Growth and IGF-I response of juvenile Nile tilapia (Oreochromis niloticus) to changes in water temperature and dietary protein level

Water temperature and dietary protein level play an important role in influencing the growth and insulin-like growth factor I (IGF-I) in Nile tilapia juveniles. The combined effect of temperature (20–34 °C) and dietary protein level (25–50%) on the specific growth rate (SGR), feed efficiency (FE), serum IGF-I level and hepatic IGF-I mRNA level was examined under laboratory conditions by employing central composite design and response surface method. Results showed that the linear effects of temperature and dietary protein level on the SGR, FE, serum IGF-I and hepatic IGF-I mRNA level were significant (P<0.05); the quadratic effects of temperature and dietary protein level on the FE and serum IGF-I were significant (P<0.05). The interaction of temperature and dietary protein level on the FE, serum IGF-I and hepatic IGF-I mRNA level all proved significant (P<0.05). The optimal temperature/dietary protein level combination was determined, i.e., 29.9 °C/40.3%, at which the greatest SGR (2.748%/d) and FE (0.775) were simultaneously arrived. Both SGR and FE were linearly correlated with serum IGF-I or hepatic IGF-I mRNA level. These results suggested that optimum combination of temperature and dietary protein level would enhance tilapia growth efficiency and IGF-I would regulate growth and FE.     

Cloning and expression of boule and dazl in the Nile tilapia (Oreochromis niloticus)

The Deleted in Azoospermia (DAZ) family of RNA binding proteins consists of highly conserved genes boule, daz and daz-like (dazl) essential for germ cell development. boule is known for its unisexual meiotic expression in invertebrates and mammals, but meiotic-specific female expression plus meiosis-preferential male expression in trout, and meiosis-preferential bisexual expression in medaka. dazl shows highly conserved bisexual expression throughout gametogenesis in diverse species. Here we report the cloning and expression of boule and dazl in the Nile tilapia (Oreochromis niloticus), an important aquaculture fish. Molecular cloning and sequence analysis led to the identification of tilapia boule and dazl cDNAs. The predicted partial Boule contains a conserved RRM motif and Dazl has the C-terminal sequence. On a phylogenetic tree, tilapia Boule and Dazl are in separate clades of Boule and Dazl homologs from other species, indicating their divergence during early vertebrate evolution. By RT-PCR analysis, boule and dazl showed bisexual gonad-specific expression. By in situ hybridization analysis, both boule and dazl RNAs were restricted to female and male germ cells of adult gonads but absent in gonadal soma. In the ovary, boule and dazl RNAs were abundant in oocytes. In the testis, boule and dazl RNAs were prominent in meiotic spermatocytes but barely detectable in meiotic products. These data show that boule and dazl are expressed bisexually in germ cells and provide useful markers to study gametogenesis in the adult tilapia.     

Effects of water temperature on growth and sex ratio of juvenile Nile tilapia Oreochromis niloticus (Linnaeus) reared in geothermal waters in southern Tunisia

Growth of juvenile Nile tilapia, Oreochromis niloticus (Maryut strain) was studied under laboratory conditions. Four thermal regimes (22, 26, 30, and 34 °C) were tested on 480 20-day-old fry.     

Nile Tilapia Neu3 sialidases: Molecular cloning,functional characterization and expression in Oreochromis niloticus

Mammalian Neu3 is a ganglioside specific sialidase. Gangliosides are involved in various physiological events such as cell growth, differentiation and diseases. Significance of Neu3 and gangliosides is still unclear in aquaculture fish species. To gain more insights of fish Neu3 sialidases, molecular cloning and characterization were carried out in tilapia (Oreochromis niloticus). A tilapia genome-wide search for orthologues of human NEU1, NEU2, NEU3 and NEU4 yielded eight putative tilapia sialidases, five of which were neu3-like and designated as neu3a, neu3b, neu3c, neu3d and neu3e. Among five neu3 genes, neu3a, neu3d and neu3e were amplified by PCR from adult fish brain cDNA with consensus sequences of 1227 bp, 1194 bp and 1155 bp, respectively. Multiple alignments showed conserved three Asp-boxes (SXDXGXTW), YRIP and VGPG motifs. The molecular weights for Neu3a, Neu3d and Neu3e were confirmed using immunoblotting analysis as 45.9 kDa, 44.4 kDa and 43.6 kDa, respectively. Lysate from neu3 genes transfected HEK293 cells showed sialidase activity in Neu3a towards ganglioside mix optimally at pH 4.6. Using pure gangliosides as substrates, highest sialidase activity for Neu3a was observed towards GD3 followed by GD1a and GM3, but not GM1. On the other hand, sialidase activities were not observed in Neu3d and Neu3e towards various sialoglycoconjugates. Indirect immunofluorescence showed that tilapia Neu3a and Neu3d are localized at the plasma membrane, while most Neu3e showed a cytosolic localization. RT-PCR analyses for neu3a showed significant expression in the brain, liver, and spleen tissues, while neu3d and neu3e showed different expression patterns. Based on these results, tilapia Neu3 exploration is an important step towards full understanding of a more comprehensive picture of Neu3 sub-family of proteins in fish.     

Molecular characterization and expression analysis of ubiquitin-activating enzyme E1 gene in Citrus reticulata

Ubiquitin-activating enzyme E1 (UBE1) catalyzes the first step in the ubiquitination reaction, which targets a protein for degradation via a proteasome pathway. UBE1 plays an important role in metabolic processes. In this study, full-length cDNA and DNA sequences of UBE1 gene, designated CrUBE1, were obtained from ‘Wuzishatangju’ (self-incompatible, SI) and ‘Shatangju’ (self-compatible, SC) mandarins. 5 amino acids and 8 bases were different in cDNA and DNA sequences of CrUBE1 between ‘Wuzishatangju’ and ‘Shatangju’, respectively. Southern blot analysis showed that there existed only one copy of the CrUBE1 gene in genome of ‘Wuzishatangju’ and ‘Shatangju’. The temporal and spatial expression characteristics of the CrUBE1 gene were investigated using semi-quantitative RT-PCR (SqPCR) and quantitative real-time PCR (qPCR). The expression level of the CrUBE1 gene in anthers of ‘Shatangju’ was approximately 10-fold higher than in anthers of ‘Wuzishatangju’. The highest expression level of CrUBE1 was detected in pistils at 7 days after self-pollination of ‘Wuzishatangju’, which was approximately 5-fold higher than at 0 h. To obtain CrUBE1 protein, the full-length cDNA of CrUBE1 genes from ‘Wuzishatangju’ and ‘Shatangju’ were successfully expressed in Pichia pastoris. Pollen germination frequency of ‘Wuzishatangju’ was significantly inhibited with increasing of CrUBE1 protein concentrations from ‘Wuzishatangju’.     

Molecular cloning and expression in vitro of a carboxylesterase gene from the Glanville fritillary butterfly (Melitaea cinxia)

Carboxylesterase (EC 3.1.1.1) is a member of the carboxyl/cholinesterase (CCE) superfamily, which is widely distributed in animals, plants and microorganisms. This enzyme has been known to be associated with insecticide resistance and detoxification. Although CCEs have been extensively studied in insects, including lepidopterans, the research on butterflies, a major subgroup in Lepidoptera, is still poor. In the present study, we cloned a CCE gene (McCCE1) from the Glanville fritillary butterfly (Melitaea cinxia, Lepidoptera: Nymphalidae). The full-length cDNA encoding McCCE1 was 1786 bp, containing a 1641 bp open reading frame encoding 546 amino acids, a 38 bp 5′-untranslated region (5′-UTR), and a 107 bp 3′-UTR with a poly(A) tail. The functionally conserved amino acids in McCCE1 shared the 55% identity with the cytoplasmic esterase CCE017a in Helicoverpa armigera (Lepidoptera: Noctuidae), which has been associated with detoxification. Assays in vitro showed that the recombinant McCCE1 could hydrolyze α- and β-naphthyl acetate. Thus, the present study adds to the body of knowledge concerning the detoxification of pesticides by lepidopterans.     

Molecular cloning,characterization and expression of myoglobin in Tibetan antelope (Pantholops hodgsonii), a species with hypoxic tolerance

The Tibetan antelope (Pantholops hodgsonii) is a hypoxia-tolerant species that lives at an altitude of 4000–5000 m above sea level on the Qinghai–Tibetan plateau. Myoglobin is an oxygen-binding cytoplasmic hemoprotein that is abundantly expressed in oxidative skeletal and cardiac myocytes. Numerous studies have implicated that hypoxia regulates myoglobin expression to allow adaptation to conditions of hypoxic stress. Few studies have yet looked at the effect of myoglobin on the adaptation to severe environmental stress on TA. To investigate how the Tibetan antelope (TA) has adapted to a high altitude environment at the molecular level, we cloned and analyzed the myoglobin gene from TA, compared the expression of myoglobin mRNA and protein in cardiac and skeletal muscle between TA and low altitude sheep. The results indicated that the full-length myoglobin cDNA is composed of 1154 bp with a 111 bp 5′ untranslated region (UTR), a 578 bp 3′ UTR and a 465 bp open reading frame (ORF) encoding a polypeptide of 154 amino acid residues with a predicted molecular weight of 17.05 kD. The TA myoglobin cDNA sequence and the deduced amino acid sequence were highly homologous with that of other species. When comparing the myoglobin sequence from TA with the Ovis aries myoglobin sequence, variations were observed at codons 21 (GGT → GAT) and 78 (GAA → AAG), and these variations lead to changes in the corresponding amino acids, i.e., Gly → Asp and Glu → Lys, respectively. But these amino acid substitutions are unlikely to effect the ability of binding oxygen because their location is less important, which is revealed by the secondary structure and 3D structure of TA myoglobin elaborated by homology modeling. However, the results of myoglobin expression in cardiac and skeletal muscles showed that they were both significantly higher than that in plain sheep not only in mRNA but also protein level. We speculated that the higher expression of myoglobin in TA cardiac and skeletal muscles improves their ability to obtain and store oxygen under hypoxic conditions. This study indicated that TA didn't improve the ability of carrying oxygen by changing the molecular structure of myoglobin, but through increasing the expression of myoglobin in cardiac and skeletal muscles.     

Molecular characterization and expression analysis of a GTP-binding protein (MiRab5) in Mangifera indica

The Rab family, the largest branch of Ras small GTPases, plays a crucial role in the vesicular transport in plants. The members of Rab family act as molecular switches that regulate the fusion of vesicles with target membranes through conformational changes. However, little is known about the Rab5 gene involved in fruit ripening and stress response. In this study, the MiRab5 gene was isolated from stress-induced Mangifera indica. The full-length cDNA sequence was 984 bp and contained an open reading frame of 600 bp, which encoded a 200 amino acid protein with a molecular weight of 21.83 kDa and a theoretical isoelectric point of 6.99. The deduced amino acid sequence exhibited high homology with tomato (91% similarity) and contains all five characteristic Rab motifs. Real-time quantitative RT-PCR analysis demonstrated that MiRab5 was ubiquitously expressed in various mango tree tissues at different levels. The expression of MiRab5 was up-regulated during later stages of fruit ripening. Moreover, MiRab5 was generally up-regulated in response to various abiotic stresses (cold, salinity, and PEG treatments). Recombinant MiRab5 protein was successfully expressed and purified. SDS-PAGE and western blot analysis indicated that the expressed protein was recognized by the anti-6-His antibody. These results provide insights into the role of the MiRab5 gene family in fruit ripening and stress responses in the mango plant.     

Molecular cloning and characterization of an Hsp90/70 organizing protein gene from Frankliniella occidentalis (Insecta: Thysanoptera,Thripidae)

The heat shock 90/70 organizing protein (Hop), also known as Sti-1 (stress-induced protein-1), is a co-chaperone that usually mediates the interaction of Hsp90 and Hsp70 and has been extensively characterized in mammals and plants. However, its role in insects remains unknown. In the present study, we isolated and characterized a Hop homologue gene from Frankliniella occidentalis (Fohop). The Fohop contains a 1659 bp ORF encoding a protein of 552 amino acids with a caculated molecular mass of approximately 62.25 kDa, which displays a reasonable degree of identity with the known Hops and shares several canonical motifs, including three tetratricopeptide repeated motif domains (TPR1, TPR2A and TPR2B) and two aspartic acid–proline (DP) repeat motifs (DP1 and DP2). As in other hops, Fohop contains introns, but the number and the position are quite variable. The mRNA expression patterns indicated that Fohop was constitutively expressed throughout the developmental stages, but was obviously upregulated by heat stress both in larvae and adults. Our studies imply that Hop, as in other Hsps, may play an important role in heat shock response of F. occidentalis.     

Molecular characterization,tissue distribution and feeding related changes of NUCB2A/nesfatin-1 in Ya-fish (Schizothorax prenanti)

The protein nucleobindin-2 (NUCB2) was identified over a decade ago and recently raised great interest as its derived peptide nesfatin-1 was shown to reduce food intake and body weight in rodents. However, the involvement of NUCB2 in feeding behavior has not well been studied in fish. In the present study, we characterized the structure, distribution, and meal responsive of NUCB2A/nesfatin-1 in Ya-fish (Schizothorax prenanti) for the first time. The full length cDNA of Ya-fish was 2140 base pair (bp), which encoded a polypeptide of 487 amino acid residues including a 23 amino acid signal peptide. A high conservation in NUCB2 sequences was found in vertebrates, however the proposed propeptide cleavage site (Arg–Arg) conserved among other species is not present in Ya-fish NUCB2A sequence. Tissue distribution analysis revealed that Ya-fish NUCB2A mRNA was ubiquitously expressed in all test tissues, and abundant expression was detected in several regions including the hypothalamus, hepatopancreas, ovary and intestines. NUCB2A mRNA expression respond to feeding status change may vary and be tissue specific. NUCB2A mRNA levels significantly increased (P < 0.05) in the hypothalamus and intestines after feeding and substantially decreased (P < 0.01) during a week food deprivation in the hypothalamus. Meanwhile, NUCB2A mRNA in the hepatopancreas was significantly elevated (P < 0.001) during food deprivation, and a similar increase was also found after short-time fasting. This points toward a potential hepatopancreas specific local role for NUCB2A in the regulation of metabolism during food deprivation. Collectively, these results provide the molecular and functional evidence to support potential anorectic and metabolic roles for NUCB2A in Ya-fish.