SNP discovery and gene annotation in the surf clam Mesodesma donacium

The main objective of this research was to identify single‐nucleotide polymorphisms (SNPs) from an Expressed Sequences Tags (EST) data set generated by 454 pyrosequencing in the soft clam Mesodesma donacium. A total of 180 159 ESTs were yielded from a M. donacium cDNA library. De novo assembly was performed using stringent calling parameters, producing 10 178 contigs and 41 765 singletons. Here, a total of 2594 SNPs were discovered related to 613 consensus sequences, achieving a frequency of 1 SNPs per 260 bp. SNP variants showed that A/G, A/T and C/T were the most abundant among the identified polymorphisms. We validated a total of 12 SNPs loci by HRMA for annotated genes such as heat shock protein‐70 and the translation elongation factor 1‐alpha. The Gene Ontology analysis regarding molecular function level revealed that sequences with SNPs were mainly classified to protein and nucleotide binding, as well hydrolase activity, ion binding and oxidoreductase activity. Further, biological processes like cellular and metabolic process, biogenesis, localization and biological regulation were highly annotated. The most expressed genes were related to the mitochondrial electron transport chain, senescence‐associated protein, ubiquitin and actin. Interestingly, some relevant genes related to immune response and biomineralization showed a high abundance, such as tumor necrosis factor (TNF)‐alpha‐receptor‐like protein, serine protease inhibitor, heat shock protein, aragonite‐binding protein and ferritin. This study contributes to relevant genes associated with functional polymorphisms and gives an overview for future genetic investigations.     

The role of polychaete Nereis diversicolor in bioremediation of wastewater and its growth performance and fatty acid composition in an integrated culture system with Huso huso (Linnaeus, 1758)

This study was undertaken to evaluate the role of polychaete Nereis diversicolor in bioremediation of waste water and its growth performance and fatty acid composition in an integrated culture system with great sturgeon, Huso huso. Three treatments consisting of N. diversicolor fed with H. huso waste (FNW), N. diversicolor fed with fish feed waste (NW), and fish waste without the worm (FW) were considered at water temperature of 23°C for 8 weeks. The obtained results demonstrated that N. diversicolor in the flow‐through system could grow via feeding with the fish waste water. The pure production and survival rate of harvested Nereis in NW treatment were significantly higher than those of FNW treatment (p < .05). However, no significant difference was observed in specific growth rate and weight gain between these two treatments (p > .05). The highest removal efficiency of waste water including total nitrogen (56%), total phosphorus (53%), NO₂‐N (91%), NH₃‐N (35%)_, PO₄‐P (47%), BOD₅ (60%) were seen in FNW treatment. Also, the highest additional efficiency of NO₃‐N occurred in FW (37%) treatment. Certain fatty acids specifically 20:5 ω3 (eicosapentaenoic acid [EPA]) and 22:6 ω6 (docosahexaenoic acid [DHA]) were also abundant in Nereis, and analysis revealed some differences due to the diet. These results demonstrated that the promotion of growth by cultured Nereis can enhance the decomposition rate of organic matter in enriched sediment and minimize negative effects in fish farms. These results also suggest that the use of N. diversicolor is an excellent potential candidate for an integrated aquaculture and nutrient recycling including the removal of organic wastes.     

Establishment of a continuous cell line from female half‐smooth tongue sole (Cynoglossus semilaevis)

A new cell line (TSHC) derived from heart tissues was established from female half‐smooth tongue sole (Cynoglossus semilaevis), an economically important marine fish species in China. The cell line had been subcultured for more than 30 times over a period of 200 days. The cell line was optimally maintained at 24°C in minimum essential medium (MEM) medium containing foetal bovine serum (FBS), 2‐mercaptoethanol (2‐Me), sodium pyruvate, basic fibroblast growth factor (bFGF) and antibiotics. The TSHC cells were mostly composed of fibroblast‐like cells. Chromosome analysis revealed that the TSHC cell line had a normal diploid karyotype with 2n = 42, containing the heterogametic W chromosome. The TSHC cell line was susceptible to infection by flounder Lymphocystis disease virus (LCDV). Although an atypical cytopathic effect and only few of virus particles in the cytoplasm was observed, it provides a research material on the cell–pathogen interaction research about the viral infection of non‐host species.     

Age dependency of nervous necrosis virus infection in barramundi Lates calcarifer (Bloch)

Age‐dependent susceptibility to nervous necrosis virus (NNV) was demonstrated for barramundi (Lates calcarifer). The experiment used juvenile barramundi produced from a single spawning that were challenged consecutively by immersion with a redspotted grouper nervous necrosis virus (RGNNV) isolate. The dose and environmental conditions (35 ppt salinity and 30 °C) were constant. Fish and water were sampled longitudinally for histopathology and RT‐qPCR analysis to examine the evolution of the disease, virus replication, immune response and release of virus into water. Viral nervous necrosis (VNN) disease occurred in barramundi challenged at 3 and 4 weeks of age while fish challenged at 5, 7 and 9 weeks of age developed subclinical infection. Replication of NNV occurred faster and the concentration of virus reached higher concentrations in the younger fish with clinical disease. Virus isolation and qPCR tests indicated that infectious NNV was released from carcasses into water when fish were affected with clinical disease but not when NNV infection was subclinical. Based on these observations, we consider that carcasses from clinically infected fish have a potentially important role in the horizontal transmission of NNV, and barramundi juveniles should be protected from exposure to NNV until they are 5 weeks of age and reach the disease resistance threshold.     

Ultrastructural morphogenesis of salmonid alphavirus 1

Studies on the ultrastructural morphogenesis of viruses give an insight into how the host cell mechanisms are utilized for new virion synthesis. A time course examining salmonid alphavirus 1 (SAV 1) assembly was performed by culturing the virus on Chinook salmon embryo cells (CHSE‐214). Different stages of viral replication were observed under electron microscopy. Virus‐like particles were observed inside membrane‐bound vesicles as early as 1 h following contact of the virus with the cells. Membrane‐dependent replication complexes were observed in the cytoplasm of the cells, with spherules found at the periphery of late endosome‐like vacuoles. The use of intracellular membranes for RNA replication is similar to other positive‐sense single‐stranded RNA (+ssRNA) viruses. The number of Golgi apparatus and associated vacuoles characterized by ‘fuzzy’‐coated membranes was greater in virus‐infected cells. The mature enveloped virions started to bud out from the cells at approximately 24 h post‐infection. These observations suggest that the pathway used by SAV 1 for the generation of new virus particles in vitro is comparable to viral replication observed with mammalian alphaviruses but with some interesting differences.     

Analysis of viral load between different tissues and rate of progression of white spot syndrome virus (WSSV) in Penaeus monodon

At present the most common and most devastating disease of shrimp is caused by the white spot syndrome virus (WSSV), which has spread throughout the world mainly by different species of crustaceans carrying the virus. After experimental injection of Penaeus monodon with a known copy number of WSSV in the abdominal muscle, the rate of viral progression in different tissues at 12, 24, 36 and 48 hpi (hours post infection) was assessed using quantitative real‐time PCR. At 12 hpi the viral load was highest in haemocytes followed by pleopod, muscle and gills whereas at 48 hpi, the gills, the main target of WSSV, showed the highest viral load followed by pleopod, muscle and haemocytes. Viral copy number in the haemocytes was the lowest beyond 12 hpi indicating a remarkable reduction in the rate of viral replication in haemocytes compared with other tissues. The viral load in haemocytes, though increased again beyond 36 hpi, never surpassed the load in the other tissues. The real‐time PCR assay with its high sensitivity and wide dynamic range make it ideal for detecting low‐level WSSV infections that can occur in apparently healthy P. monodon.     

Effect of chemical and physical treatments on the inactivation of Macrobrachium rosenbergii nodavirus and extra small virus

White tail disease (WTD) is found to cause immense economic losses in hatcheries, with mortalities often reaching 100% within 4 or 5 days. The pathogenic agents have been identified as Macrobrachium rosenbergii nodavirus (MrNV) associated with extra small virus (XSV), which are 27 and 15 nm in diameter respectively. The effects of some chemical disinfectants hydrogen ions (pH), heat and ultraviolet (UV) irradiation on the inactivation of MrNV and XSV were investigated. The viral inoculum exposed to UV irradiation for a period of 5 min and more was totally inactivated and failed to cause mortality in postlarvae of prawn. The viruses were totally inactivated by this high pH (8.5, 9 and 10). The viral suspension treated with sodium hypochloride, formalin, Benzalkonium chloride and Benzethonium chloride at the concentration of 200 ppm caused 100% mortality in postlarvae of prawn. Iodine was found to be effective to inactivate MrNV and XSV at the concentration of 100 ppm or more, whereas the viral suspension treated with iodine at the concentration of 50 ppm or less caused mortality in postlarvae. The infected postlarvae in treated and positive control groups showed positive by RT‐PCR for these viruses.     

Differential viral haemorrhagic septicaemia virus genotype IVb infection in fin fibroblast and epithelial cell lines from walleye,Sander vitreus (Mitchill), at cold temperatures

A cell line, WE‐cfin11e, with an epithelial‐like morphology was developed from a caudal fin of walleye, Sander vitreus (Mitchill), characterized as distinct from the established walleye caudal fin fibroblast‐like cell line, WE‐cfin11f, and compared with WE‐cfin11f for susceptibility to VHSV IVb. Immunocytochemistry and confocal microscopy were used to localize the intermediate filament protein, vimentin, the tight junction protein, zonula occludens‐1 (ZO‐1), the extracellular matrix protein, collagen I, and the viral protein, G. Although both cell lines contained vimentin, only WE‐cfin11e stained for ZO‐1 and only WE‐cfin11f stained for collagen I. Ascorbic acid increased the accumulation of collagen I and caused the appearance of collagen fibres only in WE‐cfin11f cultures. At 14 °C, both cell lines produced VHSV IVb, but the infection developed more rapidly in WE‐cfin11f. At 4 °C, both cell lines became infected with VHSV IVb as judged by the expression of viral proteins, N and G, but only WE‐cfin11f produced virus. The results suggest that cold temperatures can modulate viral tropism.     

Mortality from scale drop disease in farmed Lates calcarifer in Southeast Asia

In Southeast Asia, a new disease called scale drop disease (SDD) caused by a novel Megalocytivirus (SDDV) has emerged in farmed Asian sea bass (Lates calcarifer) in Singapore, Malaysia and Indonesia. We received samples from an Eastern Thai province that also showed gross signs of SDD (loss of scales). Clinical samples of 0.2–1.1 kg L. calcarifer collected between 2016 and 2018 were examined for evidence of SDDV infection. Histopathology was similar to that in the first report of SDDV from Singapore including necrosis, inflammation and nuclear pyknosis and karyorrhexis in the multiple organs. Intracytoplasmic inclusion bodies were also observed in the muscle tissue. In a density‐gradient fraction from muscle extracts, TEM revealed enveloped, hexagonal megalocytiviral‐like particles (~100–180 nm). By PCR using primers derived from the Singaporean SDDV genome sequence, four different genes were amplified and sequenced from the Thai isolate revealing 98.7%–99.9% identity between the two isolates. Since viral inclusions were rarely observed, clinical signs and histopathology could not be used to easily distinguish between SDD caused by bacteria or SDDV. We therefore recommend that PCR screening be used to monitor broodstock, fry and grow‐out fish to estimate the current impact of SDDV in Southeast Asia and to prevent its spread.     

Natural co‐infection of cultured Nile tilapia Oreochromis niloticus with Aeromonas hydrophila and Gyrodactylus cichlidarum experiencing high mortality during summer

Aeromonas hydrophila and Gyrodactylus cichlidarum are common pathogens that induce significant economic losses in farm‐reared Nile tilapia. Nowadays, the sudden appearance of fish mortalities was exaggerated due to mixed and multiple infections. During summer 2016, mass mortality among earthen pond‐farmed Nile tilapia was reported. Clinico‐pathological, bacteriological and parasitological examinations have been demonstrated. As well, the water quality parameters were assessed. The clinical and histopathological findings of the moribund and recently dead fish were characterized by generalized septicaemic signs. The water quality parameters were significantly elevated over the permissible levels, whereas there was an elevation in nitrite (0.04 mg/L), un‐ionized ammonia (0.8 mg/L), hydrogen sulphide levels (153.1 mg/L) and organic matter content (3.79 mg/L). A. hydrophila was identified based on phenotypic characterization, API 20E features and the homology of 16S rRNA gene sequence analysis. In addition, PCR data confirmed the presence of aerolysin (aerA) and haemolysin (hly) genes in the identified A. hydrophila isolates. Gene sequencing and phylogenetic analysis based on 16S rRNA sequence confirmed that A. hydrophila H/A (accession No. MN726928) of the present study displayed 98%–99% identity with the 16S rRNA gene of A. hydrophila. Furthermore, the monogenetic trematode, G. cichlidarum was identified in the wet mounts from the skin and gills of the examined fish with a high infestation rate. In this context, it was reported that the synergistic co‐infection of A. hydrophila and G. cichlidarum with deteriorated water quality parameters could induce exaggerated fish mortalities during hot weather.     

Identification of RAPD‐SCAR marker linked to white spot syndrome virus resistance in populations of giant black tiger shrimp,Penaeus monodon Fabricius

White spot disease (WSD) caused by white spot syndrome virus (WSSV) creates severe epizootics in shrimp aquaculture industry worldwide. Despite several efforts, no such permanent remedy was yet developed. Selective breeding using DNA markers would be a cost‐effective strategy for long‐term solution of this problem. In the present investigation, out of 30 random primers, only one primer produced a statistically significant (P < 0.01) randomly amplified polymorphic DNA (RAPD) marker of 502 bp, which provided a good discrimination between disease resistant and disease susceptible populations of Penaeus monodon from three geographical locations along the East coast of India. Because RAPD markers are dominant, a sequence characterized amplified region (SCAR) marker was developed by cloning and sequencing of 502 bp RAPD fragment, which generates a single 457 bp DNA fragment after PCR amplification only in the disease resistant shrimps. Challenge experiment was also conducted to validate this 457 bp SCAR marker, and the results suggested that the WSSV loads were 2.25 × 10³ fold higher in disease susceptible than that in disease resistant shrimps using real‐time PCR. Therefore, this 457 bp DNA SCAR marker will be very valuable towards the development of disease‐free shrimp aquaculture industry.     

Effects of rearing density on growth,fatty acid profile and bioremediation ability of polychaete Nereis diversicolor in an integrated aquaculture system with rainbow trout (Oncorhynchus mykiss)

The objective of this study was to assess the effects of rearing density on the growth and fatty acid profile of Nereis diversicolor and on its capability to bioremediate wastewater in an integrated culture system with rainbow trout (Oncorhynchus mykiss). To this end, a batch of juvenile N. diversicolor (0.03 ± 0.01 mg) was assigned into four different densities (T₁ = 250, T₂ = 500, T₃ = 1,000, T₄ = 2,000, besides T₀ = with no worm) in three replicates. The worm groups were fed with solid waste that was supplied from tanks containing rainbow trout (107.17 ± 13.5 g; 1.39 ± 0.18 kg/m³). During the experiment (60 days), the water temperature was 17.71 ± 0.6°C. The results revealed that SR%, SGR% and WG% of N. diversicolor in T₁ were significantly higher than those of T₂, T₃ and T₄. Both FER rate and biomass gain in T₄ were significantly higher than those in the other groups. The highest removal rate of NO₂‐N (73.72%), NH₃‐N (65.70%), PO₄‐P (59.32%), BOD₅ (69.60%) and TSS (82.33%) were observed in T₄. The major fatty acids presents were palmitic acid, oleic acid, linoleic acid, stearic acid and alpha‐linolenic acid in all worm‐treated groups, with no difference observed in the concentration of these fatty acids among them. Taken together, these findings suggest that organic wastes from rainbow trout farms could be recycled to achieve a sustainable aquaculture goal, and demonstrate that a high percentage of fatty acids in fish feed is not absorbed by rainbow trout but is done by N. diversicolor.     

Ultrastructural and biomolecular detection of Rickettsiales‐like organisms in tissues of rainbow trout with Red Mark Syndrome

Red mark syndrome (RMS) and US strawberry disease (US SD) are skin disorders affecting rainbow trout farmed in Europe and USA. The disease etiology has not yet been established. In spite of specific investigations, identifying Rickettsia‐like organism (RLO)‐ and Midichloria‐like organism (MLO)‐related DNA in affected individuals, these pathogens have never been observed. We performed histological, ultrastructural and biomolecular analysis on skin and spleen samples of trout with RMS. Examination by TEM revealed the presence of intracytoplasmic microorganisms resembling Rickettsiales within macrophages, fibroblasts and erythrocytes. The microorganisms were oval or short rod shaped (400–800 nm in length and 100–200 nm in width) and often showed a cell wall similar to Gram‐negative bacteria. PCR analysis for Rickettsiales supported these findings: 53% of affected trout were positive by both PCR and TEM The primers RiFCfw‐RiFCrev were used to anneal both the RLO 16S DNA sequence and the MLO 16S DNA sequence. For this reason, and in agreement with previous studies confirming the presence of Rickettsiales‐related DNA in trout with RMS, we assume that TEM detected microorganisms morphologically consistent with bacteria belonging to Rickettsiales order and could be considered as possible causative agents of RMS.     

Immersion challenge with low and highly virulent infectious salmon anaemia virus reveals different pathogenesis in Atlantic salmon,Salmo salar L.

The salmonid orthomyxovirus infectious salmon anaemia virus (ISAV) causes disease of varying severity in farmed Atlantic salmon, Salmo salar L. Field observations suggest that host factors, the environment and differences between ISAV strains attribute to the large variation in disease progression. Variation in host mortality and dissemination of ISAV isolates with high and low virulence (based on a previously published injection challenge) were investigated using immersion challenge. Virus dissemination was determined using real‐time PCR and immunohistochemistry in several organs, including blood. Surprisingly, the low virulent virus (LVI) replicated and produced nucleoprotein at earlier time points post‐infection compared to the virus of high virulence (HVI). This was particularly noticeable in the gills as indicated by different viral load profiles. However, the HVI reached a higher maximum viral load in all tested organs and full blood. This was associated with a higher mortality of 100% as compared to 20% in the LVI group by day 23 post‐infection. Immersion challenge represented a more natural infection method and suggested that specific entry routes into the fish may be of key importance between ISAV strains. The results suggest that a difference in virulence is important for variations in virus dissemination and pathogenesis (disease development).     

Identification of sex‐specific markers and heterogametic XX/XY sex determination system by 2b‐RAD sequencing in redtail catfish (Mystus wyckioides)

Sex‐specific markers provide significant molecular basis for sex control breeding biotechnology to produce all‐male or all‐female fish in commercial breeding. Redtail catfish (Mystus wyckioides), one of the commercial bagrid catfishes distributed in Southeast Asian, which have a long sexual maturation period that can last 3–5 years and males have apparent growth advantage over females, but its sex determination system remains unknown. In this study, we first applied 2b‐RAD‐seq approach to identify three male‐specific 2b‐RAD‐tags and one male heterogametic SNP locus and validated by blast to the genome survey sequences and PCR amplification in both wild and breeding populations. To get longer sex‐specific region, we performed genome walking and obtained a 4,630 bp of Y‐specific sequence and 4,581 bp of X‐specific sequence from the 2b‐RAD‐tag ref189950 with 92.19% nucleotide identity between them. And 9,923 bp/3,935 bp of Y‐specific sequences and 8,491 bp/5,172 bp of X‐specific sequences were also identified with 77.49% and 57.07% nucleotide identity in ref208528 and ref210837, respectively. Subsequently, three different kinds of sex‐specific primers with different length products were designed based on the detected highly sex differentiated regions and could be used to distinguish males and females both in wild and artificially bred populations. What is more, the X‐specific fragment was discovered to produce the dosage effect association in females and in males. The data suggest that male heterogametic XX/XY sex determination system should exist in the redtail catfish. More significantly, the sex‐specific markers are of great value to protect wild resources and improve the efficiency of all‐male breeding practices for aquaculture in the redtail catfish.     

Effects of crude extracellular protein and crude intracellular polysaccharides of Vibrio alginolyticus on the growth,energy metabolism regulation and WSSV resistance of Litopenaeus vannamei

This study aimed to investigate the effects of adding crude extracts of the extracellular protein (Ex‐Pro) and intracellular polysaccharides (In‐Poly) of Vibrio alginolyticus to diets, at a dosage of 10 g/kg, on the growth, energy metabolism and WSSV (White Spot Syndrome Virus) resistance of Litopenaeus vannamei (initial weight, 0.88 ± 0.04 g/shrimp). Growth and survival rate were not significantly affected by the crude extracts (p > 0.05). Shrimp fed Ex‐Pro crude extracts showed higher succinate dehydrogenase (SDH) activity in the hepatopancreas and muscle but lower lactate dehydrogenase (LDH) activity in the muscle (p < 0.05). The activities of hexokinase (HK), pyruvate kinase (PK), LDH and SDH in the hepatopancreas and the activity of SDH in the muscle were significantly increased by feeding In‐Poly crude extracts (p < 0.05). In contrast, the content of fatty acid synthase (FAS) in the muscle was significantly reduced by the crude extracts (p < 0.05). The contents of glucose and triglyceride and the activity of the electron transport system in the hepatopancreas were significantly increased by the crude extracts (p < 0.05), and the WSSV resistance of the shrimp was increased. These results indicated that the Ex‐Pro and In‐Poly crude extracts of V. alginolyticus could affect energy metabolism, and there was a correlation between WSSV resistance and energy metabolism in L. vannamei.