain isoforms in terms of tissue localisation, transcriptional response to bacterial exposure and stimulation by specific immune factors.
The mechanisms through which brown-marbled grouper accomplishes resistance to infection, particularly against Vibrios, are not yet fully understood. In this study, brown-marbled grouper fingerlings were experimentally infected with Vibrio parahaemolyticus, to identify disease resistance grouper, and the serum proteome profiles were compared between resistant and susceptible candidates, via two-dimensional gel electrophoresis (2-DE). The results showed that putative parvalbumin beta-2 subunit I, alpha-2-macroglobulin, nattectin and immunoglobulin light chain proteins were among proteins that significantly overexpressed in the resistant fish as compared to the susceptible group of fish, whereas apolipoprotein E and immunoglobulin light chain proteins were observed to be differentially overexpressed in the susceptible fish. Further analysis by peptide sequencing revealed that the immunoglobulin light chain proteins identified in the resistant and susceptible groups differed in amino acid composition. Taken together, the results demonstrated for the first time that putative parvalbumin beta-2 subunit I, alpha-2-macroglobulin, nattectin and immunoglobulin light chain are among important proteins participating to effect disease resistance mechanism in fish and were overexpressed to function collectively to resist V. parahaemolyticus infection. Most of these molecules are mediators of immune response.
The endogenous production of long-chain polyunsaturated fatty acids (LC-PUFA) in carnivorous teleost species inhabiting freshwater environments is poorly understood. Although a predatory lifestyle could potentially supply sufficient LC-PUFA to satisfy the requirements of these species, the nutrient-poor characteristics of the freshwater food web could impede this advantage. In this study, we report the cloning and functional characterisation of an elongase enzyme in the LC-PUFA biosynthesis pathway from striped snakehead (Channa striata), which is a strict freshwater piscivore that shows high deposition of LC-PUFA in its flesh. We also functionally characterised a previously isolated fatty acyl desaturase cDNA from this species. Results showed that the striped snakehead desaturase is capable of Δ4 and Δ5 desaturation activities, while the elongase showed the characteristics of Elovl5 elongases. Collectively, these findings reveal that striped snakehead exhibits the genetic resources to synthesise docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Both genes are expressed at considerable levels in the brain and the liver. In liver, both genes were up-regulated by dietary C18 PUFA, although this increase did not correspond to a significant rise in the deposition of muscle LC-PUFA. Brain tissue of fish fed with plant oil diets showed higher expression of fads2 gene compared to fish fed with fish oil-based diet, which could ensure DHA levels remain constant under limited dietary DHA intake. This suggests the importance of DHA production from EPA via the ∆4 desaturation step in order to maintain an optimal reserve of DHA in the neuronal tissues of carnivores.
Taxonomic confusion exists within the genus Epinephelus due to the lack of morphological specializations and the overwhelming number of species reported in several studies. The homogenous nature of the morphology has created confusion in the Malaysian Marine fish species Epinephelus fuscoguttatus and Epinephelus hexagonatus. In this study, the partial DNA sequence of the 16S gene and mitochondrial nucleotide sequences of two gene regions, Cytochrome Oxidase Subunit I and III were used to investigate the phylogenetic relationship between them. In the phylogenetic trees, E. fuscoguttatus was monophyletic with E. hexagonatus species and morphology examination shows that no significant differences were found in the morphometric features between these two taxa. This suggests that E. fuscoguttatus is not distinguishable from E. hexagonatus species, and that E. fuscoguttatus have been identified to be E. hexagonatus species is likely attributed to differences in environment and ability to camouflage themselves under certain conditions. Interestingly, this finding was also supported by Principal Component Analysis on Attenuated Total Reflectance-Fourier-transform Infrared (ATR-FTIR) data analysis. Molecular, morphological and meristic characteristics were combined with ATR-FTIR analysis used in this study offer new perspectives in fish species identification. To our knowledge, this is the first report of an extensive genetic population study of E. fuscoguttatus in Malaysia and this understanding will play an important role in informing genetic stock-specific strategies for the management and conservation of this highly valued fish.
Interacting effects of feeding and stress on corticoid responses in fish were investigated in common carp fed 3.0% or 0.5% body mass (BM) which received no implant, a sham or a cortisol implant (250 mg/kg BM) throughout a 168 hour post-implant period (168 h-PI). At 12h-PI, cortisol implants elevated plasma cortisol, glucose and lactate. Plasma osmolality and ions remained stable, but cortisol increased gill and kidney Na(+)/K(+) ATPase (NKA) and H(+) ATPase activities. Gill NKA activities were higher at 3%-BM, whereas kidney H(+) ATPase activity was greater at 0.5%-BM. Cortisol induced liver protein mobilization and repartitioned liver and muscle glycogen. At 3%-BM, this did not increase plasma ammonia, reflecting improved excretion efficiency concomitant with upregulation of Rhesus glycoprotein Rhcg-1 in gill. Responses in glucocorticoid receptors (GR1/GR2) and mineralocorticoid receptor (MR) to cortisol elevation were most prominent in kidney with increased expression of all receptors at 24 h-PI at 0.5%-BM, but only GR2 and MR at 0.5%-BM. In the liver, upregulation of all receptors occurred at 24 h-PI at 3%-BM, whilst only GR2 and MR were upregulated at 0.5%-BM. In the gill, there was a limited upregulation: GR2 and MR at 72 h-PI and GR1 at 168 h-PI at 3%-BM but only GR2 at 72 h-PI at 0.5%-BM. Thus cortisol elevation led to similar expression patterns of cortisol receptors in both feeding regimes, while feeding affected the type of receptor that was induced. Induction of corticoid receptors occurred simultaneously with increases in Rhcg-1 mRNA expression (gill) but well after NKA and H(+) ATPase activities increased (gill/kidney).
Pax3 and Pax7 are the regulators and markers of muscle progenitors and satellite cells that contribute to the embryonic development and postembryonic growth of skeletal muscle in vertebrates, as well as to its repair and regeneration. However, information regarding them in vertebrate genome model, torafugu Takifugu rubripes, has remained unknown. Therefore, as an initial step, here we characterized Pax3 and Pax7 from torafugu and investigated their expression patterns during different developmental stages by RT-PCR. In silico analysis with the Fugu genome database (ver. 4.0) yielded two distinct genes each for Pax3 (Pax3a and Pax3b) and Pax7 (Pax7a and Pax7b). The 75th amino acid, glutamine (Gln75), from the N-terminus was replaced by proline in the paired box domain (PD) of Pax3a. One single cDNA clone encoding Pax3a had deletion of Gln75 in PD, suggesting the presence of alternatively spliced variants (Q+/Q-). This was further supported by identification of two adjacent alternative 3' splice acceptor sites which produce Pax3b Q+ (aagCAGGGA) and Q- (aagcagGGA) variants. Interestingly, torafugu Pax7a, but not Pax7b, had an insert encoding five amino acid residues (SGEAS) in a C-terminal region of PD in two out of three cDNA clones. Genomic analysis showed two alternate splice donor sites at exon 4 of Pax7a. In synteny analysis, torafugu Pax3a showed syntenic relationship with the corresponding regions in other teleosts only, whereas Pax3b and Pax7b showed high syntenic relationship with the corresponding regions of both mammals and other teleosts. RT-PCR revealed that expression of Pax3a and Pax3b transcripts was restricted to embryonic stages only, whereas those of Pax7a and Pax7b was continued to be expressed in larvae and importantly those of Pax7a were found in adult skeletal muscles. Therefore, Pax3 appears to be most important for primary myogenesis and Pax7 for secondary myogenesis and growth by hyperplasia in fish. In this regard, the transcripts of torafugu Pax3 and Pax7 genes might be used for further investigation as a marker for identification of muscle precursor cells during different phases of growth, and this ambiguity is the next target of our research.
Despite the potential of vegetable oils as aquafeed ingredients, a major drawback associated with their utilization is the inferior level of beneficial n-3 long-chain polyunsaturated fatty acids (LC-PUFA). Echium oil (EO), which is rich in stearidonic acid (SDA, 18:4n-3), could potentially improve the deposition of n-3 LC-PUFA as the biosynthesis of LC-PUFA is enhanced through bypassing the rate-limiting ∆6 desaturation step. We report for the first time an attempt to investigate whether the presence of a desaturase (Fads2) capable of ∆4 desaturation activities and an elongase (Elovl5) will leverage the provision of dietary SDA to produce a higher rate of LC-PUFA bioconversion. Experimental diets were designed containing fish oil (FO), EO or linseed oil (LO) (100FO, 100EO, 100LO), and diets which comprised equal mixtures of the designated oils (50EOFO and 50EOLO) were evaluated in a 12-week feeding trial involving striped snakeheads (Channa striata). There was no significant difference in growth and feed conversion efficiency. The hepatic fatty acid composition and higher expression of fads2 and elovl5 genes in fish fed EO-based diets indicate the utilization of dietary SDA for LC-PUFA biosynthesis. Collectively, this resulted in a higher deposition of muscle eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) compared to LO-based diets. Dietary EO improved the ratio of n-3 LC-PUFA to n-6 LC-PUFA in fish muscle, which is desirable for human populations with excessive consumption of n-6 PUFA. This study validates the contribution of SDA in improving the content of n-3 LC-PUFA and the ratio of EPA to arachidonic acid (ARA, 20:4n-6) in a freshwater carnivorous species.
The complete mitogenome of the ray Taeniura lymma was recovered from genome skimming using the HiSeq sequencing system. The T. lymma mitogenome has 17,652 base pairs (59.13% A + T content) made up of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs and a 1906 bp non-coding AT-rich region. This mitogenome sequence is the second for a ray from Australian waters, the first for the genus Taeniura and the ninth for the family Dasyatidae.
The mitogenome of an Australian sample of the mudskipper, Periophthalmus minutus, was recovered from partial sequencing using the MiSeq sequencer. This mudskipper has a mitogenome of 16,506 base pairs (55% A + T content) made up of two ribosomal subunit genes, 13 protein-coding genes, 22 transfer RNAs, and a 838 bp non-coding AT-rich region. This is the first sequenced mitogenome for the genus Periophthalmus and the fifth for the subfamily Oxudercinae.
Immediate early response (IER) 2 gene, a member of the IER family, is a gene of unknown function which is affected by external stimuli in the brain. In the present study, the full length sequence and localization of medaka (Oryzias latipes) ier2 was investigated in the brain to understand the functions of Ier2 in the future studies. The full length sequence of medaka ier2 was identified using a 3'-, 5'- rapid amplification of cDNA ends method, and distribution in the brain was identified using in situ hybridization. The identified full length ier2 mRNA consisted of 939 nucleotides spanning along 1 exon. The deduced amino acid sequence consisted of 171 amino acid residues which contains a highly conserved sequence, nuclear localization signal. ier2 mRNA was distributed in the telencephalon, midbrain and the hypothalamus. This highly conserved primary response gene Ier2 can be used to visualize and map functionally activated neuronal circuitry in the brain of medaka.
There is a lack of understanding on how the environment and trophic niche affect the capability of long-chain polyunsaturated fatty acids (LC-PUFA) in freshwater carnivorous teleost. In this present study, we isolated and functionally characterised a fatty acyl desaturase (Fads) from the striped snakehead Channa striata. Sequence comparison and phylogenetic analysis suggested a Fads2 protein that is closely related to previously characterised Fads2 proteins from freshwater carnivorous and marine herbivorous fish species. We further demonstrated the capacity of Δ6 and Δ5 desaturation activities for this particular desaturase, with highest activities towards the conversion of omega-3 (n-3) polyunsaturated fatty acids (PUFA). Low Δ4 desaturation activity was also detected, although the significance of this at a physiological level remains to be studied. The expression of this striped snakehead Δ6/Δ5 fads2 gene was highest in brain, followed by liver and intestine. In liver, diet fortified with high LC-PUFA concentration impeded the expression of Δ6/Δ5 fads2 gene compared to vegetable oil (VO) based diets. The discovery of Δ6/Δ5 Fads2 desaturase here complements the previous discovery of a Δ4 Fads2 desaturase and an Elovl5 elongase, lending proof to the existence of all the required enzymatic machinery to biosynthesise LC-PUFA from C18 PUFA in a freshwater carnivorous species.
The complete mitochondrial genome of the conservationally significant Macquarie perch (Macquaria australasica) was obtained from low-coverage shotgun sequencing using the MiSeq sequencer. The M. australasica mitogenome has 16,496 base pairs (55% A + T content) made up of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a 819 bp non-coding AT-rich region. This is the first mitogenome sequence for the genus Macquaria, and the third to be reported for the family Percichthyidae.
A feeding trial was performed to assess the effects of dietary Medlar (Mespilus germanica) leaf extract (MLE) on the growth performance, skin mucus non-specific immune parameters as well as mRNA levels of immune and antioxidant related genes in the skin of common carp (Cyprinus carpio) fingerlings. Fish were fed diets supplemented with graded levels (0, 0.25, 0.50, and 1.00%) of MLE for 49 days. The results revealed an improvement to the growth performance and feed conversion ratio in MLE fed carps (P fish fed 1% MLE (P 0.05) in case protease activity in the skin mucous or tumor necrosis factor alpha and interleukin 1 beta gene expression in the skin of carps (P > 0.05). The expression of genes encoding glutathione reductase and glutathione S-transferase alpha were remarkably increased in MLE fed carps compared to the control group (P