Identification and functional characterization of Histone‐like DNA‐binding protein in Nocardia seriolae (NsHLP) involved in cell apoptosis

Nocardia seriolae, a facultative intracellular bacterium, is the main pathogen of fish nocardiosis. Bioinformatic analysis showed that the histone‐like DNA‐binding protein (HLP) gene of N. seriolae (nshlp) encoded a secreted protein and might target the mitochondria in the host cell. To further study the preliminary function of HLP in N. seriolae (NsHLP), the gene cloning, extracellular products identification, subcellular localization, overexpression and apoptosis detection assay were carried out in this study. Mass spectrometry analysis of the extracellular products from N. seriolae showed that NsHLP was a secreted protein. Subcellular localization of HLP‐GFP fusion proteins mainly assembled in the nucleus, which indicated that the NsHLP was co‐located with the nucleus rather than mitochondria in fathead minnow (FHM) cells. Notably, the expression of NsHLP had changed the distribution of mitochondria into lumps in the FHM cell. In addition, apoptotic features were found in the transfected FHM cells by overexpression of NsHLP. Quantitative assays of mitochondrial membrane potential value, caspase‐3 activity and pro‐apoptotic genes mRNA (Bad, Bid and Bax) expression level demonstrated that the cell apoptosis was induced in the transfected FHM cells. All the results presented in this study provided insight on the function of NsHLP, which suggested that it may participate in the cell apoptosis regulation and play an important role in the pathogenesis of N. seriolae.     

Identification of a secreted superoxide dismutase (SOD) from Nocardia seriolae which induces apoptosis in fathead minnow (FHM) cells

Fish nocardiosis is a chronic systemic granulomatous disease, and Nocardia seriolae is the main pathogen. The pathogenesis and virulence factors of N. seriolae are not fully understood. Secreted superoxide dismutase (SOD) may be a virulence factor found by a comparative bioinformatics analysis of the whole genome sequence of N. seriolae and the virulence factor database (VFDB). In order to determine the subcellular localization and study the preliminary function of SOD from N. seriolae (NsSOD), gene cloning, secreted protein identification, subcellular localization in fish cells, and apoptosis detection of NsSOD were carried out in this study. Subcellular localization research revealed that NsSOD‐GFP fusion proteins were evenly distributed in the cytoplasm. Furthermore, apoptotic bodies were observed in the transfected FHM cells by the overexpression of protein NsSOD. Then, assays of mitochondrial membrane potential (ΔΨm) value, caspase‐3 activity and apoptosis‐related genes (Bax, Bid, Bad and Bcl‐2) mRNA expression were conducted. The results showed that ΔΨm was decreased, and caspase‐3 was significantly activated. The mRNA expression of the Bad gene showed significant up‐regulated expression at 24 h.p.t., while Bid and Bax genes showed significant up‐regulated expression at 72 and 96 h.p.t. and anti‐apoptotic gene (Bcl‐2) was down‐regulated in NsSOD overexpressed cells. Taken together, the results indicated that the protein NsSOD might be involved in apoptosis regulation. This study may lay the foundations for further studies on the function of NsSOD and promote the understanding of the virulence factors and the pathogenic mechanisms of N. seriolae.     

Identification of a mitochondrial‐targeting secretory protein from Nocardia seriolae which induces apoptosis in fathead minnow cells

Nocardia seriolae is the main pathogen responsible for fish nocardiosis. A mitochondrial‐targeting secretory protein (MTSP) 3141 with an N‐terminal transit peptide (TP) from N. seriolae was predicted by bioinformatic analysis based on the genomic sequence of the N. seriolae strain ZJ0503. However, the function of the MTSP3141 and its homologs remains totally unknown. In this study, mass spectrometry analysis of the extracellular products from N. seriolae proved that MTSP3141 was a secretory protein, subcellular localization research showed the MTSP3141‐GFP fusion protein co‐localized with mitochondria in fathead minnow (FHM) cells, the TP played an important role in mitochondria targeting, and only the TP located at N‐terminus but not C‐terminus can lead to mitochondria directing. Moreover, quantitative assays of mitochondrial membrane potential (ΔΨm) value, caspase‐3 activity and apoptosis‐related gene (Bcl‐2, Bax, Bad, Bid and p53) mRNA expression suggested that cell apoptosis was induced in FHM cells by the overexpression of both MTSP3141 and MTSP3141ΔTP (with the N‐terminal TP deleted) proteins. Taken together, the results of this study indicated that the MTSP3141 of N. seriolae was a secretory protein, might target mitochondria, induce apoptosis in host cells and function as a virulence factor.     

一种(鱼师)鱼诺卡氏菌酪氨酸蛋白磷酸酶基因的克隆及功能初步研究

這鱼诺卡氏菌是鱼类诺卡氏菌病的主要病原,可导致鱼类慢性系统性肉芽肿疾病.這鱼诺卡氏菌全基因组序列分析发现了一个酪氨酸蛋白磷酸酶(protein tyrosine phosphtase,PTP)基因,生物信息学分析显示该基因很可能编码一个靶向定位于宿主细胞线粒体的分泌蛋白.本实验对這鱼诺卡氏菌PTP进行了基因克隆、分泌蛋白鉴定、亚细胞定位、过表达和线粒体膜电位检测,结果显示,在這鱼诺卡氏菌胞外产物中质谱鉴定到了PTP肽段,证实其为分泌蛋白.亚细胞定位研究观察到PTP-GFP融合蛋白均匀地分布在FHM细胞中,与线粒体分布不重合,说明這鱼诺卡氏菌PTP蛋白并未靶向定位于线粒体.亚细胞定位和过表达研究都显示PTP蛋白在FHM细胞中表达后,细胞核出现固缩浓染、凋亡小体等明显的细胞凋亡特征.通过线粒体膜电位检测表明,在pcDNA-PTP转染后48 h,线粒体跨膜电位被明显破坏,说明這鱼诺卡氏菌PTP很可能是一种可诱导细胞凋亡的细菌蛋白.通过对這鱼诺卡氏菌PTP开展基因克隆和功能初步研究,为进一步揭示该基因的功能和深入了解這鱼诺卡氏菌的分子致病机理奠定了基础.     

Immune response of largemouth bass, <Emphasis Type="Italic">Micropterus salmoides</Emphasis>, to whole cells of different <Emphasis Type="Italic">Nocardia seriolae</Emphasis> strains

Largemouth bass Micropterus salmoides were immunized with four different N. seriolae strains—two α-glucosidase-positive (961113, KU040801) and two α-glucosidase-negative (94260, OTTS) strains—along with Freund’s incomplete adjuvant. After primary immunization (week 0), a booster was administered at weeks 4 and 8. Nonspecific immune responses to multiple immunizations with the different N. seriolae strains were determined based on serum lysozyme activity and nitroblue tetrazolium (NBT)-positive cells in peripheral blood. The serum lysozyme activity and NBT-positive cells in peripheral blood were not significantly increased even after the two booster immunizations. Specific antibody responses against N. seriolae cells were investigated by enzyme-linked immunosorbent assay. At 4 weeks after immunization, all groups immunized with N. seriolae antigens showed significant increases in their specific antibody levels. The sera from fish immunized with different N. seriolae strains exhibited reactivity with N. seriolae sonicated antigens of 28, 30, 36 and 84 kDa by western blot analysis. After two boosters, fish were challenged with live N. seriolae to assess the vaccine’s efficacy; however, multiple injections of the N. seriolae strains did not reduce mortality, irrespective of the bacterin.     

Live vaccine trials against nocardiosis in yellowtail Seriola quinqueradiata

In an attempt to develop a vaccine against Nocardia seriolae, related species of live bacteria N. soli, N. fluminea, and N. uniformis were injected into yellowtail Seriola quinqueradiata. In addition, fish were challenged with a low virulence strain of N. seriolae to model the concept of use of a live vaccine. The fish injected with live N. soli and N. fluminea cells showed slight resistance against an artificial challenge with N. seriolae. On the other hand, the fish that survived the N. seriolae infection showed complete resistance to the N. seriolae challenge. These results suggest that protective immune responses against N. seriolae are induced in yellowtails.     

Sequencing of 16S−23S rRNA internal transcribed spacer and its application in the identification of Nocardia seriolae by polymerase chain reaction

A method for the diagnosis of nocardiosis in yellowtail (Seriola quinqueradiata), using polymerase chain reaction (PCR), was developed in this study. Primers specific for Nocardia seriolae were synthesized based on the alignment of 16S?23S rRNA internal transcribed spacer region sequences of N. seriolae. The primers did not amplify specific PCR product from other fish pathogens. However, two and three fishes could be diagnosed as infected with N. seriolae by clinical signs and bacterial isolation. PCR amplification of N. seriolae by specific primers detected six infected fishes. Thus, the primers used in this study are useful in detecting nocardiosis in fish.     

Modified resazurin microtiter assay for in vitro and in vivo assessment of sulfamonomethoxine activity against the fish pathogen <Emphasis Type="Italic">Nocardia seriolae</Emphasis>

Resazurin microtiter assay (REMA) was carried out using four sulfonamides, three culture media, and four inoculum sizes as a first screening step to establish an easy-to-interpret sulfonamides susceptibility testing method for Nocardia seriolae. The in vitro activity of sulfamonomethoxine (SMM) against 190 clinical N. seriolae isolates was then examined, and in vivo experimental treatment was performed. When the culture medium and the inoculum size were considered in tandem, a 0.5× the original concentration of cation-adjusted Mueller–Hinton broth and an inoculum size of 10² CFU/well showed the clearest endpoint reading for all tested drugs, and the REMA-generated data were in excellent agreement with those generated by the reference Etest method. SMM activity showed minimum inhibitory concentration (MIC) values of 4–32 μg/ml against all tested N. seriolae isolates. Treatment of amberjack groups experimentally infected with N. seriolae isolates having SMM MICs of 4 and 32 μg/ml, resulted in survival rates of 100% and 87.5% in the two groups, respectively. In this study, we developed a simple visual method to test SMM activity against N. seriolae.     

Amberjack <Emphasis Type="Italic">Seriola dumerili</Emphasis> interleukin-10 negatively suppresses host cell-mediated immunity

Interleukin-10 (IL-10) is an anti-inflammatory cytokine and negatively regulates cell-mediated immunity (CMI) induction by inhibiting cytokine production in type 1 T helper cells. IL-10 genes have been isolated from several fish, and inflammatory cytokine inhibition by IL-10 has been well examined. However, a CMI regulator of IL-10 in fish has not yet been identified. In this study, we cloned the IL-10 gene in amberjack Seriola dumerili and analyzed its function using its recombinant protein (rIL-10). In an in vitro culture experiment, gene expression of inflammatory cytokines was suppressed in leukocytes incubated with rIL-10 compared with cells that only received Nocardia seriolae stimulation. This result suggests amberjack IL-10 has conserved function as an inflammatory cytokine inhibitor. Bactericidal activity of amberjack cells against intracellular pathogen stimulation was decreased in a rIL-10 dose-dependent manner. Furthermore, a significant reduction in the T-bet/GATA-3 ratio was observed in N. seriolae living cell (LC)?+?rIL-10-injected fish. Taken together, these results suggest amberjack rIL-10 suppresses CMI induction both in vitro and in vivo. In addition, the number of IgM⁺ cells among spleen leukocytes in N. seriolae?+?rIL-10-injected fish was higher than in only N. seriolae LC, suggesting that Th2-dominant immunity was induced by adding rIL-10.     

Application of α-glucosidase activity and drug susceptibility tests to epidemiological studies on the fish pathogen <Emphasis Type="Italic">Nocardia seriolae</Emphasis>

This study determined the minimum inhibitory concentrations (MICs) for oxytetracycline hydrochloride (OTC) and erythromycin (Em), along with the α-glucosidase (α-glu) activities in 110 Nocardia seriolae strains isolated in Miyazaki and Kagoshima prefectures in 2008–2009. The strains were examined for the presence of the tet(K), tet(L), tet(M), tet(O), tet(S), erm(A), erm(B), mph(A), mef(A), and msr(D) genes. All the α-glu-positive strains (n = 15) were OTC resistant and Em sensitive, with MICs of 32–64 and <0.125 μg/ml, respectively. All the α-glu-negative strains (n = 95) were OTC sensitive, with MICs of 2–4 μg/ml, and most of them (93 of 95) were Em resistant, with MICs of >128 μg/ml. The MICs for Em in the remaining 2 α-glu-negative strains were <0.125 μg/ml. The 15 OTC-resistant strains possessed the tet(K) and/or tet(L) gene(s), and all of the 93 Em-resistant strains possessed both the mef(A) and msr(D) genes. The relationship between α-glu activity and drug sensitivity of the N. seriolae strains may explain the difference in prevalence of each phenotype. Nevertheless, the relationship should be further explored using N. seriolae isolates collected from more prefectures and farms.     

Effect of cyclical feeding on compensatory growth,nitrogen and phosphorus budgets in juvenile Litopenaeus vannamei

This study assessed the effect of cyclical feeding on compensatory growth, nitrogen (N) and phosphorus (P) budgets of juvenile Litopenaeus vannamei. A 36‐day growth trial was performed with four different feeding protocols. The control group (S0) was fed to satiation twice every day during the whole experimental period; treatment groups S1, S2 and S3 were fed by the 9, 5 and 3 cycles of 1:3, 2:5 and 3:9 (fasting days:feeding days) respectively. Fasting in S1, S2 and S3 groups did not change the specific growth rate in wet weight (SGR_w), but the feed conversion efficiency (FCE) and protein efficiency ratio (PER) were significantly increased (P < 0.05) in comparison with control. The N and P consumed per unit wet weight gain of shrimp in S1, S2, S3 groups were significantly lower (P < 0.05) than control group by 15.39%, 15.96%, 19.33% for N, and 15.16%, 15.98%, 19.26% for P respectively. The total discharge of N and P (including N and P discharged by faeces (F_N/P), non‐faecal excretions (U_N/P) and exuviations (E_N/P); ) was significantly lower (P < 0.05) in the experiment groups by 19.91–22.07% for N and 18.68–26.37% for P respectively. Overall, the results suggest that the L. vannamei can reach completely compensatory growth, and the total discharge of N and P per unit wet weight gain of L. vannamei significantly decreased by cyclical feeding, which could have a positive effect on the reduced of environmental N and P loading due to the cultured of L. vannamei.     

Influence of native catfish mucus on Flavobacterium columnare growth and proteolytic activity

Flavobacterium columnare causes columnaris disease of farmed and wild freshwater fish. Skin mucus is an important factor in early stages of columnaris pathogenesis, albeit little studied. Our objectives were to (a) characterize the terminal glycosylation pattern (TGP) of catfish mucus, (b) determine the growth of F. columnare in formulated water (FW)‐containing channel catfish (Ictalurus punctatus) or hybrid catfish (Ictalurus punctatus X Ictalurus furcatus) mucus and (c) examine extracellular protease activity of two F. columnare isolates differing in virulence. The TGP of catfish mucus by lectin binding was as follows: alpha‐D‐mannose/alpha‐D‐glucose >N‐acetyl‐beta‐D‐glucosamine >N‐acetyl‐beta‐D‐glucosamine/N‐acetylneuraminic acid >N‐acetyl‐D‐galactosamine >alpha‐D‐galactose/N‐acetyl‐alpha‐D‐galactosamine >beta‐D‐galactose = alpha‐L‐fucose. Virulence studies demonstrated isolate AL‐02‐36 was highly virulent in channel catfish fry (0.1 g) with cumulative mortality of 90%‐100% versus 60% for isolate ALG‐00‐530 at equivalent doses (~3 × 10⁶ CFU/ml); a similar result was observed in larger (0.7 g) catfish. In multiple experiments, F. columnare replicated (2‐3 logs) and survived (28 days) in formulated water‐containing catfish mucus. Highly virulent isolate AL‐02‐36 possessed at least 2.5‐ to fivefold higher protease activity following growth in mucus than the less virulent ALG‐00‐530. Flavobacterium columnare utilized catfish mucus as a nutrient source and mucus presence modulated extracellular protease production.     

四川彭州地区养殖虹鳟传染性造血器官坏死病病毒的分离、鉴定及病理学观察

近期,四川省成都彭州市某虹鳟养殖场暴发流行疾病,导致养殖虹鳟死亡率高达90%。现场采样观察发现患病鱼主要症状为背部发黑,鳔壁、腹膜严重出血,心包积液,空肠、空胃和显著肠炎。同时对病鱼进行细菌学与组织病理学检测,细菌学检查结果为阴性,病理学观察发现脾脏有典型凝固性坏死,肝脏组织广泛变性、坏死,肠道黏膜下层水肿,肠上皮充血及上皮细胞脱落坏死,脑膜和心外膜水肿。将病鱼的脾组织研磨过滤除菌后,腹腔注射60尾健康虹鳟,注射组均表现为急性死亡(累计死亡率达85%),试验鱼出现与自然发病鱼相同的症状而对照组无异常。取病鱼的脾脏组织研磨过滤后接种胖头鲤细胞(fathead minnow cell,FHM),细胞感染3 d后出现典型的细胞病变效应(CPE)。针对编码IHNV糖蛋白(Glycoprotein,G)基因进行逆转录-聚合酶链反应(RTPCR)检测显示,患病鱼、人工感染病鱼和病变细胞均为IHNV阳性,扩增序列与IHNV糖蛋白基因同源性为98.2%。对该病毒分离株的G基因进行系统发育分析,结果显示,该分离株与亚洲分离株聚为一簇,属于JRt基因型。     

Preliminary investigation into the killing effect of kingfish (Seriola lalandi) serum and mucus against the monogenean parasites Benedenia seriolae and Zeuxapta seriolae

The survival of two economically important monogenean parasites, Benedenia seriolae and Zeuxapta seriolae, exposed to either naïve or infected yellowtail kingfish Seriola lalandi serum and mucus was observed over an 8-h period. Infection status of the host had no effect on survival of monogeneans in either serum or mucus therefore data were grouped according to each parasite. Serum exposure at the highest concentration of 1:5 did not have any effect on Z. seriolae, however, 50% killing activity against B. seriolae was observed within 3 h at concentrations greater than 1:100. This killing activity was completely abolished following heat treatment and inactivation of the serum. Prior incubation of parasites with heat-treated serum for 2 h in order to potentially coat the parasites with antibodies did not enhance the activity of fresh serum against either species. Addition of 5 mM EDTA, but not 5 mM EGTA-Mg, inhibited the killing ability of fresh serum against B. seriolae, suggesting that previously observed activity against this parasite was most likely mediated via the alternative complement pathway. Cutaneous mucus was not found to have any effect on B. seriolae or Z. seriolae survival. Overall these results suggest that the blood-feeding gill parasite Z. seriolae is considerably more resistant to host immune responses compared with skin- and mucus-dwelling B. seriolae.     

L‐carnitine exerts a cytoprotective effect against H2O2‐induced oxidative stress in the fathead minnow muscle cell line

This study was conducted to investigate the protective effect of L‐carnitine (LC) against H₂O₂‐induced oxidative stress in the fathead minnow muscle cell line (FHM). The FHM cells were stimulated with 1 mM H₂O₂ for 1 h after LC pre‐treatment, and the cell viability and the activity and mRNA relative expression of antioxidant enzyme were measured to assess the antioxidant properties of LC. The results showed that the toxic effect of H₂O₂ on the viability of FHM cells was both dose‐ and time‐dependent. Furthermore, the viability of the 0.01–1 LC mM groups was significantly higher than those of the 1 mM H₂O₂ group. L‐carnitine protected the cells from H₂O₂‐induced oxidative damage, which was demonstrated by a significant reduction in the malondialdehyde and reactive oxygen species levels and increases in the intracellular total glutathione levels and the activities of total superoxide dismutase, catalase, glutathione peroxidase (GPx) and gamma‐glutamyl‐cysteine synthetase (γ‐GCS) in FHM cells pre‐treated with LC for 6 h compared with the 1 mM H₂O₂ group. In addition, the mRNA relative expression levels of the γ‐GCS catalytic subunit and nuclear factor nuclear factor erythroid 2‐related factor 2 were significantly higher than those of the 1 mM H₂O₂ group. It could be concluded that LC exerts a beneficial antioxidant effect against oxidative stress induced by H₂O₂ in FHM cells and that the appropriate treatment is 0.1–1 mM for 6 h in this study.     

Comparative assessment of Vibrio virulence in marine fish larvae

Vibrionaceae infections are a major obstacle for marine larviculture; however, little is known about virulence differences of Vibrio strains. The virulence of Vibrio strains, mostly isolated from vibriosis outbreaks in farmed fish, was tested in larval challenge trials with cod (Gadus morhua), turbot (Scophthalmus maximus) and halibut (Hippoglossus hippoglossus) using a multiwell dish assays with single‐egg/larvae cultures. The strains differed significantly in virulence as some caused a high mortality of larva reaching 100% mortality after a few days, while others had no or only marginal effects on survival. Some Vibrio strains were pathogenic in all of the larva species, while some caused disease only in one of the species. Twenty‐nine of the Vibrio anguillarum strains increased the mortality of larvae from at least one fish species; however, pathogenicity of the strains differed markedly. Other Vibrio species had no or less pronounced effects on larval mortalities. Iron uptake has been related to V. anguillarum virulence; however, the presence or absence of the plasmid pJM1 encoding anguibactin did not correlate with virulence. The genomes of V. anguillarum were compared (D. Castillo, P.W. D'Alvise, M. Middelboe & L. Gram, unpublished data) and most of the high‐virulent strains had acquired virulence genes from other pathogenic Vibrio.     

N and P budgets of Haliotis discus hanai,Apostichopus japonicas,and Sebastes schlegeli in a polyculture system

The viability of placing abalones (Haliotis discus hannai), sea cucumbers (Apostichopus japonicas) and rockfish (Sebastes schlegeli) in a polyculture system, the effect of this mixed species group on the system's nitrogen (N) and phosphorous (P) budgets, and the growth and food intake of the organisms in the system were examined using a recirculating aquaculture system. Four replicates were set up for each of three treatment groups (abalone only (C), abalone‐sea cucumber (AS) and abalone‐sea cucumber‐rockfish (ASF)) with an experimental period of 60 d. Compared with the C group, in the AS group the abalone survival rate and specific growth rate (SGR) of body weight increased and the harvested abalones from the polyculture system became the main source of N and P output of the polyculture system. However, the N and P output in the water layer did not differ significantly from that in the C group (p > 0.05), and the N utilization rate was significantly higher than that in the C and ASF groups (p < 0.05). Compared with the AS group, in the ASF treatment the SGR of body weight as well as the protease and amylase activities of sea cucumbers were significantly higher (p < 0.05), the water layer and faeces became the main sources of N and P output in the system. These results showed that the AS polyculture mode significantly improved the N and P utilization rates in the system and led to increased aquaculture production.     

病毒感染过程中青鳉irf2基因过表达的双面性

为了探究干扰素调节因子2 (interferon regulatory factor,IRF2)如何通过调控干扰素(IFN)表达影响鱼类的免疫,实验从青鳉中克隆了irf2 (Olirf2),发现该基因在青鳉各个组织中均有表达;将构建的真核表达载体pTol2/CMV-IRF2/IE1-pr转染到胖头鱥肌肉细胞系(FHM)后,发现瞬时过表达Olirf2能够显著促进鲤春病毒血症病毒(spring viremia of carp virus,SVCV)的复制,并抑制抗病毒相关基因mx1、ifn和irf3的表达。进一步通过双荧光素报告系统发现,Olirf2能够显著抑制NF-κB和ISRE的活性,说明Olirf2可能通过抑制细胞的天然免疫应答进而促进病毒的增殖。然而持续过表达Olirf2则增强了细胞的抗病毒能力,同时促进干扰素相关基因mx1、ifn和irf3的表达。因此,Olirf2基于表达的持续时间不同而具有抗病毒或者促病毒的双面效果。实验通过研究Olirf2在抗病毒信号通路中发挥的作用,为通过基因编辑或者转基因手段来构建抗病毒的鱼类提供了理论基础。     

患腹水病牙鲆病原菌分离、鉴定及病原菌的特性

为确定引起河北北戴河地区养殖牙鲆患腹水病死亡的病原,本实验从患腹水病牙鲆体内分离到3株优势菌,对分离株进行生理生化鉴定和16S rRNA序列比对来确定分离株的生物学地位,进一步通过毒力基因(toxR、vhhA、vhhB)鉴定及组织病理学分析其病理特征,并通过人工回感实验分析分离株毒性。结果显示,通过生理生化实验及16S rRNA序列比对,确定此次从患病牙鲆中分离到的3株菌株均为哈维氏弧菌;经鉴定,3株分离株毒力基因(toxR、vhhA、vhhB)结果均为阳性,病理组织切片结果显示,该分离株对牙鲆多器官(肠、肾脏、脾脏和肝脏)均可造成不同程度的病理损伤,呈全身性感染;人工回感实验显示,菌株BDHYPFS-Y1G对牙鲆的半致死浓度为LD50=5.88×106 CFU/mL,低于自然状态毒性;药敏实验表明,3株分离株均对呋喃妥因高度敏感。本实验确认了此次牙鲆腹水病的病原菌,并初步研究了病原菌致病性以及药物敏感性,以期为牙鲆工厂化养殖疫病防控提供科学依据。     

Effects of addition of Navicula sp. (diatom) in different densities to postlarvae of shrimp Litopenaeus vannamei reared in a BFT system: Growth,survival, productivity and fatty acid profile

The objective of this study was to evaluate the effect of the addition of Navicula sp. on the growth and fatty acids profile of Litopenaeus vannamei postlarvae in a biofloc system (BFT). Four treatments were used: BFT; BFT 2.5N (addition of 2.5 × 10⁴ cells/ml of Navicula sp.); BFT 5N (addition of 5 × 10⁴ cells/ml of Navicula sp.) and BFT 10N (addition of 10 × 10⁴ cells/ml of Navicula sp.), all in triplicate. The shrimp (1 ± 0.01 mg) were stocked at a density of 3,000 postlarvae/m³ and fed with commercial feed. The diatom was added every 10 days, and at the end of 42 days, shrimp performance, water quality and proximal composition were evaluated. The BFT 5N and BFT 10N treatments had higher performance values, highlighting the values of productivity (2.30 and 2.42 kg/m³) and specific growth rate (15.92 and 16.08%/day), which were higher than the other treatments. In addition, the highest levels of fatty acids were observed in treatments with diatom (BFT 5N and BFT 10N), indicating the benefits of Navicula sp. on growth enhancement and fatty acid content of L. vannamei postlarvae grown in biofloc systems.