The Genetic and Phenotypic Diversity of Bacillus spp. from the Mariculture System in China and Their Potential Function against Pathogenic Vibrio.

Bacillus spp. could be one of the most suitable substitutes for the control and prevention of aquatic diseases. The occurrence of species population, antimicrobial character, and virulence diversity in Bacillus spp. recovered from the mariculture system in China between 2009 and 2021 were investigated, screening for probiotic Bacillus strains with good biological safety that can inhibit Vibrio parahaemolyticus, V. alginolyticus, V. harveyi, V. owensii, V. campbellii. The results showed that 116 Bacillus isolates were divided into 24 species, and the top three species were B. subtilis (37/116), B. velezensis (28/116), and B. amyloliquefaciens (10/116). Among the 116 Bacillus isolates, 32.8% were effective against V. parahaemolyticus, 30.1% for V. alginolyticus, 60.3% for V. harveyi, 69.8% for V. owensii and 74.1% for V. campbellii. More than 62% of Bacillus isolates were susceptible to florfenicol, doxycycline and tetracycline, etc., and 26/116 Bacillus isolates were found to be multiple-antibiotic-resistant (MAR), with MARI values ranging from 0 to 0.06. Eighteen kinds of antibiotic resistance genes were tested; only tetB, blaTEM, and blaZ were detected. And 9 isolates in 2 Bacillus species were excluded by 6/10 kinds of Bacillus-related toxin gene (hblA, hblC, nheB, nheC, entFM, cykK). Bio-safety testing indicated that three kinds of probiotics were good probiotic candidates to prevent Vibriosis. These results provide comprehensive genetic diversity, potential risks, and probiotic characteristics of Bacillus in the mariculture system in China, and provide basic support for green and healthy development of aquatic industry.

Virulence and antimicrobial resistance characteristics assessment of Vibrio isolated from shrimp (Penaeus vannamei) breeding system in south China.

The diseases caused by Vibrio during shrimp breeding program have the risk of spreading in different aquatic areas through larvae transportation between different regions. Therefore, the population distribution and the virulence and antibiotic resistance risk of 5 pathogenic Vibrio in shrimp (Penaeus vannamei) breeding system in China were evaluated for the first time. A total of 418 isolates were recovered from shrimp, breeding water and biological baits samples, and 312 isolates were identified as Vibrio genus based on 16s rDNA, among which V. alginolyticus, V. harveyi, V. parahaemolyticus, V. cholerae and V. campbellii were the dominant species. And 10/20 kinds of virulence genes (chiA, luxR, vhh, tlh, chxA, sepro, flaA, vch, VAC and rpoS) were detected among the 5 Vibrio species. Multiple antibiotic resistance (MAR) index of the 5 dominant Vibrio isolates were 0.13-0.88 %, and 36.5 % isolates with MAR < 0.2. But the antibiotic resistance pattern abundance (ARPA) index ranged from 0.25 to 0.56, which indicated the antibiotic phenotypes of Vibrio species in the shrimp breeding system in China were homogeneity. Furthermore, resistance quotients (RQs) calculation results displayed that the dominant Vibrio species in the shrimp breeding system in China showed no or low selection pressure for resistance to cefoperazone/sulbactam, enrofloxacin, ciprofloxacin, fluoroquine, florfenicol, tetracycline and doxycycline. But only 5 resistance genes were detected, which were strA (43.8 %), strB (11.7 %), QnrVC (2.9 %), sul2 (8.8 %) and Int4 (8.8 %), respectively, and the antimicrobial resistance genotypes were not previously correlated with their phenotypes. The relevant research results provide theoretical basis for epizootic tracking in aquatic system in China, and targeting its final risk in aquatic ecosystem and public health perspectives.

Complete genome analysis of a virulent Vibrio scophthalmi strain VSc190401 isolated from diseased marine fish half-smooth tongue sole, Cynoglossus semilaevis.

BACKGROUND: Vibrio scophthalmi is an opportunistic bacterial pathogen, which is widely distributed in the marine environment. Earlier studies have suggested that it is a normal microorganism in the turbot gut. However, recent studies have confirmed that this bacterial strain can cause diseases in many different marine animals. Therefore, it is necessary to investigate its whole genome for better understanding its physiological and pathogenic mechanisms. RESULTS: In the present study, we obtained a pathogenic strain of V. scophthalmi from diseased half-smooth tongue sole (Cynoglossus semilaevis) and sequenced its whole genome. Its genome contained two circular chromosomes and two plasmids with a total size of 3,541,838 bp, which harbored 3185 coding genes. Among these genes, 2648, 2298, and 1915 genes could be found through annotation information in COG, Blast2GO, and KEGG databases, respectively. Moreover, 10 genomic islands were predicted to exist in the chromosome I through IslandViewer online system. Comparison analysis in VFDB and PHI databases showed that this strain had 334 potential virulence-related genes and 518 pathogen-host interaction-related genes. Although it contained genes related to four secretion systems of T1SS, T2SS, T4SS, and T6SS, there was only one complete T2SS secretion system. Based on CARD database blast results, 180 drug resistance genes belonging to 27 antibiotic resistance categories were found in the whole genome of such strain. However, there were many differences between the phenotype and genotype of drug resistance. CONCLUSIONS: Based on the whole genome analysis, the pathogenic V. scophthalmi strain contained many types of genes related to pathogenicity and drug resistance. Moreover, it showed inconsistency between phenotype and genotype on drug resistance. These results suggested that the physiological mechanism seemed to be complex.

The intestine of artificially bred larval turbot (Scophthalmus maximus) contains a stable core group of microbiota.

Generally speaking, fish intestinal microbiota is easily affected by food or water environment, and it may be dynamically changed along with body growth. However, it remains unclear whether fish gut microbiota can be affected under any conditions. In the present study, we focused on cultured larval turbot (Scophthalmus maximus) and tracked its artificial breeding process from eggs to larvae in two farms located in different regions of China. Through continuous sampling, we analyzed and compared characteristics of intestinal microbiota in turbot larvae and its correlation with the bacteria in water and food at different developmental stages. The results showed that there was a steady group of microbiota in larval gut, and the highest relative abundance of strain was same between the two farms. This microbiota was established soon after hatching of fertilized eggs. Particularly, the structure of this microbiota was nearly not changeable afterward 3-4 months of development. The bacteria carried by fertilized eggs might play an important role during the formation of this microbiota. In conclusion, our findings suggested that there was a core microbiota represented by Lactococcus sp. in gut of artificially bred turbot larvae. The relative proportion of such strain in gut was higher than 30% at the initial stage of turbot life.

First report of isolation and complete genome of Vibrio rotiferianus strain SSVR1601 from cage-cultured black rockfish (Sebastes schlegelii) associated with skin ulcer.

Vibrio rotiferianus is an important marine pathogen of various aquatic organisms and can be found widely distributed in the marine environment. To further characterize this pathogen, the pathogenic properties and genome of V. rotiferianus SSVR1601 isolated from Sebastes schlegelii with skin ulcer were analysed. SSVR1601 was shown to be short rod-shaped cell with a single polar flagellum. Different degrees of pathological changes in fish kidney, intestine, gills and liver were observed after SSVR1601 challenge. The SSVR1601 genome consists of two chromosomes and two plasmids with a total of 5,717,113 bp, 42.04%-44.93% GC content, 5,269 predicted CDSs, 134 tRNAs and 40 rRNAs. The common virulence factors including OMPs, haemolysin, flagellin, DNase, entF, algU, tcpI, acfB and rfaD were found in strain SSVR1601. Furthermore, factors responsible for iron uptake (fur, fepC and ccmC) and types II, IV and VI secretion systems were detected, which are likely responsible for the pathogenicity of SSVR1601. The antimicrobial resistance genes, bacA, tet34 and norM, were detected based on Antibiotic Resistance Genes Database. The phylogenetic analysis revealed SSVR1601 to be most closely related to V. rotiferianus strains CAIM577 and B64D1.

Transcriptome Analysis and Discovery of Genes Involved in Immune Pathways from Coelomocytes of Sea Cucumber (Apostichopus japonicus) after Vibrio splendidus Challenge.

Vibrio splendidus is identified as one of the major pathogenic factors for the skin ulceration syndrome in sea cucumber (Apostichopus japonicus), which has vastly limited the development of the sea cucumber culture industry. In order to screen the immune genes involving Vibrio splendidus challenge in sea cucumber and explore the molecular mechanism of this process, the related transcriptome and gene expression profiling of resistant and susceptible biotypes of sea cucumber with Vibrio splendidus challenge were collected for analysis. A total of 319,455,942 trimmed reads were obtained, which were assembled into 186,658 contigs. After that, 89,891 representative contigs (without isoform) were clustered. The analysis of the gene expression profiling identified 358 differentially expression genes (DEGs) in the bacterial-resistant group, and 102 DEGs in the bacterial-susceptible group, compared with that in control group. According to the reported references and annotation information from BLAST, GO and KEGG, 30 putative bacterial-resistant genes and 19 putative bacterial-susceptible genes were identified from DEGs. The qRT-PCR results were consistent with the RNA-Seq results. Furthermore, many DGEs were involved in immune signaling related pathways, such as Endocytosis, Lysosome, MAPK, Chemokine and the ERBB signaling pathway.

Ingestion, egestion and physiological effects of polystyrene microplastics on the marine jellyfish Rhopilema esculentum.

Jellyfish are planktonic predators that may be susceptible to ingesting microplastics. However, the effects of MP exposure on jellyfish are poorly understood. In this study, the ingestion and egestion of polystyrene microbeads, and its chronic physiological effects on Rhopilema esculentum at an environmental concentration (100 items/L) and a predicted concentration (1000 items/L) were evaluated. The results showed that the ingestion amount of juvenile medusae was relatively low. The MP egestion rates reached 100 % within 9 h of clearance. Chronic exposure (15 days) to MPs at environmental concentrations led to no adverse impacts. Nevertheless, the predicted concentration of MP exposure induced growth inhibition, a reduction in assimilation efficiency, oxygen consumption increase, and lipase enzyme activity reduction in the jellyfish, indicating that MPs can cause adverse effects on the energy budget of jellyfish in the near future. Our study provides new insights into the potential risk of MPs in marine environments.

Antibiotic resistance, virulence and genetic characteristics of Vibrio alginolyticus isolates from aquatic environment in costal mariculture areas in China.

Vibrio alginolyticus has been the second most common Vibrio species in the world and mainly grows in the ocean or estuary environment, which can induce epidemics outbreaks under marine organisms, and causing serious economic losses in aquaculture industry. In this study, the genetic populations and evolutionary relationship analysis of V. alginolyticus isolated from different geographical locations in China with typical interannual differences were exhibited originally genetic diversity. Then the virulence genes prevalence, antibiotic resistance phenotype, and antimicrobial resistance genes risk diversity of V. alginolyticus were analyzed by phenotypic and molecular typing methods. And they were complex correlations among antibiotic phenotypes, resistance and virulence genes under different genotype of V. alginolyticus. The results provide a theoretical foundation for further understanding the genetic and metabolic diversity among V. alginolyticus in China, and lay a theoretical foundation for the transmission risk assessment and regional diagnosis of Vibrio in aquatic animals.

Differential expression of miRNAs in the body wall of the sea cucumber Apostichopus japonicus under heat stress.

MicroRNAs, as one of the post-transcriptional regulation of genes, play an important role in the development process, cell differentiation and immune defense. The sea cucumber Apostichopus japonicus is an important cold-water species, known for its excellent nutritional and economic value, which usually encounters heat stress that affects its growth and leads to significant economic losses. However, there are few studies about the effect of miRNAs on heat stress in sea cucumbers. In this study, high-throughput sequencing was used to analyze miRNA expression in the body wall of sea cucumber between the control group (CS) and the heat stress group (HS). A total of 403 known miRNAs and 75 novel miRNAs were identified, of which 13 miRNAs were identified as significantly differentially expressed miRNAs (DEMs) in response to heat stress. A total of 16,563 target genes of DEMs were predicted, and 101 inversely correlated target genes that were potentially regulated by miRNAs in response to heat stress of sea cucumbers were obtained. Based on these results, miRNA-mRNA regulatory networks were constructed. The expression results of high-throughput sequencing were validated in nine DEMs and four differentially expressed genes (DEGs) by quantitative real-time polymerase chain reaction (RT-qPCR). Moreover, pathway enrichment of target genes suggested that several important regulatory pathways may play an important role in the heat stress process of sea cucumber, including ubiquitin-mediated proteolysis, notch single pathway and endocytosis. These results will provide basic data for future studies in miRNA regulation and molecular adaptive mechanisms of sea cucumbers under heat stress.

Development of new microsatellite DNA markers from Apostichopus japonicus and their cross-species application in Parastichopus parvimensis and Pathallus mollis.

Twenty microsatellite DNA markers were developed for sea cucumber and used to investigate polymorphisms of 60 wild Apostichopus japonicus individuals collected from China. It revealed that all the markers were polymorphic. A total of 164 alleles were detected at 20 loci. The number of alleles per locus varied from 3 to 17 with an average of 8.2, and the expected heterozygosities of each locus ranged from 0.03 to 0.89 with an average of 0.64. Cross-species amplification was also conducted in Parastichopus parvimensis collected from the United States and Pathallus mollis collected from Peru. The result showed that 17 loci amplified Parastichopus parvimensis DNAs while only 4 loci could amplify Pathallus mollis DNAs. All of the polymorphic markers would be useful for future genetic breeding and the assessment of genetic variation within sea cucumbers.

Morphological and molecular evidence of a new species of Melanochlamys (Gastropoda: Heterobranchia) from the Bohai Sea, China.

Increasing discoveries of new species of the genus Melanochlamys Cheeseman, 1881 in the recent years indicate that the biodiversity of this group remains underestimated. Recently, several aglajid sea slugs were collected from Laizhou Bay, Shandong Province, China. Morphological observation of their external and internal anatomy revealed that they represent an undescribed species of Melanochlamys. This new species, Melanochlamys aquilina sp. nov., is closely related to Melanochlamys fukudai Cooke, Hanson, Hirano, Ornelas-Gatdula, Gosliner, Chernyshev Valdés, 2014 in body size and dark external coloration, but can be differentiated from the latter by features of both shell and male reproductive organ. Phylogenetic analyses of two mitochondrial (COI, 16S rRNA) and a nuclear (H3) genes using Bayesian inference, maximum likelihood, and species delimitation analysis also support the separation of Melanochlamys aquilina sp. nov. from its related congeners.

The complete mitochondrial DNA sequence of Heterochaerus australis (Acoela, Convolutidae).

One complete mitochondrial genomes (mitogenomes) was determined for Heterochaerus australis (Acoela, Convolutidae). Its mitochondrial genome size was 13,885 bp. The sequence contains 2 ribosomal RNA genes (rrnL and rrnS), 20 tRNA genes, and 12 protein-coding genes (PCGs). The A + T content of the complete mitochondrial genome sequence was 70.8%. The base composition showed a tendency of high AT. The resulted maximum likelihood (ML) tree supported that Acoela had a distant relationship with other orders of Turbellaria and the Xenacoelomorpha.

Complete sequence of mitochondrial DNA of a deep-sea holothurian species of the genus Synallactes (Synallactida: Synallactidae).

One complete mitochondrial genome (mitogenome) was determined for a deep-sea holothurian species of the genus Synallactes (Synallactida: Synallactidae). The mitochondrial genome size of the sea cucumber was 15,920 bp. The sequence contains 2 ribosomal RNA genes (12S and 16S), 22 transfer RNA genes, and 13 protein-coding genes, as found in most previously determined holothurian mitogenomes. The A + T content of the complete mitochondrial genome sequence was 64.45%. The base composition showed a tendency of AT. The resulted maximum likelihood (ML) tree of Holothuroidea supported that Synallactes sp. is a species of Synallactida.

Responses of microbial community structure in turbot (Scophthalmus maximus) larval intestine to the regulation of probiotic introduced through live feed.

Various bacteria that adhere to the gut are important for the health of fish. Regulating the microbial community in a desirable direction may be beneficial in aquaculture for preventing and controlling the diseases caused by pathogenic microbes. In this study, we investigated the changes in the microflora in the intestinal tracts of turbot (Scophthalmus maximus) larvae after introducing a probiotic (Bacillus amyloliquefaciens) after the first feed. B. amyloliquefaciens was added as part of a nutrient enrichment system in live feed (Branchionus plicatilis or Artemia sinica), so it passed into the intestinal tracts of the newly hatched turbot larvae. The turbot larvae were fed on live feed containing B. amyloliquefaciens in the experimental group, whereas live feed without the probiotic was provided to larvae in the control group. The total bacterial genomic DNA in the larval guts was extracted and sequenced with an Illumina HiSeq PE250 system. According to the sequencing results, the abundances of microbial species and the microflora diversity were lower in the intestines in the experimental group than the control. Throughout development, the microflora structure in the intestines was mainly constructed before the first feed and the composition of the dominant operational taxonomic units (OTUs) was stable, where the abundances of OTU8, OTU124, OTU150, OTU107, and OTU17 were always high. Compared with the control, the structures of the microflora in the intestines were similar on different days during the development and the growth of larvae in the experimental group. However, the similarity of the microflora structure between different treatments was low on the same day. Furthermore, the mean proportion of common OTUs was only 74.7% in different treatments on each day, which indicates that the introduction of B. amyloliquefaciens in the live feed changed the microflora structure in the intestine. During the early development stage (days 3-30), the average abundance of Pseudomonas was reduced by 0.8% whereas that of Lactococcus increased by 3.5% in the experimental group. Pseudomonas spp. are considered potentially pathogenic bacteria but there is no direct evidence for the pathogenicity of Lactococcus in turbot. Moreover, several Lactococcus species are regarded as probiotics in aquaculture. Therefore, the use of B. amyloliquefaciens could be beneficial for optimizing the microbial community structure in the intestines of turbot larvae, which may explain the probiotic effect of B. amyloliquefaciens. This study provides a theoretical basis for the biological regulation of the microflora structure in the intestinal tract during turbot breeding.

Complete Genome Sequence of Photobacterium damselae Subsp. damselae Strain SSPD1601 Isolated from Deep-Sea Cage-Cultured Sebastes schlegelii with Septic Skin Ulcer.

Photobacterium damselae subsp. damselae (PDD) is a Gram-negative bacterium that can infect a variety of aquatic organisms and humans. Based on an epidemiological investigation conducted over the past 3 years, PDD is one of the most important pathogens causing septic skin ulcer in deep-sea cage-cultured Sebastes schlegelii in the Huang-Bohai Sea area and present throughout the year with high abundance. To further understand the pathogenicity of this species, the pathogenic properties and genome of PDD strain SSPD1601 were analyzed. The results revealed that PDD strain SSPD1601 is a rod-shaped cell with a single polar flagellum, and the clinical symptoms were replicated during artificial infection. The SSPD1601 genome consists of two chromosomes and two plasmids, totaling 4,252,294 bp with 3,751 coding sequences (CDSs), 196 tRNA genes, and 47 rRNA genes. Common virulence factors including flagellin, Fur, RstB, hcpA, OMPs, htpB-Hsp60, VasK, and vgrG were found in strain SSPD1601. Furthermore, SSPD1601 is a pPHDD1-negative strain containing the hemolysin gene hlyAch and three putative hemolysins (emrA, yoaF, and VPA0226), which are likely responsible for the pathogenicity of SSPD1601. The phylogenetic analysis revealed SSPD1601 to be most closely related to Phdp Wu-1. In addition, the antibiotic resistance phenotype indicated that SSPD1601 was not sensitive to ceftazidime, pipemidic, streptomycin, cefalexin, bacitracin, cefoperazone sodium, acetylspiramycin, clarithromycin, amikacin, gentamycin, kanamycin, oxacillin, ampicillin, and trimethoprim-sulfamethoxazole, but only the bacitracin resistance gene bacA was detected based on Antibiotic Resistance Genes Database. These results expand our understanding of PDD, setting the stage for further studies of its pathogenesis and disease prevention.

Development of a new protocol for freeze-drying preservation of Pseudoalteromonas nigrifaciens and its protective effect on other marine bacteria

Background: Freeze-drying is known as one of the best methods to preserve bacterial strains. Protectant is the key factor affecting the survival rate of freeze-dried strains. In addition, salinity, bacterial suspension concentration, drying time, and other factors can also affect the survival rate of strains to varying degrees. At present, there are relatively few studies on freeze-drying preservation of marine bacteria. In the present study, we performed the freeze-drying protectant screening and optimized the preservation conditions for Pseudoalteromonas nigrifaciens, which is widely distributed in marine environment. The protective effects of the screened protectants were verified by 18 other marine bacterial strains. Results: The results indicated that the combination of 5.0% (w/v) lactose, 5.0% (w/v) mannitol, 5.0% (w/v) trehalose, 10.0% (w/v) skim milk powder, 0.5% (w/v) ascorbic acid and 0.5% (w/v) gelatin was the best choice for the preservation of P. nigrifaciens. The suggested salinity and concentration of initial cell suspension were 10 g/L NaCl and 1.0 × 109 CFU/mL, respectively. Furthermore, stationary-phase cells were the best choice for the freeze-drying process. The highest survival rate of P. nigrifaciens reached 52.8% when using 5­10% (w/v) skim milk as rehydration medium. Moreover, the other 18 marine strains belonging to Pseudoalteromonas, Vibrio, Photobacterium, Planomicrobium, Edwardsiella, Enterococcus, Bacillus, and Saccharomyces were freezedried under the abovementioned conditions. Their survival rates were 2.3­95.1%. Conclusion: Collectively, our results supported that the protectant mixture and parameters were beneficial for lyophilization of marine bacteria

Molecular cloning and differential expression of three GnRH genes during ovarian maturation of spotted halibut, Verasper variegatus.

In this study, the gonadotropin-releasing hormone (GnRH) genes in spotted halibut were cloned and sequenced by isolating their cDNAs. The species expressed three molecular forms of GnRH in the brain: chicken-type GnRH-II (cGnRH-II), seabream-type GnRH (sbGnRH), and salmon-type GnRH (sGnRH). Phylogenetic analysis divided the molecular forms of GnRHs into three branches: cGnRH-II branch, sGnRH branch, and fish-specific GnRH branch. The spatial expression showed that they had the highest expression levels in the brain. cGnRH-II was exclusively detected in the brain, while sbGnRH had a global expression pattern in all examined organs. sGnRH was detected in the brain, pituitary, and ovary. The temporal changes of brain GnRH mRNA expression levels were examined during ovarian maturation and postspawning, and the serum steroid hormones and gonadosomatic index (GSI) were recorded. Amounts of sbGnRH mRNA substantially elevated (P < 0.05) during ovarian maturation, which concomitant with considerable elevation of GSI and serum steroids levels. On the contrary, neither sGnRH nor cGnRH-II mRNA levels showed significant changes during ovarian maturation in this study. These results suggested that these three GnRH genes are the important regulators for the differential expression of GnRH in spotted halibut, and would help us better understand the reproductive endocrine mechanism of spotted halibut.