The hepatopancreas microbiome of velvet crab, Necora puber.

Crustaceans are a valuable resource globally, both ecologically and economically, and investigations into their health are becoming increasingly important as exploitation rises. The microbiome plays a crucial role in crustacean immunity, and understanding its composition and structure can provide insights into the health of an organism and its interactions with various factors. In this study, we investigated the hepatopancreas microbiome of the velvet swimming crab, Necora puber, and compared its composition and structure with several study factors, including two different sampling points and infection with a paramyxid parasite, Paramarteilia canceri. To our knowledge, we provide the first description of a velvet crab microbiome, highlighting the dominance of a single microorganism, Candidatus hepatoplasma. We identified variations in microbiome composition between sampling points and discussed the possible processes affecting microbiome assembly. We also outline a core microbiome for the velvet crab hepatopancreas, consisting of 12 core phyla. Our study adds to the growing literature on crustacean microbiomes and provides a baseline for future investigations into the velvet crab microbiome and the health of this crustacean species.

Biological and genomic characterization of the novel bacteriophage vB_VpM-pA2SJ1, which infects Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease.

Vibrio parahaemolyticus is a major seafood-borne zoonotic pathogen that causes gastroenteritis in humans and acute hepatopancreatic necrosis disease (AHPND) in shrimp. In this study, we isolated and characterized Vibrio phage vB_VpM-pA2SJ1, which infects clinical and AHPND-associated strains of V. parahaemolyticus. The phage genome is a linear dsDNA 51,054 bp in length with a G + C content of 43.7%, and it contains 89 open reading frames. Genome comparisons revealed basal similarity to other Vibrio phages, particularly Vibrio phage vB_VpP_1, with 84.2% identity and 46% coverage. Phylogenetic analysis based on the whole genome, the terminase large subunit, and the major capsid protein revealed that phage vB_VpM-pA2SJ1 did not cluster with other known phage families, thus indicating its uniqueness.

Responses of survival, antioxidant system and intestinal microbiota of native snail Bellamya purificata to the invasive snail Pomacea canaliculata.

Pomacea canaliculata is one of the most successful invader in worldwide, adversely affecting native ecosystem through direct predation or indirect competition, while the mechanism of indirect effects on native species remain poorly understood. To clarify the effects of P. canaliculata on the native near-niche species, Bellamya purificata, a widespread freshwater gastropod in China, was selected as the research subject. The changes of mortality, histology, antioxidant system as well as the intestinal flora diversity of B. purificata were explored in present study. The results showed that the median lethal dose of P. canaliculata culture solution for B. purificata was 23.76 ind/L and a concentration-dependent damage of both the gonad and hepatopancreas were observed, the gonadal villi were dissolved and the hepatopancreas cells were broken at 20 ind/L. Furthermore, different concentrations of P. canaliculata culture solution leading to the antioxidant damage on the enzyme or non-enzyme systems of B. purificata at various degrees. Additionally, a decrease in the diversity of the intestinal flora was observed, accompanied by an increase in the abundance of pathogenic bacteria such as Pseudomonas and Aeromonas after the exposure of the culture solution of P. canaliculata. Last, after being recovered in freshwater for 24 h, the antioxidant damage of B. purificata and the disturbance of intestinal flora diversity were still not recovered especially in the high concentration group. The indirect competitive mechanism of P. canaliculata culture solution on B. purificata were explored from the aspects of tissue, biochemical level and intestinal flora, which enriched the research of P. canaliculata invasion on native snails in China, and provided new insights for the study of the invasion strategy of P. canaliculata.

Genome sequencing analysis and validation of infestation-related functional genes of Vibrio parahaemolyticus LG2206 isolated from the hepatopancreas of diseased mud crab (Scylla paramamosain) in South China.

Vibrio parahaemolyticus (V. parahaemolyticus) is a major bacterial pathogen found in brackish environments, leading to disease outbreaks and great economic losses in the mud crab industry. This study investigated the molecular mechanism of V. parahaemolyticus infecting mud crabs through genome sequencing analysis, survival experiments, and the expression patterns of related functional genes. A strain of V. parahaemolyticus with high pathogenicity and lethality was isolated from diseased mud crab in South China. The genome sequencing results showed that the genome size of V. parahaemolyticus was a circular chromosome of 3,357,271 bp, with a GC content of 45 %, containing 2985 protein-coding genes, denoted as V. parahaemolyticus LG2206. Genome analysis data revealed that a total of 113 adherence coding genes were obtained, including 120 virulence factor coding genes, 37 type III secretion system (T3SS) coding genes, and 277 sequences of T3SS effectors. Survival experiments showed that the mortality was 20 % within 96 h in the 1 × 104 CFU/mL infection group, 90 % in the 3.2 × 105 CFU/mL treatment group, and 100 % in the 1 × 106 CFU/mL treatment group. The LD50 of V. parahaemolyticus LG2206 was determined as 4.6 × 104 CFU/mL. Six genes of znuA and fliD (flagellin encoding genes), yscE and yscR (T3SS encoding genes), and nfuA and htpX (virulence factor encoding genes) were selected and validated by quantitative real-time PCR analysis after infection with 4.6 × 104 CFU/mL of V. parahaemolyticus LG2206 for 96 h. The expression of the six genes exhibited a significant up-regulation trend at all tested time points. The results indicated that the infestation-related genes screened in the experiment play important roles in the infestation process. This study provides timely and effective information to further analyze the molecular mechanism of V. parahaemolyticus infection and develop comprehensive measures for disease prevention and control.

Shewanella putrefaciens as an emerging pathogen of hepatopancreas necrosis disease in Chinese mitten crab Eriocheir sinensis.

Shewanella putrefaciens has been recognized as an emerging important pathogen in aquaculture. However, scarce information is available on the characterization and microbial control of S. putrefaciens as a causal agent of hepatopancreas necrosis disease in Chinese mitten crab Eriocheir sinensis. In this study, a multi-resistant S. putrefaciens isolate (DZ-A) was identified as a causal pathogen of hepatopancreas necrosis disease in Chinese mitten crabs. It showed a lethal dose (LD50) of 2.20 × 105 CFU ml-1 in Chinese mitten crabs, and multiple resistance to aminoglycoside, chloramphenicol, macrolide, penicillin, peptide, and tetracycline antimicrobials. In addition, Bdellovibrio powder exhibited a significant antibacterial effect against the pathogenic S. putrefaciens, and conferred significant protection to challenged Chinese mitten crabs with relative percentage survivals of 80.00% to 93.33% via significant improvement in their immune response and antioxidant capability. The findings of this study provide valuable insights into the phenotypic characterization and biological control of pathogenic S. putrefaciens in Chinese mitten crabs.

Characteristics and pathogenicity of Vibrio alginolyticus SWS causing high mortality in mud crab (Scylla serrata) aquaculture in Hong Kong.

Introduction: Vibrio alginolyticus is a Gram-negative, rod-shaped bacterium belonging to the family of Vibrionaceae, a common pathogen in aquaculture animals, However, studies on its impact on Scylla serrata (mud crabs) are limited. In this study, we isolated V. alginolyticus SWS from dead mud crab during a disease outbreak in a Hong Kong aquaculture farm, which caused up to 70% mortality during summer. Methods: Experimental infection and histopathology were used to investigate the pathogenicity of V. alginolyticus SWS in S. serrata and validate Koch's postulates. Comprehensive whole-genome analysis and phylogenetic analysis antimicrobial susceptibility testing, and biochemical characterization were also performed. Results: Our findings showed that V. alginolyticus SWS caused high mortality (75%) in S. serrata with infected individuals exhibiting inactivity, loss of appetite, decolored and darkened hepatopancreas, gills, and opaque muscle in the claw. Histopathological analysis revealed tissue damage and degeneration in the hepatopancreas, gills, and claw muscle suggesting direct and indirect impacts of V. alginolyticus SWS infection. Conclusions: This study provides a comprehensive characterization of V. alginolyticus SWS as an emerging pathogen in S. serrata aquaculture. Our findings underscore the importance of ongoing surveillance, early detection, and the development of targeted disease management strategies to mitigate the economic impact of vibriosis outbreaks in mud crab aquaculture.

Effects of a phytobiotic-based additive on the growth, hepatopancreas health, intestinal microbiota, and Vibrio parahaemolyticus resistance of Pacific white shrimp, Litopenaeus vannamei.

Vibrio genus is a common pathogen in aquaculture and causes acute hepatopancreatic necrosis disease (AHPND) and massive mortality of shrimp. Many studies have suggested that a single functional ingredient such as plant extract or organic acid can reduce the dependence on antibiotics and promote the growth and immunity of aquatic animals. In this study, we evaluated the effects of a phytobiotic-based compound additive (Sanacore® GM, SNGM), which had a successful trajectory of commercial application in fish farming. However, its effects on the hepatopancreas health and intestinal microbiota of shrimp after Vibrio challenge have not been well evaluated. In the present study, Pacific white shrimp were fed diets with or without supplementation of SNGM, and the SNGM grades were 0-g/kg (CON), 3-g/kg (SNGM3), and 5-g/kg (SNGM5) diets. The feed trial lasted 60 days, after which a Vibrio parahaemolyticus challenge was performed. The results showed that compared to the CON group, both the SNGM3 and SNGM5 groups had a significantly higher weight gain and a lower feed conversion ratio as well as higher survival after Vibrio parahaemolyticus challenge. In the growth trial, the SNGM3 group had a significantly increased total protein, albumin concentration, and acid phosphatase activity in hemolymph compared to the CON group. In the challenge experiment, the SNGM3 and SNGM5 groups had increased albumin and glucose contents as well as the activities of phenoloxidase, lysozyme, alkaline phosphatase, and superoxide dismutase in hemolymph. Both the SNGM3 and SNGM5 groups had improved morphology of the hepatopancreas and intestine. The SNGM5 group had alleviated gut microbiota dysbiosis induced by Vibrio infection by increasing the potential probiotic bacterium abundance (Shewanella) and decreasing the potential pathogenic bacteria abundance (Vibrio, Photobacteriuma, Pseudoalteromonas, and Candidatus_Bacilloplasma). In conclusion, the dietary phytobiotic-based additive at 3-g/kg level increased the growth and Vibrio parahaemolyticus resistance of Pacific white shrimp by promoting immune-related enzyme activities and improving the morphological structure of the hepatopancreas and intestine and the intestinal microbiota composition.

Unveiling the Bacterial Community across the Stomach, Hepatopancreas, Anterior Intestine, and Posterior Intestine of Pacific Whiteleg Shrimp.

The gastrointestinal (GI) tract of shrimp, which is comprised of the stomach, hepatopancreas, and intestine, houses microbial communities that play crucial roles in immune defense, nutrient absorption, and overall health. While the intestine's microbiome has been well-studied, there has been limited research investigating the stomach and hepatopancreas. The present study addresses this gap by profiling the bacterial community in these interconnected GI segments of Pacific whiteleg shrimp. To this end, shrimp samples were collected from a local aquaculture farm in South Korea, and 16S rRNA gene amplicon sequencing was performed. The results revealed significant variations in bacterial diversity and composition among GI segments. The stomach and hepatopancreas exhibited higher Proteobacteria abundance, while the intestine showed a more diverse microbiome, including Cyanobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Chloroflexi, and Verrucomicrobia. Genera such as Oceaniovalibus, Streptococcus, Actibacter, Ilumatobacter, and Litorilinea dominated the intestine, while Salinarimonas, Sphingomonas, and Oceaniovalibus prevailed in the stomach and hepatopancreas. It is particularly notable that Salinarimonas, which is associated with nitrate reduction and pollutant degradation, was prominent in the hepatopancreas. Overall, this study provides insights into the microbial ecology of the Pacific whiteleg shrimp's GI tract, thus enhancing our understanding of shrimp health with the aim of supporting sustainable aquaculture practices.

Glycosaminoglycan lyase: A new competition between bacteria and the pacific white shrimp Litopenaeus vannamei.

Horizontal gene transfer (HGT) is an important evolutionary force in the formation of prokaryotic and eukaryotic genomes. In recent years, many HGT genes horizontally transferred from prokaryotes to eukaryotes have been reported, and most of them are present in arthropods. The Pacific white shrimp Litopenaeus vannamei, an important economic species of arthropod, has close relationships with bacteria, providing a platform for horizontal gene transfer (HGT). In this study, we analyzed bacteria-derived HGT based on a high-quality genome of L. vannamei via a homology search and phylogenetic analysis, and six HGT genes were identified. Among these six horizontally transferred genes, we found one gene (LOC113799989) that contains a bacterial chondroitinase AC structural domain and encodes an unknown glycosaminoglycan (GAG) lyase in L. vannamei. The real-time quantitative PCR results showed that the mRNA expression level of LOC113799989 was highest in the hepatopancreas and heart, and after stimulation by Vibrio parahaemolyticus, its mRNA expression level was rapidly up-regulated within 12 h. Furthermore, after injecting si-RNA and stimulation by V. parahaemolyticus, we found that the experimental group had a higher cumulative mortality rate in 48 h than the control group, indicating that the bacteria-derived GAG lyase can reduce the mortality of shrimp with respect to infection by V. parahaemolyticus and might be related to the resistance of shrimp to bacterial diseases. Our findings contribute to the study of the function of GAGs and provide new insights into GAG-related microbial pathogenesis and host defense mechanisms in arthropods.

Proteome analysis of outer membrane vesicles from Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease.

A specific strain of Vibrio parahaemolyticus (VpAHPND) causes acute hepatopancreatic necrosis disease (AHPND), leading to significant losses in shrimp aquaculture. Outer membrane vesicles (OMVs) are naturally secreted by Gram-negative bacteria, and their significant roles in host-pathogen interactions and pathogenicity have been recognized. In the present study, OMVs were isolated from VpAHPND by differential-ultracentrifugation and used for proteomics analysis. In the Nano-HPLC-MS/MS analysis, totally 645 proteins were determined, including virulence factors, immunogenic proteins, outer membrane protein, bacterial secretory proteins, ribosomal proteins, protease, and iron regulation proteins. Furthermore, GO and KEGG annotations indicated that proteins identified in VpAHPND-OMVs are involved in metabolism, regulation of multiple biological processes, genetic information processes, immunity and more. Meanwhile, toxin proteins PirAvp and PirBvp, associated with VpAHPND pathogenicity, were also identified in the proteome of VpAHPND-OMVs. Our objective is to identify the protein composition of OMVs released by VpAHPND, analyzing the potential for cytotoxicity and immunomodulatory activity of these granule hosts. This study is crucial for understanding the roles played by bacterial-derived vesicles in the disease process, given that these vesicles carry relevant activities inherent to the bacteria that produce them.

The assembly of gut microbiota implicates shrimp acute hepatopancreas necrosis disease progression.

Ample evidence shows dysbiosis in the gut microbiota when comparing healthy shrimp with those affected by severe acute hepatopancreatic necrosis disease (AHPND). However, the static comparison used in available studies leads to the uncertainties regarding how and to what extent the gut microbiota responds to the progressive severity of AHPND. In addition, shrimp AHPND is featured by rapid and massive mortality, thus the initiation of AHPND must be diagnosed for preemptive therapy. For these reasons, we explored the ecological assembly of gut microbiota over shrimp AHPND progression. Increasing AHPND severity was associated with linear increase in the copies of pirAB genes, relative abundance of gut Vibrio and potentially pathogenic, and reduction in the gut bacterial diversity, stability, and relative abundance of Bdellovibrio. Negative and significant association between gut Vibrio and Bdellovibrio were noted, indicating that compromised predation exerts a role in AHPND progression. Notably, the extents of departure to the healthy shrimp gut microbiota were positively coupled with the increasing severity of AHPND. After controlling the temporal variation in the gut microbiota as healthy shrimp age, we constructed a diagnosis model that accurately diagnosed the initial, progressed or moribund stages of AHPND, with an overall accuracy of 86.5%. Shrimp AHPND induced more stochastic gut microbiotas as a consequence of the attenuated ability of diseased shrimp to select their commensals, resulting in convergent bacterial communities between gut and rearing water over AHPND progression. Collectively, our findings provide important step toward the ecological assembly of gut microbiota implicating in AHPND etiology and in diagnosing AHPND stages. KEY POINTS: • The departure of shrimp gut microbiota positively linked with AHPND severity. • The diagnosis model accurately diagnosed the stages of AHPND. • Shrimp AHPND induced more stochastic gut microbiota.

Effect of functional diets on intestinal microbiota and resistance to Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND) of Pacific white shrimp (Penaeus vannamei).

AIMS: The present study evaluated the effect of four functional diets and a reference diet on the survival and intestinal bacterial community of shrimp Penaeus vannamei infected with acute hepatopancreatic necrosis disease (AHPND). METHODS AND RESULTS: After 42 days of feeding trail, shrimp were inoculated with a Vibrio parahaemolyticus (CIB-0018-3) carrying the plasmid encoding for the PirAB toxins responsible for AHPND. After 120 h postinfection (hpi), shrimp fed with a diet containing 2% of a mix with Curcuma longa and Lepidium meyenii (TuMa) and a diet containing 0.2% of vitamin C (VitC) showed a significantly higher survival (85%) compared to the remaining treatments (50%-55%) (p < 0.05). Infected shrimp fed with TuMa diet, showed a significant reduction of Vibrionales, and VitC diet promoted an increase of Alteromonadales. CONCLUSIONS: Our findings suggest that the TuMa diet conferred protection against AHPND and could be attributed to a combined effect of antibacterial properties against Vibrionales, and promoting a desirable bacterial community in the shrimp intestine, while the VitC diet protection could be attributed to their antioxidant capacity and in a lower proportion to a bacterial modulation in shrimp gut. SIGNIFICANCE AND IMPACT OF THE STUDY: Acute hepatopancreatic necrosis disease is a devastating disease that significantly affects aquaculture production of shrimps. Therefore, the use of functional diets that promote resistance to AHPND represents a valuable tool to reduce the mortality of farmed shrimp.

Microbiome of the Successful Freshwater Invader, the Signal Crayfish, and Its Changes along the Invasion Range.

Increasing evidence denotes the role of the microbiome in biological invasions, since it is known that microbes can affect the fitness of the host. Here, we demonstrate differences in the composition of an invader's microbiome along the invasion range, suggesting that its microbial communities may affect and be affected by range expansion. Using a 16S rRNA gene amplicon sequencing approach, we (i) analyzed the microbiomes of different tissues (exoskeleton, hemolymph, hepatopancreas, and intestine) of a successful freshwater invader, the signal crayfish, (ii) compared them to the surrounding water and sediment, and (iii) explored their changes along the invasion range. Exoskeletal, hepatopancreatic, and intestinal microbiomes varied between invasion core and invasion front populations. This indicates that they may be partly determined by population density, which was higher in the invasion core than in the invasion front. The highly diverse microbiome of exoskeletal biofilm was partly shaped by the environment (due to the similarity with the sediment microbiome) and partly by intrinsic crayfish parameters (due to the high proportion of exoskeleton-unique amplicon sequence variants [ASVs]), including the differences in invasion core and front population structure. Hemolymph had the most distinct microbiome compared to other tissues and differed between upstream (rural) and downstream (urban) river sections, indicating that its microbiome is potentially more driven by the effects of the abiotic environment. Our findings offer an insight into microbiome changes during dispersal of a successful invader and present a baseline for assessment of their contribution to an invader's overall health and its further invasion success. IMPORTANCE Invasive species are among the major drivers of biodiversity loss and impairment of ecosystem services worldwide, but our understanding of their invasion success and dynamics still has many gaps. For instance, although it is known that host-associated microbial communities may significantly affect an individual's health and fitness, the current studies on invasive species are mainly focused on pathogenic microbes, while the effects of the remaining majority of microbial communities on the invasion process are almost completely unexplored. We have analyzed the microbiome of one of the most successful crayfish invaders in Europe, the signal crayfish, and explored its changes along the signal crayfish invasion range in the Korana River, Croatia. Our study sets the perspective for future research required to assess the contribution of these changes to an individual's overall health status and resilience of dispersing populations and their impact on invasion success.

Therapeutic effect and immune mechanism of chitosan-gentamicin conjugate on Pacific white shrimp (Litopenaeus vannamei) infected with Vibrio parahaemolyticus.

To explore the disease resistance mechanism of chitosan conjugates, chitosan-gentamicin conjugate (CS-GT) was synthesized and systematically characterized, the immune mechanism of CS-GT on Litopenaeus vannamei infected with Vibrio parahaemolyticus was further explored. The results showed that imine groups in CS-GT were effectively reduced. Dietary supplementation of CS-GT can significantly increase the survival rate, total hemocyte counts, the antioxidant and immune related enzyme activity levels of shrimps (P < 0.05), which are all dose-dependent under the experimental conditions. In addition, CS-GT can protect the hepatopancreas from invading bacteria and alleviate inflammation. Particularly, CS-GT promotes the expressions of legumain (LGMN), lysosomal acid lipase (LIPA) and Niemann-Pick type C2 (NPC2) up-regulated. It is speculated that CS-GT may stimulate the lysosome to phagocytose pathogens more effectively. In conclusions, shrimps fed with CS-GT can produce immune response via lysosome and greatly improve the disease resistance to Vibrio parahaemolyticus.

Etiological characteristics of "tail blister disease" of Australian redclaw crayfish (Cherax quadricarinatus).

In November 2019, an acute disease outbreak in Australian redclaw crayfish (Cherax quadricarinatus) occurred in a farm in Hubei, China, with a cumulative mortality rate of over 80%. One of the characteristic symptoms of the disease was blisters on the tail. This symptom is also common in diseased Procambarus clarkii every year in this country, but the causative agent has not been determined. This study analyzed the etiological characteristics of this disease. Bacterial isolation and identification combined with high-throughput sequencing analysis were conducted to obtain the microbiota characteristics in the hemolymph, hepatopancreas, and intestines. Results showed that this outbreak was caused by infection from Aeromonas hydrophila and Aeromonas veronii. The underlying cause was stress imposed on crayfish during transferring from outdoor pond to indoor pond because of temperature drops. Aeromonas infection caused remarkable changes in the structure of the microbial composition in the hemolymph, hepatopancreas, and intestines of the crayfish. The abundance of Aeromonas in the hemolymph of the sick crayfish was as high as 99.33%. In particular, KEGG metabolic pathway analysis showed that some antibiotic synthesis, enterobactin biosynthesis, and myo-inositol degradation pathways were abundant in healthy crayfish hemolymphs, which may be the mechanism of maintaining crayfish health. Conversely, inhibition of these pathways led to the disorder of microbiota structure, finally leading to the occurrence of diseases. To the knowledge of the authors, this study was the first to use high-throughput amplicon sequencing targeting the 16S rRNA gene to find the causative bacteria in aquatic animals. This protocol can provide more comprehensive and reliable evidence for pathogen identification, even if the pathogenic bacteria are anaerobes or other hard-to-culture bacteria.

Differentially Expressed Genes in Hepatopancreas of Acute Hepatopancreatic Necrosis Disease Tolerant and Susceptible Shrimp (Penaeus vannamei).

Acute hepatopancreatic necrosis disease (AHPND) is a lethal disease in marine shrimp that has caused large-scale mortalities in shrimp aquaculture in Asia and the Americas. The etiologic agent is a pathogenic Vibrio sp. carrying binary toxin genes, pirA and pirB in plasmid DNA. Developing AHPND tolerant shrimp lines is one of the prophylactic approaches to combat this disease. A selected genetic line of Penaeus vannamei was found to be tolerant to AHPND during screening for disease resistance. The mRNA expression of twelve immune and metabolic genes known to be involved in bacterial pathogenesis were measured by quantitative RT-PCR in two populations of shrimp, namely P1 that showed susceptibility to AHPND, and P2 that showed tolerance to AHPND. Among these genes, the mRNA expression of chymotrypsin A (ChyA) and serine protease (SP), genes that are involved in metabolism, and crustin-P (CRSTP) and prophenol oxidase activation system 2 (PPAE2), genes involved in bacterial pathogenesis in shrimp, showed differential expression between the two populations. The differential expression of these genes shed light on the mechanism of tolerance against AHPND and these genes can potentially serve as candidate markers for tolerance/susceptibility to AHPND in P. vannamei. This is the first report of a comparison of the mRNA expression profiles of AHPND tolerant and susceptible lines of P. vannamei.

Dietary supplementation with probiotic Rhodobacter sphaeroides SS15 extract to control acute hepatopancreatic necrosis disease (AHPND)-causing vibrio parahaemolyticus in cultivated white shrimp.

Cultivation of Penaeus vannamei (Pacific white shrimp) is faced with the serious problem of acute hepatopancreatic necrosis disease (AHPND), caused by Vibrio parahaemolyticus that carries plasmids containing binary toxin genes. The disease is typically moderated by the use of antibiotics. To investigate the control of AHPND and maintenance of water quality without the use of antibiotics, the supplementation of shrimp feed with anti-vibrio compounds from a crude extract of probiotic Rhodobacter sphaeroides SS15 was evaluated. The experimental design comprised four treatments: two that were challenged with AHPND-causing V. parahaemolyticus SR2 at a density of 6.0 × 105 cells mL-1 and two that were not challenged. The unchallenged groups comprised a control group that received commercial feed only (CF) and a group that received CF supplemented with 0.27% (w/w) of the extract of R. sphaeroides SS15 (modified CF: MCF). The treatments challenged with V. parahaemolyticus SR2 comprised a challenge group that received CF only (challenge CF: CF-SR2) and a challenge group that received modified CF (challenge MCF: MCF-SR2). V. parahaemolyticus SR2 was inoculated at the start of cultivation and at day 48 at the same cell density. No significant difference in growth performance was found among all treatments. All water quality parameters were better in the two treatments that received modified CF but excess nitrite, due to overfeeding in low salinity (5-8 ppt), caused shrimp mortality in all treatments. Vibrio populations were much higher in the CF treatments than in the modified CF treatments. After the first challenge, the survival rate was about 67% in both the CF-SR2 and MCF-SR2 treatments, compared with approximately 83% in the unchallenged treatments. One day after the second challenge, mortality in the CF-SR2 treatment was 100%, whereas 16.67% survived in the MCF-SR2 treatment. The survival rate was roughly 27% higher in the MCF treatment than in the CF treatment. The hepatopancreas and gut of both modified CF treatments showed no sign of AHPND. Via better water quality and trained immunity, the anti-vibrio compounds in the modified CF have great potential to increase the survival of cultivated shrimp infected with AHPND-causing strain SR2.

OTUs and ASVs Produce Comparable Taxonomic and Diversity from Shrimp Microbiota 16S Profiles Using Tailored Abundance Filters.

The interplay between shrimp immune system, its environment, and microbiota contributes to the organism's homeostasis and optimal production. The metagenomic composition is typically studied using 16S rDNA profiling by clustering amplicon sequences into operational taxonomic units (OTUs) and, more recently, amplicon sequence variants (ASVs). Establish the compatibility of the taxonomy, α, and ß diversity described by both methods is necessary to compare past and future shrimp microbiota studies. Here, we used identical sequences to survey the V3 16S hypervariable-region using 97% and 99% OTUs and ASVs to assess the hepatopancreas and intestine microbiota of L. vannamei from two ponds under standardized rearing conditions. We found that applying filters to retain clusters >0.1% of the total abundance per sample enabled a consistent taxonomy comparison while preserving >94% of the total reads. The three sets turned comparable at the family level, whereas the 97% identity OTU set produced divergent genus and species profiles. Interestingly, the detection of organ and pond variations was robust to the clustering method's choice, producing comparable α and ß-diversity profiles. For comparisons on shrimp microbiota between past and future studies, we strongly recommend that ASVs be compared at the family level to 97% identity OTUs or use 99% identity OTUs, both using tailored frequency filters.

Crayfish (Cherax quadricarinatus) susceptibility to acute hepatopancreatic necrosis disease (AHPND).

Acute hepatopancreatic necrosis disease (AHPND) is an OIE-listed enteric disease that has continued to plague the shrimp aquaculture industry since its first discovery in 2009. AHPND is one of the biggest disease threats to the shrimp aquaculture industry along with white spot disease (WSD) which has severely impacted both crayfish and shrimp aquaculture. AHPND is caused by specific marine Vibrio spp. which carry plasmid-borne binary toxins PirAVp and PirBVp. This research investigated if crayfish are susceptible to AHPND-causing Vibrio parahaemolyticus (VpAHPND) to discern the potential risk that AHPND may pose to the crayfish aquaculture industry. Susceptibility was investigated by challenging Cherax quadricarinatus (Australian red claw crayfish) and Penaeus vannamei (Pacific white shrimp) with VpAHPND in a cohabitation immersion bioassay. Upon termination of the bioassay, crayfish survival was significantly higher than shrimp survival (87% vs. 33%). Hepatopancreas dissected from experimentally challenged animals were screened for the binary toxin genes pirAVp and pirBVp by real-time and duplex conventional PCR assays, and also were examined by H&E histology for the detection of characteristic AHPND pathology. Although AHPND toxin genes pirAVp and pirBVp were detected in a subset of crayfish samples, histopathology did not reveal any pathognomonic lesions that are characteristic of AHPND in any crayfish samples examined. These findings suggest that crayfish are likely resistant to AHPND.