Dynamic pathology and pathogen distribution of the yeast Metschnikowia bicuspidata in the Chinese mitten crab (Eriocheir sinensis).

In this study, the "milky disease" model of Eriocheir sinhensis was constructed via intramuscular injection with the pathogenic yeast Metschnikowia bicuspidata. The dynamic pathological changes of E. sinensis after injection were elucidated with two staining methods (haemotoxylin-eosin and alcian blue periodic acid-Schiff) and fluorescence in situ hybridization technology. Anatomical observation revealed three stages of the "milky disease": no clinical signs (1-4 days after infection), the appearance of signs of disease (5-7 days), and significant liquefaction (10 days). Histological observation also revealed three stages of the disease: yeast diffusion (1-2 days after infection), yeast slow development (3-4 days), and yeast rapid proliferation (5 days). And FISH technique was suitable for the early detection of infection with M. bicuspidata in E. sinensis. We found that M. bicuspidata spread to the whole body of the crab through the haemolymph and developed into fungal septicaemia. These results elucidate the systemic pathological characteristics of "milky disease" in E. sinensis and suggest the pathogenic mechanism of M. bicuspidata.

First case of Coxiella burnetii infection in Palaemonetes sinensis cultured in Liaoning, China.

The Palaemonetes sinensis aquaculture industry in Panjin City, Liaoning Province, China, experienced heavy losses in October 2018. Morbidity of cultured shrimp reached 50% and was characterized by cloudiness of muscle and the gradual spread of disease within the population. When the infection was mild, histopathological examinations revealed that the muscle cells contained a considerable number of microorganisms. In extreme cases, the structure of the hepatopancreatic glandular and muscle fiber was obscured or even vanished. Electron microscope observations revealed the presence of granular cytoplasmic inclusions in cells from hepatopancreas and muscle tissues. The 16S rDNA sequence of the intracytoplasmic organism was 94.7% identity to that of Coxiella burnetii. This is the first report of infection by C. burnetii in P. sinensis.

Metschnikowia bicuspidata isolated from milky diseased Eriocheir sinensis: Phenotypic and genetic characterization, antifungal susceptibility and challenge models.

A severe disease occurred in farmed Eriocheir sinensis characterized by milky liquid accumulation in the pectoral cavity, in the province of Liaoning, China, during October 2018-April 2019. Diseased crabs moved sluggishly, exhibited appetite loss and readily lost legs. Under the microscopic analysis, it was observed that the milky liquid contained a large number of yeastlike microorganisms (0.8-1.2 µm × 1.5-1.9 µm), which were also present in the muscle, hepatopancreas and gills. A dominant strain was isolated from the milky liquid and other tissues of diseased crabs. It grew on nutrient agar and formed 1- to 3-mm white opaque colonies, each with a protuberance in the centre. Besides, the results of TEM and SEM also demonstrate a typical multilateral budding model of the yeast clearly. We identified the strain, which we named 2EJM001, as Metschnikowia bicuspidata based on 18S rDNA, ITS and 26S rDNA sequence analyses and on its morphological, physiological and biochemical characteristics. Phylogenetic analysis revealed that 26S rDNA of 2EJM001 was clustered with M. bicuspidata (LNES0119) as reported by Bao et al. In addition, unlike Bao et al., two challenge experiments (injection and immersion) were used in this study. The results of challenge experiments show that 2EJM001 was pathogenic to E. sinensis and caused signs similar to those found in the naturally infected crabs. At the same time, the minimum inhibitory concentrations (MIC80 and MIC90 ) were determined. This study further confirms that M. bicuspidata 2EJM001 was the pathogen responsible for 'milky disease' in E. sinensis from Liaoning Province.

Delineating the key virulence factors and intraspecies divergence of Vibrio harveyi via whole-genome sequencing.

Vibrio harveyi is one of the major pathogens in aquaculture. To identify the key virulence factors affecting pathogenesis of V. harveyi towards fish, we conducted a field investigation for three representative fish farms infected with V. harveyi. Multilocus sequence typing (MLST) and whole-genome sequencing were conducted to delineate the phylogenetic relationship and genetic divergence of V. harveyi. A total of 25 V. harveyi strains were isolated from the diseased fish and groundwater and were subtyped into 12 sequence types by MLST. Five virulence genes, mshB, pilA, hutR, ureB, and ureG, were variably presented in the sequenced strains. The virulence gene profiles strongly correlated with the distinct pathogenicity of V. harveyi strains, with a strain harboring all five genes exhibiting the highest virulence towards fish. Phenotype assay confirmed that reduced virulence correlated with decreased motility and biofilm formation ability. Additionally, three types of type VI secretion system, namely T6SS1, T6SS2, and T6SS3, were identified in V. harveyi strains, which can be classified into six, four, and 12 subtypes, respectively. In conclusion, the results indicated that the virulence level of V. harveyi is mainly determined by the above virulence genes, which may play vital roles in environmental adaptation for V. harveyi.

First case of non-O1/O139 Vibrio cholerae infection in the pink-tailed chalceus Chalceus macrolepidotus in China.

A pathogen was isolated from diseased pink-tailed chalceus Chalceus macrolepidotus during a high-mortality outbreak in a freshwater culture farm in Liaoyang, China. The diseased fish were characterized by disoriented behaviors, exophthalmos, and redness and swelling of the top of the head. A Gram-negative, pure strain of bacteria (CM0428) was isolated from the brain, kidney, and liver. The isolate was identified as Vibrio cholerae based on ompW gene amplification and 16S rRNA and gyrB gene sequence analysis. Serogroup testing indicated that CM0428 was a non-O1/O139 strain of V. cholerae. The challenge test showed that CM0428 exhibited strong virulence to pink-tailed chalceus, and hlyA and toxR virulence-related genes were detected. The isolate was sensitive to multi-class antibiotics, but resistant to tetracycline. Histological examination revealed that V. cholerae CM0428 infection caused multiple organ and tissue lesions, and typical pathological features were cell degeneration and necrosis.

A novel research on isolation and characterization of Photobacterium damselae subsp. damselae from Pacific white shrimp, Penaeus vannamei, displaying black gill disease cultured in China.

In June 2019, massive mortalities of cultured Penaeus vannamei occurred in a local farm in Hainan Province, China. The diseased shrimp displayed evident black gills. Three bacterial strains 20190611001, 20190611007 and 20190611022 were isolated from hepatopancreas and gills of the diseased shrimp and identified as Photobacterium damselae subsp. damselae based on the sequence analysis of 16S rRNA and toxR genes. These three isolates showed haemolytic activities. Of them, strain 20190611022 isolated from hepatopancreas was selected and processed for pathogenic analysis. The calculated median lethal dose (LD50 ) was 9.75 ± 4.29 × 105 CFU/g (body weight) by challenging P. vannameivia reverse gavage. The diseased shrimp displayed enlarged hepatopancreatic tubules and sloughing of epithelial cells in tubular lumens. The strain 20190611022 was also characterized by the testing of API 20NE systems and antibiotic susceptibility. The results of disc diffusion test showed that strain 20190611022 was sensitive to chloramphenicol, compound sulfamethoxazole, cefoperazone, ceftriaxone, ceftazidime and cefuroxime. To our knowledge, this is the first report of isolation and characterization of Photobacterium damselae subsp. damselae from natural diseased P. vannamei. Our findings can serve as a basis for further studies of its pathogenicity and provide technological support for disease controlling in shrimp aquaculture.

Localization and characterization of hematopoietic tissues in adult sea cucumber, Apostichopus japonicus.

Sea cucumber Apostichopus japonicus rely on the efficient innate immune mechanisms against invaders, in which the consumption and regeneration of coelomocytes take place at the same time. In the present study, histological features of putative hematopoietic tissues (HPTs) including the rete mirabile, the respiratory tree, the polian vesicle and the coelomic epithelium were characterized. The distribution of transcription factor GATA1 in coelomocytes and putative HPTs was examined by immunohistochemistry. In addition, cell proliferation using EdU labeling and coelomocyte distribution in different tissues using monoclonal antibody labeling were analyzed to further confirm the HPTs. The results showed that two homologs of GATA1 were detected with molecular weight of 43 and 90 kDa in coelomocytes, rete mirabile, respiratory tree and polian vesicle, whereas no signals were detected in the coelomic epithelium. A few cells were detected to be EdU-positive for coelomocytes, which accounted for approximately 9.5%. In the rete mirabile and the respiratory tree, the EdU signals were strong in cells of the tube wall. In the polian vesicle, numerous EdU-positive cells were detected in the cyst wall. In the coelomic epithelium, little EdU signaling was detected. Immunohistochemistry analysis by mAb 3F6 against A. japonicus coelomocytes showed that positive signals were observed in the tube wall of the rete mirabile, respiratory tree, cyst wall of the polian vesicle and in the coelomocyte antrum of coelomic epithelium. These results suggest that the rete mirabile, respiratory tree and polian vesicle are the HPTs of A. japonicus.

Delineating the origins of the multidrug-resistant pathogens in ornamental fish farms by multilocus sequence typing and identification of a novel multidrug-resistant plasmid.

To evaluate the overuse of antibiotics and to identify the origin of pathogens in the ornamental fish industry, we conducted a field investigation of three representative fish farms in Liaoning province, China. Drug-resistant pathogens in the fishponds and groundwater were isolated and subtyped by multilocus sequence typing (MLST). In total, 33 pathogenic strains, including Aeromonas veronii and five other pathogens, were isolated from diseased fish and from groundwater. MLST revealed that A. veronii obtained from diseased fish in three fish farms can be subtyped into four sequence types, which were also identified in the corresponding groundwater. All of the isolates obtained from diseased fish showed resistance to at least four antibiotics. Notably, Citrobacter freundii JY-17 exhibited resistance to the majority of the antibiotics and was a carrier of a megaplasmid with 15 drug resistance genes. PCR assays targeting ß-lactam, kanamycin, macrolide, phenicol, sulfonamide, and trimethoprim resistance genes in the pathogens from the diseased fish and groundwater were also conducted. The results revealed strong correlations between antibiotic treatment and increased antimicrobial resistance in fish pathogens. The results suggested that groundwater is the origin of the pathogens in ornamental fish. Antibiotic treatment of ornamental fish promoted the emergence of resistant pathogens.

Regeneration of coelomocytes after evisceration in the sea cucumber, Apostichopus japonicus.

Sea cucumber, Apostichopus japonicus, is one of the most important holothurian species cultured in China. Severe evisceration induced by various natural and artificial factors commonly occurs during transport and culture of A. japonicus. Evisceration causes higher mortality and lower yield. Along with the visceral regeneration process, sea cucumbers also regenerate coelomocytes in order to recover immune function. In this study, evisceration of A. japonicus was induced by intracoelomic injection of 0.35 M KCl. Regeneration of coelomocytes was investigated by time course cell counting as well as detection of DNA replication by the EdU labeling technique. Coelomic fluid volume was restored to the pre-evisceration level within 2 h after evisceration. Total coelomocyte count (TCC) reached a peak at 6 h post-evisceration, followed decreased and then increased with a slight fluctuation, restored to the pre-evisceration level at 35 d post-evisceration. The change in different subtypes of coelomocytes was consistent with that of total coelomocytes. However, there were some variations in the regeneration of coelomocyte subtypes. At the end of the study, only the counts of amoebocytes and morula cells recovered to the pre-evisceration level. DNA replication assay showed EdU-positive cells accounted for 9.5% before evisceration and 4.7% at 6 h post-evisceration. However, the percentage of EdU-positive cells significantly increased, reaching 18.6% at 3 d after evisceration, then declined. Therefore, we analyzed the observed increase in coelomocytes at 6 h post-evisceration, which may be due to coelomocyte migration from the water-vascular system into the coelom rather than de novo cell proliferation.

Development of a Colloidal Gold-Based Immunochromatographic Assay for the Rapid Detection of Edwardsiella Ictaluri.

Edwardsiella Ictaluri is known as the etiological agent of enteric septicaemia of channel catfish, causing heavy economic losses in the aquaculture industry. In this study, a colloidal gold-based immunochromatography assay (GICA) was developed for rapid detection of E. ictaluri. Briefly, monoclonal antibody (MAbs) and polyclonal antibody (PAbs) against E. ictaluri were prepared. Sensitivity of MAbs and PAbs to E. ictaluri was analyzed by Dot ELISA. Mouse MAb5D11 against E. ictaluri was conjugated with the 20 nm colloidal gold particles as the detector. Rabbit PAbs of E. ictaluri and goat anti-mouse IgG antibody was sprayed on nitrocellulose membranes as test line (T) and control line (C) respectively. The minimum detectable amount of this method to E. ictaluri was 5 × 106 CFU/mL. Cross reactions wouldn't occur when detecting E. tarda, Aeromonas hydrophila, V. parahaemolyticus and other several common standard strains. The result could be got in only 5 to 10 minutes. It didn't need professional technologies and testing experience. So this assay was very suitable for basic departments of aquatic product companies.

First characterization of three cyclophilin family proteins in the oyster, Crassostrea ariakensis Gould.

Cyclophilins (CyPs) are a family of proteins that bind the immunosuppressive agent cyclosporin A (CsA) with high-affinity and belong to one of the three superfamilies of peptidyl-prolyl cis-trans isomerases (PPIase). In this report, three cyclophilin genes (Ca-CyPs), including Ca-CyPA, Ca-CyPB and Ca-PPIL3, were identified from oyster, Crassostrea ariakensis Gould in which Ca-CyPA encodes a protein with 165 amino acid sequences, Ca-CyPB encodes a protein with 217 amino acid sequences and Ca-PPIL3 encodes a protein with 162 amino acid sequences. All of the three Ca-CyPs genes contain a typical CyP-PPIase domain with its signature sequences and Ca-CyPB contains an N-signal peptide sequences. Tissue distribution study revealed that Ca-CyPs were ubiquitously expressed in all examined tissues and the highest levels were observed in hemocytes. RLO incubation upregulated the mRNA expression levels of Ca-CyPs, indicating that three Ca-CyPs might be involved in oyster immune response against RLO infection.

Characterization and expression analysis of a caspase-2 in an invertebrate echinoderm sea cumber Apostichopus japonicus.

Caspase-2 is the most evolutionarily conserved member of the caspase family which mediates the programmed cell death and plays crucial roles in key cellular processes. In this study, a caspase-2 homolog was identified and functionally characterized in sea cucumber Apostichopus japonicus, which we named AjCASP. The full-length cDNA consists of 2100 bp with an ORF encoding a protein of 378 amino acids. The deduced amino acid sequence shows that AjCASP consists of a conserved CARD-CASP2 domain and a CASs domain containing two active residues, two proteolytic cleavage residues, a substrate pocket and a dimer interface as well. In addition, a p20 large subunit with a characteristic five-peptide motif (QACRG) and a p10 small subunit in C-terminal were identified in CASs domain. Above data demonstrated that AjCASP is similar to CED-3 (the caspase-2 homolog of nematode Caenorhabditis elegans), which is further confirmed by phylogenetic tree analysis. AjCASP was ubiquitously expressed in sea cucumber and the obviously higher expression level was observed in coelomocyte, respiratory tree and intestine. Real-time PCR analyses further demonstrated that AjCASP was significantly induced by LPS. Taken together, these results strongly suggest that AjCASP is a caspase-2 homolog and it may be involved in invertebrate immune response, especially in eliminating and degrading invading pathogens.

Enhanced immune response and resistance to edwardsiellosis following dietary chitooligosaccharide supplementation in the olive flounder (Paralichthys olivaceus).

This study was conducted to evaluate the effects of dietary chitooligosaccharide (COS) supplementation on peripheral leukocyte count, head kidney leukocyte phagocytic rate, phagocytic index, respiratory burst activity, serum lysozyme activity, and immune protection in Paralichthys olivaceus. A total of 300 flounder with an average body weight of 80-100 g were randomly assigned into four dietary groups: (I) basic diet (control), basic diet containing (II) 0.5% COS, and (III) 1% COS, fed continuously for 28 d, and (IV) basic diet containing 1% COS fed in 14 d intervals. Continuous feeding of 0.5% and 1% COS diets for 28 d significantly increased the number of peripheral leukocytes, head kidney leukocyte phagocytic rate, phagocytic index, respiratory burst activity, and serum lysozyme activity (P < 0.05 or P < 0.01). After a 10 d Edwardsiella tarda challenge, the immune protection rates in the 0.5% and 1% COS groups were 30% and 60%, respectively. No control fish survived the E. tarda challenge treatment. Most immune indices were slightly lower after removal of COS from the diet for 14 d, but all immune indices were observed to recover after another 14 d of COS supplementation. This study demonstrates that supplementation of a basic diet with COS enhances the non-specific immune response and improves survival rates following infection with E. tarda in P. olivaceus. An optimized interval feeding strategy with diets containing 1% COS may have potential applications in the prevention of disease in aquacultured P. olivaceus.

Ontogenesis of coelomocytes in sea cucumber (Apostichopus japonicus) studied with probes of monoclonal antibody.

Monoclonal antibodies (MAbs) specifically against coelomocytes of Apostichopus japonicus were employed to study the ontogenesis of coelomocytes by indirect immunofluorescence assay technique (IIFAT). Different developmental stages were identified by histochemical staining method. Stages including blastula, gastrula, auricularia (small-auricular larvae, mid-auricular larvae and big-auricular larvae), doliolaria, pentactula and juvenile were examined. The positive reactions with both MAb1C2 against all the types of coelomocytes and MAb3F6 specific to spherulocytes, were observed firstly at the blastula stage of the embryos. The positive reaction with MAb1E2 against lymphoid cells was observed from the big-auricular larvae, which indicated that lymphoid cells may not be progenitor cells or stem cells for A. japonicus. An increase of fluorescence intensity for each cell may imply a possible enhancement of the innate defensive mechanism as the embryogenesis progress.

Antiparasitic efficacy of flavonoids identified from Psoralea corylifolia against Tetrahymena piriformis in guppy (Poecilia reticulate).

Tetrahymena piriformis belongs to the ciliated protists (ciliates), causing severe economic losses in aquaculture. Chemical drugs currently used usually have toxic side effects, and there is no specific drug against Tetrahymena. Therefore, it is an urgent need to identify new antiparasitic lead compounds. In the present study, the in vitro parasiticidal activity of ethyl acetate (EtOAc) extracts and water extracts from 22 selected traditional Chinese medicines (TCMs) were evaluated against T. piriformis. The EtOAc extract of P. corylifolia turned out to be the most active with the minimum parasiticidal concentration of 100 mg/L within 3 h. Thus, it was separated into 12 fractions by the first-dimensional (D1) normal phase liquid chromatography (NPLC), meanwhile combining with in vitro antiparasitic tests for activity tracking. Subsequently, 8 flavonoids were identified in the active fractions by the second-dimensional (D2) reverse phase liquid chromatography (RPLC) tandem high-resolution mass spectrometry. According to the results, 5 flavonoids were selected for in vitro antiparasitic test, of which isobavachalcone showed the minimum parasiticidal concentration of 3.125 mg/L in 2 h. Bathing treatment of infected guppies with isobavachalcone could significantly reduce the burden of T. piriformis, obtaining a 24-h median effective concentration (24-h EC50) value of 1.916 mg/L. And the concentration of isobavachalcone causing guppies to die within 24 h is 39 times than that of 24-h EC50. The results demonstrated that isobavachalcone has the potential to be developed into a novel commercial fish drug against T. piriformis.

Characterization and function of an E2-17 kDa (UBE2D) in an invertebrate Haliotis diversicolor supertexta.

Ubiquitin-conjugating enzymes (UBE2s or E2s) are characterized by the presence of a highly conserved ubiquitin-conjugating (UBC) domain, which predominantly determines the type of ubiquitin chains and directly controls the cellular fate of the substrate. In this study, an E2 homolog was identified and functionally characterized in abalone, which we named ab-UBE2D. The full-length cDNA consists of 1005 bp with an ORF encoding a protein of 147 amino acids. The deduced amino acid sequence shows ab-UBE2D shares conserved UBC domain with other E2 proteins and belongs to class I E2 enzyme family, which are further confirmed by phylogenetic tree analysis. Real-time PCR and western blot analyses showed that ab-UBE2D was ubiquitously expressed in abalone and the expression level of ab-UBE2d was significantly induced by LPS and Poly (I:C). Immunofluorescence microscopy staining demonstrated that native ab-UBE2D was mainly distributed in the cytoplast. Ubiquitination assay showed that ab-UBE2D had ubiquitin conjugating activity to form the enzyme-(Ub)n conjugates. Taken together, these results strongly suggest that ab-UBE2D is an E2 homolog and it may be involved in the immune response of abalone, Haliotis diversicolor supertexta.

Comparison of cells free in coelomic and water-vascular system of sea cucumber, Apostichopus japonicus.

The sea cucumber, Apostichopus japonicus possesses a variety of cells populating in both the coelomic (cells in the coelomic are called coelomocytes) and water-vascular system. In this study, we compared cells in these two systems of A. japonicus on total cell number, cell types and surface antigens through monoclonal antibodies against coelomocytes. The results demonstrated that the cell types observed in coelomic also could be found in water-vascular system, but the total cell number and percentages of each type were different. The total number of coelomocytes was 2-3 times of that in water-vascular system. Lymphoid cells were numerically dominant in coelomic system, while spherulocytes with pseudopods in water-vascular system. Results of indirect immunofluorescence assay technique showed that both coelomocytes and cells in water-vascular system could be recognized by the corresponding MAbs, and the distribution of its positive signals was not different. In conclusion, cell types and surface antigens in coelomic and water-vascular system were same, but the total cell number and percentages of each type were different. And further researches are needed on whether there are differences in functions of the different composition.

Molecular characterization of a novel tetraspanin from the oyster, Crassostrea ariakensis: variation, localization and relationship to oyster host defense.

We identified a tetraspanin family member gene, named Ca-TSP, in the oyster Crassostrea ariakensis and found that the transcription profiles of Ca-TSP were variable in the oyster hemocytes. Three distinct patterns of variation of Ca-TSP were observed. Using immunofluorescence and immunoelectron microscopy, we show that Ca-TSP was present in granules and in vesicular structures of the oyster hemocyte. Sequence analysis, structural features and immunogold electron microscopy showed that Ca-TSP is an integral membrane glycoprotein of granules of hemocyte and may be a novel CD63-like gene of the tetraspanin family of molluscs. The gene expression analysis of Ca-TSP using isolated oyster hemocytes, was done following challenge of the oysters with LPS and Poly I:C. The Ca-TSP mRNA levels increased in hemocytes in the first 12 h after LPS and Poly I:C stimulation, and decreased after the addition of H(2)O(2). Western blot analysis using anti-Ca-TSP antibody indicated that gene expression and protein levels were similar. The recombinant Ca-TSP was found to significantly inhibit hemocytes aggregation. Our results suggested that Ca-TSP participates in the innate immunity of the oyster.

Identification of expressed genes in cDNA library of hemocytes from the RLO-challenged oyster, Crassostrea ariakensis Gould with special functional implication of three complement-related fragments (CaC1q1, CaC1q2 and CaC3).

A SMARTer™ cDNA library of hemocyte from Rickettsia-like organism (RLO) challenged oyster, Crassostrea ariakensis Gould was constructed. Random clones (400) were selected and single-pass sequenced, resulted in 200 unique sequences containing 96 known genes and 104 unknown genes. The 96 known genes were categorized into 11 groups based on their biological process. Furthermore, we identified and characterized three complement-related fragments (CaC1q1, CaC1q2 and CaC3). Tissue distribution analysis revealed that all of three fragments were ubiquitously expressed in all tissues studied including hemocyte, gills, mantle, digestive glands, gonads and adductor muscle, while the highest level was seen in the hemocyte. Temporal expression profile in the hemocyte monolayers reveled that the mRNA expression levels of three fragments presented huge increase after the RLO incubation at 3 h and 6 h in post-challenge, respectively. And the maximal expression levels at 3 h in post-challenge are about 256, 104 and 64 times higher than the values detected in the control of CaC1q1, CaC1q2 and CaC3, respectively.

The complete mitochondrial genome sequence of the Metschnikowia bicuspidata (Metschnikoff, 1884), an emerging pathogen of 'milky disease' in Chinese mitten crab.

The outbreak of milky disease of Chinese mitten crab caused by M. bicuspidata seriously restricted the development of the crab industry. In this study, the mitochondrial genome sequence of M. bicuspidata was assembled, annotated, and further analyzed. The results indicated that the complete mitochondrial genome of M. bicuspidata was 75,095 bp, which contained two rRNAs, 23 tRNAs, and 13 protein-coding genes. The phylogenetic tree of 13 yeasts based on the complete mitochondrial genome was constructed which showed that M. bicuspidata (accession number OK514652) and M. bicuspidata (accession number MW147605.1) were clustered in a clade. To sum up, our research results would further provide essential data for the systematics and evolution study of M. bicuspidata.