Ontogeny of the digestive tract in mud loach Misgurnus anguillicaudatus larvae

This study on histological and mucous histochemistry characteristics of the digestive system of loach (Misgurnus anguillicaudatus) was carried out from hatching (0 day after hatching, DAH) until 45 DAH. The peculiar development of both digestion processes and air‐breathing functions of the intestine was revealed. At 3 DAH, both the mouth and anus opened along with the first feed. At 4 DAH, lipid vacuoles appeared in the anterior part of the intestine and at 5 DAH the acidophilic supranuclear vacuoles appeared in the posterior part of the intestine. Mucous cells occurred in the buccopharynx and oesophagus after mouth opening and grew both in number and size as larvae grew. At 15 DAH, blood capillaries were found in the posterior part of the intestine. At 20 DAH, as a valve appeared in the intestine, the whole intestine could be divided into anterior, mid and posterior parts. With large numbers of blood capillaries and a very thin wall, a gas–blood barrier formed in the posterior intestine, which indicated that the dual roles of intestine were formed. These results suggested that the air‐breathing function of the digestive tract formed from 15 to 20 DAH, which is a critical period for loach larvae.     

Effects of vitamin E supplementation on growth,lipid peroxidation and fatty acid composition of Dojo loach (Misgurnus anguillicaudatus Cantor) fingerlings

A high mortality of cultured loach during their early development has constrained loach aquaculture industry in China. The objective of this study was to evaluate the effects of dietary vitamin E (VE) supplementation on growth performance, lipid peroxidation and fatty acid composition of loach Misgurnus anguillicaudatus fingerlings. Six test diets supplemented with 0 (0E), 50 (50E), 100 (100E), 150 (150E), 200 (200E) and 500 mg kg^?1 (500E) of dl‐α‐tocopheryl acetate were fed loach fingerlings (mean initial weight 24.0 ± 0.2 mg) in triplicate for 40 days. Results showed that the fish fed diet 100E and 200E had significantly higher body weight gains and specific growth ratios and significantly lower feed conversion ratios compared with fish fed diet 0E. With the increase in dietary VE level, whole‐body VE content was significantly increased (except 500E) and thiobarbituric acid reactive substances value was significantly reduced. Increasing dietary VE level increased the percentages of 20:4n‐6 and 20:5n‐3 in neutral lipid fraction, but decreased the percentages of 20:4n‐6 and 20:5n‐3 in polar lipid fraction. In conclusion, VE requirement of loach fingerlings for optimum growth and functionally preventing lipid peroxidation was 136.1 mg kg^?1 of dry diet under the present experimental conditions.     

Effects of dietary protein and lipid levels on growth performance,fatty acid composition and antioxidant‐related gene expressions in juvenile loach Misgurnus anguillicaudatus

Dojo loach (Misgurnus anguillicaudatus) is one of the most important cultured freshwater fish in several East Asian countries. However, a little information is available in its nutritional requirements. Thus, this study was conducted to evaluate the effects of feeding varying levels of dietary protein and lipid on growth, fatty acid composition and antioxidant‐related gene expressions in juvenile loach. Six practical diets at three levels of protein (30%, 40% and 50%) and two levels of lipid (6% and 12%) were fed to loach juveniles (initial weight 0.40 g) in triplicated groups (20 fish per replicated) for a period of 8 weeks. Results showed that regardless of lipid level, body weight gain of fish was significantly increased with incremental dietary protein level. Meanwhile, feed conversion ratio was significantly decreased by dietary protein levels, and the lowest value was observed in fish fed dietary protein levels of 50%, regardless of dietary lipid level. Moreover, the percentage of 22:6n‐3 in viscera was significantly increased by different protein levels. The expression level of catalase was significantly increased with incremental dietary protein level with both lipid levels. Meanwhile, the expression level of hepatic nuclear factor erythroid 2‐related factor 2 (Nrf2) was downregulated with incremental dietary protein level with 6% of lipid level, but the expression was upregulated with incremental dietary protein level with 12% of lipid level. In conclusion, these data suggested that 6% lipid and 50% protein in diet was optimal for loach during early development stage.     

Effects of dietary highly unsaturated fatty acid levels on growth,fatty acid profiles,antioxidant activities,mucus immune responses and hepatic lipid metabolism related gene expressions in loach (Misgurnus anguillicaudatus) juveniles

A 9‐week rearing trail was conducted to examine the effects of different dietary highly unsaturated fatty acid (HUFA) levels on the growth performance, fatty acid profiles, antioxidant activities, mucus immune responses and lipid metabolism‐related gene expressions of loach (Misgurnus anguillicaudatus) juveniles. Five test diets supplemented with 0%, 0.32%, 0.64%, 0.96% and 1.28% HUFA were used here. The loaches fed no HUFA diets had the lowest specific growth rate and survival rate. The loaches fed 0.32% HUFA diets had the lowest feed conversion rate and while no significant differences were found among the other four diet groups. Contents of hepatic eicosapentaenoic acid, docosahexaenoic acid and total polyunsaturated fatty acids were significantly increased with incremental dietary HUFA levels. Activities of hepatic superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in 0% HUFA group were lowest, while malondialdehyde was just the opposite. The lysozyme activity was significantly increased with incremental dietary HUFA levels. However, the activities of alkaline phosphatase and acid phosphatase reached a peak in 0.32% HUFA group. Loaches fed diets with HUFA significantly up‐regulated expressions of SOD, GPx, CAT and two lipid metabolism related genes. In conclusion, the optimal dietary HUFA level for loach juveniles was 0.64%–0.96%.     

Effect of farming patterns on growth,antioxidant activity and nonspecific immunity of loach,Paramisgurnus dabryanus

This study was carried out to compare growth, intestinal enzymes activity, antioxidation and nonspecific immunity of loach between farmed and imitative ecological farming loach, Misgurnus anguillicaudatus. Loach from farmed (average body weight 15.03 ± 0.36 g) and imitative ecological farming (average body weight 12.44 ± 0.62 g) population were used to analyse growth, whole-body composition, intestinal enzymes, antioxidant enzymes activity and resistance to bacteria. The results showed specific growth rate, average daily gain and protease, lipase, amylase and γ-GT activities in intestine and hepatopancreas of farmed loach were significantly higher (p < 0.05) than of imitative ecological farming loach. However, total protein, lysozyme and albumin contents and superoxide dismutase, catalase and glutathione peroxidase activities of imitative ecological farming loach were significantly higher (p < 0.05) than of farmed loach. Moreover, resistance to Aeromonas hydrophila of imitative ecological farming loach was higher than of farmed loach (p < 0.05). Taken together, ecological farming loach possesses higher antioxidant capacity and resistance to bacteria than farmed loach.     

Winter kill in intensively stocked channel catfish (Ictalurus punctatus): Coinfection with Aeromonas veronii,Streptococcus parauberis and Shewanella putrefaciens

Unusual persistent natural mortality occurred in a floating in‐pond raceway system intensively stocked with channel and hybrid catfish beginning in early November 2016 up until March 2017. The temperature during the period of outbreak ranged from 7.2 to 23.7°C. Gross examination of freshly dead and moribund fish revealed pale gills, slight abdominal distension and swollen inflamed vents. Comprehensive necropsy of 20 fish demonstrated vast amounts of bloody ascitic fluid in the coelomic cavity, visceral congestion, splenomegaly and pale friable livers but macroscopically normal kidneys, suggesting systemic bacterial infection. Bacterial cultures were initiated from skin, gills and major internal organs. Following incubation, a mixture of three bacterial colony phenotypes was observed on agar plates. Presumptive biochemical characterization of the isolates followed by 16S‐rRNA sequence analysis resulted in the identification of Aeromonas veronii, Streptococcus parauberis and Shewanella putrefaciens. Channel catfish juveniles were experimentally infected with the recovered isolates to fulfil Koch's postulates. Moreover, an antibiogram was used to evaluate the susceptibility of the isolates to antimicrobial drugs approved for use in aquaculture. Aquaflor was used successfully for treatment. Here, we report bacterial coinfection lead by A. veronii and the first identification of S. parauberis and S. putrefaciens from cultured catfish in North America.     

Ploidy and growth performance of hybrid progeny between tetraploid Misgurnus anguillicaudatus and Paramisgurnus dabryanus

Experimental crossings were made between tetraploid loach (Misgurnus anguillicaudatus, T for short) and diploid large‐scale loach (Paramisgurnus dabryanus, P for short), resulting in T×T, T×P and P×P crosses (female before male). Meanwhile, a part of T×P cross was cold shocked to inhibit second meiosis (T×P‐0 for short). Fertility rate, hatching rate and survival rate of T×T, T×P, P×P and T×P‐0 were measured. Flow cytometry and chromosome count were taken to identify ploidy statuses of the four crosses. Results showed that T×T, T×P and P×P were, respectively, tetraploidy (4n = 100), triploidy (3n = 74) and diplody (2n = 48), while 89.19 per cent of T×P‐0 of 12 months old were pentaploidy (5n = 124, T×P‐0‐5n for short) and the others were triploidy. Morphological data of the four crosses of 12 months old showed that morphological characteristics of T×P and T×P‐0‐5n loach were between those of their parents, while T×P‐0‐5n loach presented a smallest head length/body length. Total lengths of T×P and T×P‐0‐5n were significantly larger than those of T×T and P×P, showing hybrid vigour. This study will provide valuable information for production of polyploidy loach and breeding programmes of the two loaches and other closely related species.     

Growth parameters,innate immune response and resistance to Listonella (Vibrio) anguillarum of Dicentrarchus labrax fed carvacrol supplemented diets

The research was aimed to assess the effect of dietary carvacrol (0.025% and 0.05%) on sea bass (Dicentrarchus labrax) growth, immune response and resistance to Listonella anguillarum. Fish (69.2 ± 0.22 g) were fed the experimental diets for 9 weeks. Dietary carvacrol did not negatively affect fish survival, growth performance, feed intake and feed conversion ratio (P > 0.05) nor carcass yield and viscerosomatic, hepatosomatic and mesenteric fat index (P > 0.05). Serum and head kidney leucocytes were collected after 1, 4 and 8 weeks of feeding. Carvacrol significantly reduced serum proteins, immunoglobulins and lysozyme activity (P < 0.01) and moderately increased phagocytosis and pinocytosis of head kidney macrophages. The release of reactive oxygen species by leucocytes was reduced in carvacrol‐fed fish, even if significantly (P < 0.05) only in those fed 0.05% carvacrol for 1 week. Dietary carvacrol did not significantly affect the aspecific immune response, although a potential antioxidant activity might be speculated. Moreover, feeding carvacrol provided an appreciable resistance to a challenge with L. anguillarum, when a bacterial dose lower than the Lethal Dose₅₀ was used. Cumulative mortality in fish fed 0.025% carvacrol was significantly lower than that of untreated controls (75% Relative Per cent Survival).     

First report of Fusarium oxysporum species complex infection in zebrafish culturing system

Fusarium oxysporum species complex (FOSC) is a highly diverse fungus. Recently, F. oxysporum infection was identified from zebrafish (Danio rerio) culturing system in Korea. Initially, a rapid whitish smudge was appeared in the water with the fungal blooming on walls of fish tanks. Microscopic studies were conducted on fungal hyphae, colony pigmentation and chlamydospore formation and the presence of macro‐ and microspores confirmed that the isolated fungus as F. oxysporum. Furthermore, isolated F. oxysporum was confirmed by internal transcribed spacer sequencing which matched (100%) to nine F. oxysporum sequences available in GenBank. Experimental hypodermic injection of F. oxysporum into adult zebrafish showed the development of fungal mycelium and pathogenicity similar to signs observed. Histopathologic results revealed a presence of F. oxysporum hyphae in zebrafish muscle. Fusarium oxysporum growth was increased with sea salt in a concentration‐dependent manner. Antifungal susceptibility results revealed that F. oxysporum is resistant to copper sulphate (up to 200 μg mL^?1) and sensitive to nystatin (up to 40 μg mL^?1). This is the first report of FOSC from zebrafish culture system, suggesting it appears as an emerging pathogen, thus posing a significant risk on zebrafish facilities in the world.     

Changes in the gut microbiome of the Chinese mitten crab (Eriocheir sinensis) in response to White spot syndrome virus (WSSV) infection

Intestinal microorganisms play important roles in maintaining host health, but their functions in aquatic animal hosts have yet to be fully elucidated. The Chinese mitten crab, Eriocheir sinensis, is one such example. We attempted to identify the shift of gut microbiota that occurred in response to infection of white spot syndrome virus (WSSV), an emerging viral pathogen in the crab aquaculture industry. The microbiota may exert some control over aspects of the viral pathogenesis. We investigated the changes in composition and structure of the crab gut microbiome during various WSSV infection stages of 6 h post‐infection (hpi) and 48 hpi, using a 16S rRNA approach on the MiSeq Illumina sequencing platform. Four phyla (Firmicutes, Proteobacteria, Tenericutes and Bacteroidetes) were most dominant in the gut of E. sinensis regardless of the WSSV infection stages. However, further analysis revealed that over 12 bacterial phyla, 44 orders and 68 families were significantly different in abundance at various states of WSSV infection. Several intriguing aspects of E. sinensis gut bacteria that had not been previously reported were also uncovered, such as class Mollicutes was dominant here, but absent in crabs from Yangtze River estuary and Chongming Islands. Overall, this study provided the first evidence that changes in gut microbiome were closely associated with the severity of WSSV infection and that indicator taxa could be used to evaluate the crab health status.     

Assessment of the bacterial community diversity associated with the queen conch Strombus gigas (Linnaeus, 1758) from the Caribbean coast of Colombia using denaturing gradient gel electrophoresis and culturing

The biotic diversity of Strombus gigas has not been thoroughly studied despite the status of the queen conch as an important ecological resource. The bacteria associated with the conch influence their host in several ways, including through the metabolism of nutrients, protection against invasive bacteria and the regulation of the physical conditions. In this study, conventional microbiological methods and molecular tools such as denaturing gradient gel electrophoresis (DGGE) were used to assess the composition of the bacterial communities associated with the queen conch (S. gigas) in wild and captive habitats in Colombia. A genetic analysis of the bacterial communities revealed a high level of diversity based on the large number of bands detected using DGGE. In addition, differences in bacterial community structure were found between the conchs in captivity and the wild populations. The dominant phylogenetic affiliations of the bacteria, as determined using 16S rRNA gene sequencing, were grouped into four classes, namely, Betaproteobacteria (16%), Gammaproteobacteria (70%), Firmicutes (7%) and Actinobacteria (2%). These groups are related to host defence processes and the decomposition of organic matter. The 16S rDNA sequence analysis of the cultured bacteria and the resulting DGGE profiles are useful tools for characterizing the diversity of the bacteria associated with the analyzed conchs.     

Targeted species substitution in giant freshwater prawn trade revealed by genotyping

This study presents the molecular barcoding results of giant freshwater prawns and allied products collected from inland landing centres, markets and stores of Vembanad Lake (Kerala state, India) which is recognized as a natural abode for Macrobrachium rosenbergii. Prawns collected from landing centres of the lake could be easily identified as the above with their large size and morphological characters. There were certain ‘alien’ prawns and prawn products (headless shell on, peeled and deveined) traded in the markets and stores of this region as M. rosenbergii. Genotyping of all these samples using COI and 16S rRNA gene sequences confirmed the speciation of individuals from inland landing centres as M. rosenbergii, ‘alien’ prawns and certain prawn products as M. malcolmsonii. To ensure the same and to detect the presence of any other congeners of genus Macrobrachium inhabiting Vembanad Lake, additional homologous COI and 16S rRNA sequences available in NCBI were acquired and incorporated for molecular analyses. Results generated from NJ tree and genetic distance data confirmed the trade of non‐indigenous Macrobrachium species M. malcolmsonii in Kerala for the first time and its use as a species substituent for M. rosenbergii.     

Genotypic and phenotypic characterization of Edwardsiella isolates from different fish species and geographical areas in Asia: Implications for vaccine development

The genus Edwardsiella is one of the major causes of fish diseases globally. Herein, we examined 37 isolates from ten different fish species from India, South Korea and Taiwan to gain insight into their phenotypic and genotypic properties, of which 30 were characterized as E. tarda with phenotypic homology estimated at 85.71% based on API‐20E biochemical tests. Genotyping using 16S rRNA put all isolates together with E. anguillarum, E. hoshinae, E. tarda, E. piscicida and E. ictaluri reference strains in a monophyletic group. In contrast, the gyrB phylogenetic tree clearly separated E. ictaluri, E. tarda and E. hoshinae reference strains from our isolates and put our isolates into two groups with group I being homologous with the E. anguillarum reference strain while group II was homologous with the E. piscicida reference strain. Hence, our findings point to E. piscicida and E. anguillarum as species infecting different fish species in Asia. Homology of the ompW protein suggested that strains with broad protective coverage could be identified as vaccine candidates. This study underscores the importance of combining genotyping with phenotyping for valid species classification. In addition, it accentuates the importance of phylogenetic comparison of bacterial antigens for identification of potential vaccine candidates.     

Correlation between gut microbial diversity and flatulence of silver pomfret (Pampus argenteus) during ontogenesis

Gut microorganisms play an important role in host health and have susceptibility to diseases, undergoing a distinct transformation at early stages of development. This study aimed to determine the regulation of gut microorganism development from fertilized eggs to 150 DAH (days after hatching) and the differences between flatulent and healthy silver pomfret (Pampus argenteus). High‐throughput sequencing was used to analyze the diversity and relative abundance of microorganisms. PICRUSt was used to predict functional alterations of dominant microorganisms. The results showed that from all 42 samples, a total of 3,746,895 reads were obtained, representing 6,081 operational taxonomic units (OTUs). Top 3 phyla were Proteobacteria, Firmicutes and Bacteroidetes. From 0 to 32 DAH, the relative abundance of Proteobacteria continuously increased and peaked at 94.99%. After that, the abundance of Proteobacteria decreased, but Bacteroidetes and Firmicutes increased instead. Alpha diversity is the lowest at 32 DAH, the difference is significant compared with others, p < .05. Comparing flatulent fish with healthy fish, the former has more specific OTUs, higher diversity and abundance. There were four genera Ethanoligenens, Methanobacterium, Caproiciproducens and Leptolinea that only obtained from the flatulent samples, but almost none in the healthy samples. All of these four genera are directly or indirectly involved in gas metabolism. This study enriched the basic research of silver pomfret, for the first time, focused on flatulent individuals which would provide a theoretical basis for the subsequent breeding.     

Aeromonas hydrophila and Aeromonas veronii cause motile Aeromonas septicaemia in the cultured Chinese sucker,Myxocyprinus asiaticus

In August 2017, a serious disease causing high mortality occurred in a Myxocyprinus asiaticus aquaculture farm. According to necropsy findings, bacteriology experiments and phylogenetic analysis based on 16S rRNA, cpn60, gyrB and rpoB genes and concatenated alignment sequences (cpn60, gyrB and rpoB genes), two isolates, that is, BBAh1 and BBAv1, were identified as Aeromonas hydrophila and Aeromonas veronii respectively. Artificial infection experiments were carried out, showing that the BBAh1 and BBAv1 strains can cause similar symptoms and have LD50 values of 1.93×10⁵ and 8.77×10⁵ cfu/g respectively. In addition, some virulent genes coding for AerA, Alt, Ast, AscV, AexT, AspA, HlyA, OmpA, Lip and FlaA were detected in the two strains. Furthermore, BBAh1 and BBAv1 showed the same sensitivities to 28 antibiotics, some of which, such as cefotaxime, aztreonam, ceftriaxone and tetracycline, may be used to control the disease. However, the strains were also resistant to many antibiotics. These results provide a scientific reference for the diagnosis, prevention and treatment of motile Aeromonas septicaemia caused by A. hydrophila or A. veronii in cultured Chinese sucker.