Expression of dmrt1 and sox9 during gonadal development in the Siberian sturgeon (Acipenser baerii)

The sex differentiation period of the Siberian sturgeon was investigated through expression profiling of two testicular markers (dmrt1 and sox9). At the molecular level, a clear sexual dimorphism of dmrt1 and sox9 was observed in 3-year-old fish with immature gonads, in which males showed higher expression of these genes. Among 16-month-old sturgeons cultured in Uruguay, gonad morphology analyses showed one group of fish with undifferentiated gonads and a second group which had started their histological differentiation into ovaries or testes. dmrt1 showed a significantly higher expression in testes of recently differentiated fish, but this was not the case for sox9. In undifferentiated fish, we observed two clearly different groups in terms of expression: one group of fish over-expressing male markers (dmrt1, sox9) and another group of fish showing very low expression of these genes. This suggests that fish undergoing male differentiation can be identified by their profiles of gene expression before they undergo morphological differentiation.     

鲤神经球蛋白基因序列与低氧表达分析

为深入了解鲤珠蛋白家族的基因组成和表达模式,及其与低氧适应能力的相关性,本实验通过鲤基因组框架图比对和全长cDNA文库筛查,获得鲤神经球蛋白(neuroglobin,Ngb)基因完整序列,证实鲤不仅具有独特的脑组织特异表达的II型肌红蛋白(myoglobin-2,Mb-2)基因,也具有Ngb珠蛋白基因,实时荧光定量PCR实验显示,该基因在脑组织特异性表达,并呈现出低氧应答特征。基因结构和系统发生分析表明,鱼类Ngb基因高度保守,鲤Ngb蛋白可能与斑马鱼直系同源蛋白具有相似的结构及功能,而与鲤Mb-2存在明显差异。鲤Ngb基因表达量在两个品系间存在显著差异,耐低氧能力强的散鳞镜鲤Ngb基因表达量高于荷包红鲤抗寒品系。研究表明,鲤Ngb基因可能以与Mb-2基因分工协作的方式共同实现脑组织供氧,执行应对低氧胁迫的神经保护功能,在鲤低氧适应中具有重要作用。     

Dimorphic expression of sex-related genes in different gonadal development stages of sterlet, <Emphasis Type="Italic">Acipenser ruthenus</Emphasis>, a primitive fish species

Molecular mechanism of sex determination and differentiation of sturgeon, a primitive fish species, is extraordinarily important due to the valuable caviar; however, it is still poorly known. The present work aimed to identify the major genes involved in regulating gonadal development of sterlet, a small species of sturgeon, from 13 candidate genes which have been shown to relate to gonadal differentiation and development in other teleost fish. The sex and gonadal development of sterlets were determined by histological observation and levels of sex steroids testosterone (T), 11-ketotestosterone (11-KT), and 17β-estradiol (E2) in serum. Sexually dimorphic gene expressions were investigated. The results revealed that gonadal development were asynchronous in 2-year-old male and female sterlets with the testes in early or mid-spermatogenesis and the ovaries in chromatin nucleolus stage or perinucleolus stage, respectively. The levels of T and E2 were not significantly different between sexes or different gonadal development stages while 11-KT had the higher level in mid-spermatogenesis testis stage. In all the investigated gonadal development stages, gene dmrt1 and hsd11b2 were expressed higher in male whereas foxl2 and cyp19a1 were expressed higher in female. Thus, these genes provided the promising markers for sex identification of sterlet. It was unexpected that dkk1 and dax1 had significantly higher expression in ovarian perinucleolus stage than in ovarian chromatin nucleolus stage and in the testis, suggesting that these two genes had more correlation with ovarian development than with the testis, contrary to the previous reports in other vertebrates. Testicular development-related genes (gsdf and amh) and estrogen receptor genes (era and erb) differentially expressed at different testis or ovary development stages, but their expressions were not absolutely significantly different in male and female, depending on the gonadal development stage. Expression of androgen receptor gene ar or rspo, which was supposed to be related to ovarian development, presented no difference between gonadal development stages investigated in this study whenever in male or female.     

Characterisation of the microbiota associated with intestine of Atlantic cod (Gadus morhua L.): The effect of fish meal, standard soybean meal and a bioprocessed soybean meal

Populations of aerobic heterotrophic bacteria present in the gastrointestinal (GI) tract of healthy Atlantic cod (Gadus morhua L.) fed three different diets, fish meal, standard or a bioprocessed soybean meal (BPSBM), were estimated using the dilution plate technique. A total of 944 isolates were characterised by biochemical and physiological properties and 425 isolates were identified further by sequencing the 16S rRNA genes. Our results showed that gut microbiota were affected by dietary manipulation. The GI tract of fish fed fish meal was dominated by Gram-positive bacteria of the genera Brochothrix and Carnobacterium. The Gram-negative bacteria Chryseobacterium spp. and Psychrobacter glacincola, and Gram-positive bacteria belonging to Carnobacterium, dominated in the digestive tract of fish fed soybean meal. In contrast to these results, genus Psychrobacter dominated in the GI tract when fish were fed BPSBM.Until recently, it was generally suggested that the gut microbiota of fish were less diverse than in homoeothermic animals. However, the present study identified several “new” bacterial species isolated from the alimentary tract of Atlantic cod. These “new” bacterial species are not normally isolated from the GI tract of fish. Based on our finding we suggest that the GI tract microbiota of fish might not be as simple as believed.Antagonistic activity of carnobacteria regarding inhibition of growth of two fish pathogens (Aeromonas salmonicida ssp. salmonicida and Vibrio anguillarum) was observed. However, some difference in the antibacterial activity of Carnobacterium spp. was observed. Whether this antagonistic activity has any effect in challenge studies will be discussed, especially in relation to the finding that the digestive tract is one of the major infection routes in fish.     

Hsp60/10 and sHsp families of heat shock protein genes in rainbow trout (Oncorhynchus mykiss) and their expression under heat stress

Heat shock proteins (Hsps) are highly conserved proteins whose expression can be induced by high temperature and play an important role in a variety of biological processes. However, systematic identification of the Hsp60/10 and small Hsp (sHsp) gene family in rainbow trout has not yet been reported, and there is little available information about its roles in evolution in rainbow trout, a typical economical cold-water fish. In this study, we performed a comprehensive analysis of the rainbow trout Hsp60/10 and sHsp gene family and to investigate their expression profiles. A total of one Hsp60 gene, one Hsp10 gene, and ten sHsp genes were identified. According to RNA-seq analysis of rainbow trout liver and head kidney under heat stress, a total of six out of ten sHsp genes were significantly upregulated in liver and head kidney. Real-time RT-PCR (RT-qPCR) was used to quantitatively analyze the expression levels of these genes in different tissues of rainbow trout. Results showed that the expression of hspe1 and hspd1 was lowest in liver and gill, respectively, and highest in brain. In sHsp gene family, all genes are highly expressed in the liver and head kidney, but relatively low in the heart, spleen, brain, gills, and muscles. This systematic analysis provided valuable information about the diverse roles of Hsp60/10 and sHsp in the evolution of teleost fish, which will contribute to the functional characterization of Hsp60/10 and sHsp genes in further research.

     

Expression profiles of interferon gamma genes in response to immunostimulants and alloantigen in ginbuna crucian carp Carassius auratus langsdorfii

Interferon gamma (IFNγ), a Th1-derived cytokine is one of the key molecules inducing cell-mediated immune responses in mammals. The expression of 2 distinct IFNγ (ifng1 and ifng2) and IFNγrel (ifngrel) genes was examined in kidney leukocytes from clonal ginbuna crucian carp Carassius auratus langsdorfii. The expression of IFNγ and IFNγrel genes was induced in kidney leukocytes by stimulation with lipopolysaccharide or phytohemagglutinin in a dose-dependent manner. The expression levels of IFNγ and IFNγrel genes in kidney leukocytes from allo-sensitized fish cocultured with allogeneic cells were higher than those from fish cocultured with isogeneic cells. The highest expression of ifng1 was observed at day 1, whereas that of ifng2 and ifngrel was detected at day 2 after cocultivation with allogeneic cells. However, the expression pattern of ifng2 and ifngrel in kidney leukocytes from allo-sensitized fish by scale-grafting was similar to those from non-sensitized fish. These results indicate that ifng1 is a major isoform of IFNγ related to antigen-specific cell-mediated immunity (CMI), and will be a useful marker to evaluate induction of CMI in ginbuna crucian carp. In addition, IFNγ would be a crucial type-II interferon compared to IFNγrel, relevant to cell-mediated immunity in fish as in mammals.     

A novel murrel Channa striatus mitochondrial manganese superoxide dismutase: gene silencing,SOD activity,superoxide anion production and expression

We have reported the molecular characterization including gene silencing, superoxide activity, superoxide anion production, gene expression and molecular characterization of a mitochondrial manganese superoxide dismutase (mMnSOD) from striped murrel Channa striatus (named as CsmMnSOD). The CsmMnSOD polypeptide contains 225 amino acids with a molecular weight of 25 kDa and a theoretical isoelectric point of 8.3. In the N-terminal region, CsmMnSOD carries a mitochondrial targeting sequence and a superoxide dismutases (SOD) Fe domain (28–109), and in C-terminal region, it carries another SOD Fe domain (114–220). The CsmMnSOD protein sequence shared significant similarity with its homolog of MnSOD from rock bream Oplegnathus fasciatus (96 %). The phylogenetic analysis showed that the CsmMnSOD fell in the clade of fish mMnSOD group. The monomeric structure of CsmMnSOD possesses 9 α-helices (52.4 %), 3 β-sheets (8.8 %) and 38.8 % random coils. The highest gene expression was noticed in liver, and its expression was inducted with fungal (Aphanomyces invadans) and bacterial (Aeromonas hydrophila) infections. The gene silencing results show that the fish that received dsRNA exhibited significant (P < 0.05) changes in expression when compared to their non-injected and fish physiological saline-injected controls. The SOD activity shows that the activity increases with the spread of infection and decreases once the molecule controls the pathogen. The capacity of superoxide anion production was determined by calculating the granular blood cell count during infection in murrel. It shows that the infection influenced the superoxide radical production which plays a major role in killing the pathogens. Overall, this study indicated the defense potentiality of CsmMnSOD; however, further research is necessary to explore its capability at protein level.     

Phenotypic plasticity in gene expression and physiological response in red drum <Emphasis Type="Italic">Sciaenops ocellatus</Emphasis> exposed to a long-term freshwater environment

Red drum (Sciaenops ocellatus) is a euryhaline fish commonly found in the Gulf of Mexico and along the Atlantic coast of North America. Because of high commercial demand and its euryhaline characteristics, aquaculture of this species has diversified from marine to low-salinity aquaculture systems. In recent years, interest in the feasibility of producing red drum in inland freshwater systems has grown and this prompted us to investigate its osmoregulatory capacity after rearing for 8 months in a freshwater aquaculture system. We compared the activities of several genes and enzymes involved in the osmoregulatory process in freshwater-acclimatized (FW) and seawater (SW) red drum. The gene expression profiles were variable: the expression of genes encoding Na⁺/K⁺-ATPase (NKA) and the cystic fibrosis transmembrane regulator (CFTR) was slightly higher in SW than FW fish, while phosphoenolpyruvate carboxykinase (PEPCK) and the glucocorticoid receptor messenger RNA (mRNA) levels were higher in FW red drum. The total plasma K concentration was 60.3% lower, and gill NKA activity was 63.5% lower in FW than in SW fish. PEPCK activity was twofold higher in FW than in SW red drum. Similarly, liver glycogen was 60% higher in FW fish. In summary, both gene expression and the enzyme activity data support the phenotypic plasticity of red drum and suggest that the limited capacity for ion homeostasis observed, in particular the low plasma K concentration, was due to the composition of freshwater and does not necessarily reflect a physiological inability to osmoregulate.     

尼罗罗非鱼雌雄鱼肌肉组织差异表达基因的筛选

应用引物退火控制技术(ACP)筛选尼罗罗非鱼雌雄鱼肌肉组织差异表达基因,寻找与雌雄鱼肌肉生长发育相关的候选基因。本实验从同等条件下养殖的尼罗罗非鱼群体中随机选取雌、雄鱼各5尾组成RNA池,采用引物退火控制技术,分析了两组个体肌肉组织差异表达基因。利用20对随机引物差异显示扩增,共获得8条ESTs,其中5个已知的ESTs分别为转录变体3(LOC100691543)、60S核糖体蛋白(RL3)、小白蛋白β样蛋白、肌型肌酸激酶M2-CK和转录因子Sox4,其余3个为未知的ESTs。实时定量PCR分析各差异表达基因在尼罗罗非鱼雌雄肌肉组织中的表达发现,8个差异表达基因中转录变体3与ACP6-Y在尼罗罗非鱼雄鱼肌肉组织中的表达均极显著高于雌鱼(P0.01),ACP3-X、60S核糖体蛋白(RL3)、小白蛋白β样蛋白、ACP15-X、肌型肌酸激酶M2-CK与转录因子Sox4在尼罗罗非鱼雌鱼肌肉组织中的表达均极显著高于雄鱼(P0.01)。结果表明,应用引物退火控制技术筛选获得了8个可能参与了雌雄鱼肌肉生长发育调控的ESTs,为进一步筛选雌雄鱼肌肉生长发育相关候选基因奠定了基础。     

Identification of Wap65, a human homologue of hemopexin as a copper-inducible gene in swordtail fish, <Emphasis Type="Italic">Xiphophorus helleri</Emphasis>

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.     

暗纹东方鲀心型脂肪酸结合蛋白H-FABP基因克隆与表达分析

为深入了解心型脂肪酸结合蛋白(heart-type fatty acid-binding protein,H-FABP)的结构与功能,采用RACE技术克隆得到暗纹东方鲀H-FABP的全长cDNA序列,共772 bp,开放阅读框为402 bp,编码133个氨基酸。生物信息学分析表明,暗纹东方鲀H-FABP基因具有一个脂质转运蛋白家族保守结构域和多个蛋白激酶磷酸化位点。NJ法系统进化分析显示,暗纹东方鲀与红鳍东方鲀亲缘关系最近,其次是鲈形目。经荧光定量PCR检测,H-FABP基因在暗纹东方鲀所有被检测的组织中都有表达,其中肝脏中的表达量最高,其次为肌肉和心脏,预示H-FABP主要参与脂肪酸氧化分解过程。为探究豆油作为脂肪源的可行性,采用不同比例(0、25%、50%和75%)豆油替代鱼油的饲料投喂初始体质量为(8.31±0.20)g的暗纹东方鲀8周。结果显示,全鱼油组肝脏中H-FABP基因的表达量显著高于50%豆油组和75%豆油组,但与25%豆油组无显著性差异,且随着饲料中豆油比例的增加,暗纹东方鲀肝脏中H-FABP基因的表达量呈下降趋势。研究表明,富含n-3多不饱和脂肪酸的鱼油可以促进脂肪酸氧化分解,能预防脂肪肝。因此,为不影响暗纹东方鲀肝脏脂肪代谢,饲料中豆油替代鱼油的比例应控制在50%以下。     

水族箱气单胞菌的鉴定及致病特性分析

为了确定南京市某渔场发病鱼感染的病原,本研究采集患鱼脏器,采用平板培养、生化实验和特异性gyrB基因扩增测序等方法进行细菌的分离鉴定;利用PCR技术检测分离株毒力基因的分布,分析其生物学特性,并进一步通过动物实验确定菌株致病力.结果分离到1株水族箱气单胞菌,命名为LK-25.该菌株携带5种主要毒力基因:气溶素(aer)、细胞毒性肠毒素(act)、细胞兴奋性肠毒素(alt)、温敏胞外蛋白酶(epr)和丝氨酸蛋白酶(ahp),其溶血性和溶蛋白能力较强,对斑马鱼的半数致死量为1.02×103 CFU/尾,确定为强毒株.进化树分析表明,水族箱气单胞菌与嗜水气单胞菌达卡亚种亲缘关系较近.本研究在国内首次报道发现水族箱气单胞菌,为进一步预防该菌所引起的相关疾病的发生和传播提供理论基础.     

Genetic population structure in the Chilean jack mackerel, Trachurus murphyi (Nichols) across the South-eastern Pacific Ocean

The Chilean jack mackerel Trachurus murphyi, is a pelagic fish from the Carangidae family that is distributed in the South Pacific Ocean. Because this species constitutes an important economic resource across the South Pacific and plays an important ecological role in this ecosystem there is a growing interest in determining its population structure. In this study, we used molecular markers (mitochondrial DNA sequences and microsatellites) from Chilean jack mackerel samples to investigate its genetic population structure across the South Pacific Ocean. The mitochondrial DNA did not detect a genetic structure in T. murphyi populations in the Pacific Ocean, but revealed very low haplotype diversity and a short genealogy history compared to other small-pelagic species. The same general pattern of a lack of genetic structure was found with microsatellite loci; however, a large genetic diversity was revealed with microsatellite markers. The present results did not support the existence of different stock units for T. murphyi across the South Pacific Ocean but a more holistic approach will be necessary to determine an adequate management strategy for this fishery.     

New type of heat shock protein 70 homologue gene abounds in the genomic sequence of kuruma shrimp Marsupenaeus japonicus

Heat shock proteins (HSPs) are a group of highly conserved molecular chaperones. Shrimp HSPs have recently been a topic of increasing interest because of their roles in shrimp immunity and homeostasis. In penaeid shrimp, HSP70s and the cognate forms, heat shock cognate (HSC) 70s, have been reported, but their responses towards various stimulations are different. We found a novel type HSP70 (MjHSP70-2) from the hyperexpansion of the large segmental duplication that is present in kuruma shrimp Marsupenaeus japonicus, which shows about 60 % identity with reported shrimp HSP70s. In a phylogenetic tree, MjHSP70-2 formed a sister clade with eukaryote HSP70 family while MjHSP70 was located close to the shrimp HSP70 and HSC70 group. MjHSP70-2 gene expression was not significantly increased by heat shock or pathogen challenge by Vibrio penaeicida, but it was significantly increased by infection with white spot syndrome virus. In contrast, MjHSP70 gene expression was increased by heat shock but decreased by infection with V. penaeicida. The kuruma shrimp genome was found to have 400-fold more copies of the MjHSP70-2 gene than the putative single-copy gene transglutaminase. In conclusion, our results reveal the presence of a novel type HSP70 gene, HSP70-2, from kuruma shrimp. There are multiple forms of HSP70 in crustaceans, and these HSP70s behave differently under various stressors.     

Analysis of plasmids encoding the tyrosine decarboxylase gene in Tetragenococcus halophilus isolated from fish sauce

In order to elucidate mechanisms of tyramine accumulation during fish sauce production, two tyramine-producing bacterial strains, referred to as TyrA and TyrB, were isolated from fish sauce mash accumulating over 141 mg of tyramine per 100 g of sample. Both strains were identified as Tetragenococcus halophilus based on phenotypic characterization and a 16S rRNA gene sequence analysis. Molecular analysis of the tyramine-producing gene in the two strains confirmed the presence of a ~30-kb plasmid encoding a single copy of the pyridoxal phosphate-dependent tyrosine decarboxylase gene (tdcA) along with three other genes related to tyramine synthesis (tdc cluster). The complete nucleotide sequences of plasmids extracted from the two strains indicated that both plasmids were almost identical, except for a 1.6-kb transposon sequence in the plasmid from the strain TyrB. Both plasmids had a replication region, a plasmid maintenance region, and two putative mobile genetic elements located upstream and downstream of the tdc cluster. This structure was identical to that of tetragenococcal plasmids encoding histidine decarboxylase (hdcA), which were sequenced previously. These results suggest a common origin for plasmids encoding hdcA and tdcA. In addition, the genes for both these biogenic amines are distributed among tetragenococcal species via this plasmid.