Long‐term low‐salinity stress affects growth,survival and osmotic related gamma‐aminobutyric acid regulation in the swimming crab Portunus trituberculatus

A 30‐day experiment was conducted to investigate the effects of long‐term low‐salinity stress on the growth performance and osmotic related chlorine ion channel ligand regulation: gamma‐aminobutyric acid (GABA) content and GABA_A receptor‐associated protein (GABARAP) expression in Portunus trituberculatus. The salinity levels of both the control group and the experimental group were 30 and 12 psu respectively. After rearing for 30 days, the specific growth rate and survival rate were compared between the two groups, and salinity 6 psu was used to test the salinity tolerance. The results were as follows: (a) Both the specific growth rate and survival rate were significant lower in the experimental group (p < 0.05) after 30 days; (b) After challenge with salinity 6 psu for 72 hr, the crabs of experimental group had a 100% survival rate, whereas the crabs of the control group were all dead within 48 hr; (c) The content of GABA and the gene expression level of GABARAP in experimental group were significant different from control group (p < 0.05) after challenge via salinity 6 psu. In the control group, the GABA content increased rapidly from 9.96 ± 2.09 to 42.00 ± 5.94 µg/g; however, in the experimental group, it only increased to 27.82 ± 2.55 µg/g; the gene expression of GABARAP in the experimental group increased to the maximum at 24 hr, then decreased and stabilized at 48 hr, suggesting that GABA and GABARAP were trigged during the early stage of low‐salinity stress resistance.     

Molecular cloning,characterization, tissue expression and nutritional regulation of O‐GlcNAc transferase gene in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂)

O‐GlcNAc transferase gene (OGT) was considered as the sole rate‐limiting enzyme in the O‐GlcNAc modification. In the present study, the OGT gene of hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) was cloned and characterized, and its expression in response to dietary carbohydrate level and acute glucose treatment was investigated. The full‐length of OGT (GenBank accession no. KY656469 ) was 4,063 bp, including a 302 bp 5′untranslated terminal region (UTR), a 3,165 bp coding region that encoded 1,054 amino acids residues and a 596 bp 3′ UTR. The highly conservation of OGT gene between fish and mammals was also observed through multiple sequences alignment and phylogenetic analysis. O‐GlcNAc transferase gene was ubiquitously expressed in all detected tissues with highest expressions in brain and liver, to a lesser degree, in eye, heart, kidney and intestine. The increasing dietary carbohydrate from 8.02% to 16.08% had no significant effect on the mRNA expression of OGT. However, the expression of OGT was slightly elevated at 6 hr post‐glucose injection, and the elevation became significant at 24 hr time‐point. These data may enhance our understanding on the nutritional regulation of OGT and O‐GlcNAc modification in fish species.     

Identification of SNPs in the 5′‐flanking region and 3′‐UTR of the MIH gene and their association with precocity of the Chinese mitten crab Eriocheir sinensis

Moult‐inhibiting hormone (MIH), an important regulator of steroidogenesis, inhibits the synthesis of ecdysteroid in Y‐organ (YO) and plays a significant role in the regulation of moulting and post‐embryonic development of crustacean. Because unsuccessful moulting have been widely observed in precocious crabs, we investigated whether genetic variants in the 5′‐flanking region and 3′‐untranslated region (3′‐UTR) of the MIH gene are associated with precocity of the Chinese mitten crab. Thirty individual DNA samples were sequenced to search for SNPs in the 5′‐flanking region and 3′‐UTR of the MIH gene. Five SNPs (g.196 T>A, g.230 C>T, g.305 T>C, g.323 C>A and g.372 C>T) in the 5′‐flanking region and 6 SNPs (g.2677 C>T, g.2759 T>A, g.2807 T>C, g.3042 A>G, g.3088 T>G and g.3295 T>G) in the 3′‐UTR of the MIH gene were selected for the individual genotyping in a two‐stage association study. We found that a SNP g.3088 T>G in the 3′‐UTR of MIH gene was consistently associated with precocity of the Chinese mitten crab in stage 1 and stage 2, with a per‐allele OR (Odds Ratio) of 1.469 (95% CI: 1.169–1.844) after two stages combined (P = 0.001). However, no significant associations were observed between the other 10 SNPs and precocity of the Chinese mitten crab. To our best knowledge, this is the first association study between various SNP genotypes and phenotype attributes in Chinese mitten crab. Our findings suggest that the SNP g.3088 T>G may be a candidate marker for effective marker‐assisted selection to decrease the precocity of the Chinese mitten crab in future studies.     

A novel 79‐bp insertion/deletion polymorphism in 3′‐flanking region of IGF‐I gene is associated with growth‐related traits in common carp (Cyprinus carpio L.)

Insulin‐like growth factor‐I (IGF‐I) participates in the modulation of the cellular development, differentiation and proliferation in various vertebrate tissues. Therefore, variations in IGF‐I gene are good candidates as growth regulators. In this study, a novel 79‐bp insertion/deletion (I/D) polymorphism was identified in 3′‐flanking region of IGF‐I gene by polymerase chain reaction (PCR) and sequencing approaches in common carp Cyprinus carpio. To gain insight into the association of the 79‐bp I/D polymorphism with growth‐related traits, a total of 747 individuals from Heilongjiang carp Cyprinus cario haematopterus (YL) (n = 263), selective line of German mirror carp Cyprinus carpio L. mirror (JL) (n = 229), and cold‐resistance line of Hebao red carp Cyprinus carpio var. wuyuanensis (HL) (n = 255) were genotyped. The DD genotype of the polymorphism was significantly associated with higher body length (135), body weight (325, 385, and 445 days), net weight (445 days) and lower overwintering body loss rate in JL. In addition, in pooled population (YL+JL+HL), fish with DD genotype also showed significantly higher body weight (445 days) and net weight (445 days) than those with II genotype. Taken together, these results suggest that the novel polymorphism in IGF‐I gene could be considered as a potential genetic marker for selection of growth performance in common carp.     

Cloning,characterization and transcription analysis of fatty acyl desaturase (Δ6Fad‐like) and elongase (Elovl4‐like,Elove5‐like) during embryos development of Strongylocentrotus intermedius

Strongylocentrotus intermedius has high nutritional value because it is rich in proteins, amino acids and long‐chain polyunsaturated fatty acids (LC‐PUFA). LC‐PUFA are essential nutrients that not only determine the nutritional value of sea urchins but also guarantee normal growth and reproduction performance. To better understand the molecular basis of LC‐PUFA biosynthesis in S. intermedius, the Δ6Fad‐like, Elovl4‐like and Elovl5‐like genes were cloned and fatty acid compositions during the early developmental stages of sea urchins were detected. The full‐length of Δ6Fad‐like was 2,199 bp, with a putative open reading frame of 1,248 bp encoding a polypeptide of 415 amino acid (AA). The Elovl4‐like and Elovl5‐like genes encoded 310 and 234 AA, respectively, which exhibited all of the characteristics of the Elovl family, such as a histidine box motif and putative transmembrane‐spanning domains. Tissue distribution analysis revealed that Δ6Fad‐like, Elovl4‐like and Elovl5‐like genes were expressed at the highest levels in the gonads and intestine, and the expression levels gradually increased in embryos during development. These results can help to understand the role of the Δ6Fad‐like, Elovl4‐like and Elovl5‐like genes in the different developmental stages of the sea urchin and to clarify the biosynthetic pathways of LC‐PUFA during the development of the sea urchin and provide a theoretical basis for improving the quality and breeding of excellent traits in sea urchins.     

Comparison of immune responses and antioxidant status of different generations of growth‐selected Portunus trituberculatus families

The purpose of this study was to investigate the effects of inbreeding on the immune responses and antioxidant status of Portunus trituberculatus juveniles. Results showed that inbreeding affected the total haemocyte count, and phagocytic, pro‐phenoloxidase (propo), phenoloxidase and antibacterial activities decreased after the seventh generation. Antioxidant status showed a similar pattern: total antioxidant capacity, superoxide dismutase (SOD) activity and GSH/GSSG in the cell‐free haemolymph and hepatopancreas decreased, while catalase activity in the cell‐free haemolymph increased (P < 0.05). There were no significant differences in α₂‐macroglobulin and bacteriolytic activities in the cell‐free haemolymph and glutathione peroxidase activity in the cell‐free haemolymph and hepatopancreas among nine inbreeding generations. Gene expression levels of proPO and crustin in haemocytes and SOD in haemocytes and hepatopancreas also decreased significantly as the inbreeding generations increased. The results suggest that a high level of inbreeding could severely reduce the physiological health of P. trituberculatus. Our obtained data would be particularly useful for P. trituberculatus breeding programmes.     

iTRAQ‐based identification of differentially expressed proteins related to growth in the swimming crab,Portunus trituberculatus

The swimming crab, Portunus trituberculatus, is an important farmed species in China, and it has been studied extensively, which requires more information on its genetic background. To date, information on the proteomics of P. trituberculatus is scarce. In this study, we used isobaric tags for relative and absolute quantitation (iTRAQ)‐based proteomics to investigate growth regulation in P. trituberculatus. Total proteins were isolated from five tissues (eyestalk, gill, heart, hepatopancreas and muscle). Equal quantities of protein from each tissue were pooled at the proteome level using the iTRAQ method. A total of 961 proteins were identified using liquid chromatography coupled with tandem mass spectrometry and de novo sequencing data. Using a 1.2‐fold change in expression as a physiologically significant benchmark, 30 differentially expressed proteins (DEPs) were found to undergo differential expression related to the growth of P. trituberculatus, and most of the growth‐related proteins, including those involved in metabolism, immune responses, DNA duplication and protein synthesis, were upregulated, indicating that conservation of energy is an important strategy to cope with growth. There was a high consistency between the expression levels determined using iTRAQ and mRNA, highlighting the high reproducibility of our proteomic approach and its great value in elucidating the molecular mechanisms of P. trituberculatus.     

Effects of γ‐aminobutyric acid supplementation on the growth performance,serum biochemical indices and antioxidant status of pharaoh cuttlefish,Sepia pharaonis

This study evaluated the effects of dietary γ‐aminobutyric acid (GABA) on the growth performance, serum biochemical indices and antioxidant status of pharaoh cuttlefish, Sepia pharaonis. Cuttlefish were cultured in open‐culturing cement pool systems for 8 weeks. Six practical diets supplemented with graded levels of GABA (0, 20, 40, 60, 80 and 100 mg/kg) were formulated. Each diet was randomly assigned to triplicate groups of 60 cuttlefish (mean weight: 10.33 g), the cuttlefish were fed two times per day to apparent satiation. The results showed that the specific growth rate (SGR), weight gain (WG) and feed efficiency (FE) significantly increased with dietary GABA supplementation (p < .05). The survival rate (SR) and protein content in muscle significantly increased when 58.9 mg/kg GABA supplied. Moreover, the nitric oxide (NO) content and acid phosphatase (ACP) activity in serum were significantly increased with dietary GABA supplementation (p < .05), while the activity of aspartate aminotransferase (AST) in serum decreased significantly when supplied with GABA at 58.9 mg/kg (p < .05). In addition, dietary GABA improved antioxidation activity by significantly increasing the activities of superoxide dismutase (SOD) and catalase (CAT) but decreasing malondialdehyde (MDA) levels in the liver and gill (p < .05). On the basis of the quadratic regression analysis of FE, the optimum content of dietary GABA in S. pharaonis was estimated to be 55.3 mg/kg. The findings of this study demonstrated that dietary GABA had a positive effect on the growth performance, serum biochemical indices and antioxidant status of S. pharaonis.     

Characterization of β‐catenin 1 during the gonad development in the common carp (Cyprinus carpio)

β‐catenin gene is a pivotal gene for gonad development and maintenance of ovarian function in mammals. However, little is known about its expression and function in gonad development of fish. In this study, a complete cDNA (3342 bp) sequence of β‐catenin 1 was cloned from the common carp, Cyprinus carpio, by RACE PCR, which encodes a 780‐amino‐acid protein. Quantitative real‐time PCR demonstrated that β‐catenin 1 mRNA expressions were high in the testis and ovary tissue and the expression increased as the testes developed and the early stage ovaries developed. Western blot results revealed a single immunoreactive band with an estimated molecular weight of 90 kDa in testes. Immunohistochemistry analysis revealed that the β‐catenin 1 protein was concentrated mainly in the cytoplasm of early development stage of oocyte cells and in the cytomembrane of developing and mature sperm cells. 17β‐Ethinylestradiol injecting intraperitoneally into the fish decreased the relative β‐catenin 1 mRNA expression level except 1 μg/g 72 hr and 5 μg/g 48 hr of treatments in the ovary by real‐time PCR. These results suggest, for the first time, that β‐catenin 1 is an essential protein in gonad development and might be involved in ovarian early development of C. carpio.     

Influence of dietary linoleic acid (18:2n‐6) and α‐linolenic acid (18:3n‐3) ratio on fatty acid composition of different tissues in freshwater fish Songpu mirror carp,Cyprinus Carpio

Six isonitrogenous and isoenergetic purified diets were formulated to feed Songpu mirror carp for 60 days. The control diet (CD) was only supplemented with soybean oil. The other five experimental diets contained soybean oil, linseed oil and lard oil blended at various inclusion levels to attain different linoleic acid (LA)/α‐linolenic acid (LNA) ratios (0.53, 1.04, 2.09, 3.95, 6.82) with a constant total C₁₈ polyunsaturated fatty acids (PUFA; LA+LNA, 2% dry weight) content. The fatty acid (FA) profiles of hepatopancreas, dorsal muscle, intestine, intraperitoneal fat (IPF), spleen and kidney reflected those of the diets, but with some differences. The spleen showed the lowest correlation with diet compared with other tissues, followed by the hepatopancreas (P < 0.05). The intestine and IPF showed relatively higher correlation. On the other hand, the control group had the lowest tissue‐diet correlation, followed by the LA/LNA0.53 group (P < 0.05), whereas the LA/LNA2.09 showed the highest. The LA/LNA ratios in the tissues were up‐regulated in the LA/LNA0.53, 1.04 groups and down‐regulated in the LA/LNA3.95, 6.82 groups. This was due to when LA (or LNA) was highly added in diet, the decrease in this FA was huge in tissue. The contents of saturated fatty acids and monounsaturated fatty acids increased in the control group, but seemed not influenced by dietary LA/LNA ratios. These results demonstrated that the FA deposition was tissue‐specific, and also influenced by the dietary FA composition in the experimental fish. Finally, we suggest that 2.09 is the optimal LA/LNA ratio (2% C₁₈ PUFA) of Songpu mirror carp for fillet FA composition.     

Comparative effect of Freund's adjuvant and Aloe vera L. gel on the expression of TNF‐α and IL‐1β in vaccinated Cyprinus carpio L. with Aeromonas hydrophila C. bacterin

The comparative effects of Freund's and Aloe vera gel as adjuvants on the expression of IL‐1β and TNF‐α genes were studied in vaccinated common carp (Cyprinus carpio) with Aeromonas hydrophila bacterin. Fishes were intraperitoneally immunized with A. hydrophila bacterin in combination with Aloe vera gel or Freund's and also without any adjuvant. At day 28 after immunization, all groups were challenged by lethal dose of A. hydrophila (10⁷ cells/fish). Changes in the expression of IL‐1β and TNF‐α genes were evaluated in anterior kidney before challenge and 12, 24, 72 and 7 days postchallenge using quantitative real‐time PCR. Higher expression levels of both genes were observed in all vaccinated groups compared with non‐immunized group. Fishes which received Aloe vera gel showed higher expression of IL‐1β and TNF‐α in relation to animals which vaccinated with or without Freund's adjuvant. We concluded that Aloe vera gel in compared with Freund's adjuvant had a more stimulatory effect on the expression of immune‐related genes in vaccinated common carp and it can use as a novel adjuvant in aquaculture.     

Artificial substratum consisting of poly‐β‐hydroxybutyrate‐based biodegradable plastic improved the survival and overall performance of postlarval tiger shrimp Penaeus monodon

The use of artificial substratum consisting of poly‐β‐hydroxybutyrate (PHB)‐based biodegradable plastic for penaeid shrimp culture was investigated in the present study. The survival of postlarval tiger shrimp Penaeus monodon (30 ± 5 mg) provided with PHB substratum made out of PHB type DP9002 (Metabolix GmbH, Köln, Germany) was 88.7 ± 3.4% and this was significantly higher as compared to postlarvae provided conventional substratum consisting of polyvinylchloride (PVC) pipes (67.3 ± 6.5%). However, no significant weight improvement was observed for the postlarval tiger shrimp indicating that PHB could not be used as growth promoter. Nevertheless, a trend of improved robustness against adverse environmental conditions (lethal ammonium chloride concentration) and increased resistance to pathogenic Vibrio was observed in postlarval tiger shrimp provided with PHB substratum as compared to postlarvae provided with PVC substratum. Results indicate higher preference by postlarvae on PHB substratum over PVC substratum. Overall, this study indicates the potential of artificial substratum consisting of PHB‐based biodegradable plastic as replacement for conventional substratum consisting of PVC pipes in enhancing the survival of postlarval tiger shrimp and improving its performance against adverse environmental conditions and disease resistance.     

Recombinant expression and comparative bioactivity of tongue sole insulin‐like growth factor (IGF)‐1 and IGF‐2 in Pichia pastoris

Insulin‐like growth factor (IGF) plays a key role in the complex system that regulates bony fish growth, differentiation, and reproduction. In the current study, recombinant tongue sole IGF‐1 and IGF‐2 were obtained using the Pichia pastoris expression system and their comparative bioactivities were investigated. Tricine–SDS–PAGE and western blot analysis showed that the recombinant tongue sole IGFs were secreted into the culture medium and had a molecular weight of 8.7 kDa. The optimal incubation time and pH for recombinant expression of IGFs were 36 hr and 5.0 respectively. Functional analysis demonstrated that both recombinant tongue sole IGF‐1 and IGF‐2 significantly promoted cell proliferation of MFC‐7 in vitro. In addition, the recombinant tongue sole IGF‐1 and IGF‐2 proteins could suppress hepatic mRNA levels of igf‐1 and igf‐2 in vitro, which showed that they have similar physiological functions. Taken together, the biologically active recombinant tongue sole IGF‐I and IGF‐II proteins will allow us to further investigate their physiological roles in growth regulation of this species. Furthermore, the present results also hinted at the potential application of these two recombinant IGF‐I and IGF‐II proteins into the tongue sole farming industry.     

Prevalence,geographic distribution and phenotypic differences of Piscirickettsia salmonis EM‐90‐like isolates

Early reports accounted for two main genotypes of Piscirickettsia salmonis, a fish pathogen and causative agent of piscirickettsiosis, placing the single isolate EM‐90 apart from the prototypic LF‐89 and related isolates. In this study, we provide evidence that, contrary to what has been supposed, the EM‐90‐like isolates are highly prevalent and disseminated across Chilean marine farms. Molecular analysis of 507 P. salmonis field isolates derived from main rearing areas, diverse hosts and collected over 6 years, revealed that nearly 50% of the entire collection were indeed typed as EM‐90‐like. Interestingly, these isolates showed a marked host preference, being recovered exclusively from Atlantic salmon (Salmo salar) samples. Although both strains produce undistinguishable pathological outcomes, differences regarding growth kinetics and susceptibility to the antibiotics and bactericidal action of serum could be identified. In sum, our results allow to conclude that the EM‐90‐like isolates represent an epidemiologically relevant group in the current situation of piscirickettsiosis. Based on the consistency between genotype and phenotype exhibited by this strain, we point out the need for genotypic studies that may be as important for the Chilean salmon industry as the continuous surveillance of antimicrobial susceptibility patterns.