Search

The dinoflagellate Noctiluca scintillans is a globally distributed bloom-forming species. Previous studies have shown that the primary reason for the frequent occurrence of N. scintillans blooms may be the proliferation of microalgae due to eutrophication, which provides a sufficient source of food. Meanwhile, N. scintillans may release nutrients into the environment, thus affecting the population dynamics of microalgae. Thus, to investigate the interaction between N. scintillans and co-occurring microalgae, this study examined the population dynamics of N. scintillans and their interaction with three representative microalgae species in response to varying nutrient levels. The findings indicate that the growth of N. scintillans is slow when co-cultured with diatom Skeletonema costatum. Moreover, a high density and rapid growth rate of S. costatum may have an inhibitory effect on the growth of N. scintillans. Conversely, the population abundance of N. scintillans increased with the rise in the population density and nutritional level of Heterocapsa steinii (dinoflagellate) and Heterosigma akashiwo (raphidophyceae). Notably, N. scintillans can discharge specific nutrients into the aquatic environment, which can subsequently be assimilated and exploited by H. steinii. Thus, the interaction between the species and population dynamics of plankton, as well as changes in nutrient levels within the ecosystem, played a significant role in influencing the growth and population dynamics of N. scintillans. The mutualistic association between N. scintillans and microalgae may establish a transient closed loop, thereby fostering the sustained proliferation and subsequent expansion of N. scintillans.

In future space-borne gravitational wave (GW) detectors, time delay interferometry (TDI) will be utilized to reduce the overwhelming noise, including the laser frequency noise and the clock noise etc., by time shifting and recombining the data streams in post-processing. The successful operation of TDI relies on absolute inter-satellite ranging with meter-level precision. In this work, we numerically and experimentally demonstrate a strategy for inter-satellite distance measurement. The distances can be coarsely determined using the technique of arm-locking ranging with a large non-ambiguity range, and subsequently TDI can be used for precise distance measurement (TDI ranging) by finding the minimum value of the power of the residual noises. The measurement principle is introduced. We carry out the numerical simulations, and the results show millimeter-level precision. Further, we perform the experimental verifications based on the fiber link, and the distances can be measured with better than 0.05 m uncertainty, which can well satisfy the requirement of time delay interferometry.

The raphidophyte Heterosigma akashiwo is a harmful algal species. The bloom of this organism has been associated with the massive mortality of fish in many coastal waters. To investigate the molecular mechanism of H. akashiwo blooms, having a reliable reference transcriptome of this species is essential. Therefore, in this study, a full-length transcriptome of H. akashiwo was obtained by single-molecule real-time sequencing. In total, 45.44 Gb subread bases were generated, and 16,668 unigenes were obtained after the sequencing data processing. A total of 8666 (52.00%) unigenes were successfully annotated using seven public databases. Among them, mostly phosphorus and nitrogen metabolism genes were detected. Moreover, there were 300 putative transcription factors, 4392 putative long non-coding RNAs, and 7851 simple sequence repeats predicted. This study provides a valuable reference transcriptome for understanding how H. akashiwo blooms at a molecular level.

The ichthyotoxic raphidophyte Heterosigma akashiwo is associated with harmful algal blooms (HABs). Although the influence of H. akashiwo on fish has been reported, little is known of its effects on other marine organisms, such as shellfish. In this study, the physiological and transcriptomic responses of the Asiatic hard clam Meretrix meretrix to H. akashiwo were investigated. The results showed that clam survival was not influenced by H. akashiwo (at a concentration of 2 × 104 cells/mL), and M. meretrix eliminated the alga effectively after 48 h in co-culture. H. akashiwo did not alter the antioxidant capacity of the clams. However, 544 unigenes were found to be differentially expressed between the control and H. akashiwo-treated clams. Interestingly, the significant up-regulation of lysosome-related genes and transport proteins suggested remodeling of the clam metabolism possibly for digestion of H. akashiwo. In summary, our results indicate that the clam M. meretrix can effectively feed on H. akashiwo, and intensification of shellfish farming may be helpful to control and mitigate H. akashiwo blooms.

In the future space-borne gravitational wave (GW) detector, the optical transponder scheme, i.e., the phase-locking scheme, will be utilized so as to maintain the signal-to-noise ratio (SNR). In this case, the whole constellation will share one common laser equivalently, which enables the considerable simplification of time delay interferometry (TDI) combinations. Recently, and remarkably, the unique combination of TDI and optical frequency comb (OFC) has shown a bright prospect for the future space-borne missions. When the laser frequency noise and the clock noise are synchronized using OFC as the bridge, the data streams will be reasonably simplified. However, in the optical transponder scheme, the weak-light phase-locking (WLPL) loops could bring additional noises. In this work, we analyze the phase-locking scheme with OFC and transfer characteristics of the noises including the WLPL noise. We show that the WLPL noise can be efficiently reduced by using the specific TDI combination, and the cooperation of phase-locking and frequency combs can greatly simplify the post-processing.

The surface mechanical properties of the Selective Laser Melting (SLM) formed Ti6Al4V samples were improved by adopting a novel laser cleaning pretreatment process combined with a jet electrodeposition process. This paper aimed to investigate the effects of different laser powers on the morphologies and adhesions of the nickel coatings. The advantages of the laser cleaning process are no grinding, no contact, high efficiency and environmental protection. The morphologies, adhesion, wear resistance, and hardness of the coatings were characterized. The results indicate that when the laser energy density reached 20% (4 J/cm2), the contaminations on the substrate and the oxide layer were removed and the crystalline grain of the coating was 15.3 nm. The shallow pits generated by laser burning increased the adhesion of the coatings. In addition, when the laser energy density increased to 6 J/cm2, a yellow oxide layer was produced on the surface of the cleaned titanium alloy. Moreover, the wear resistance of the titanium alloy after the nickel plating was improved. The wear volume was only 0.046 mm3, and the hardness increased to 1967.6 N/mm2.

Different viruses are abundant in aquatic ecosystems. There has been limited research on the viral communities in the upper reaches of the Yangtze River. Yellow catfish (Pelteobagrus fulvidraco), an important economic fish that is widely distributed in the upper reaches of the Yangtze River, was selected as the research object. Using RNA sequencing, we identified 11 viruses belonging to the Adintoviridae, Tombusviridae, Caudovirales, Microviridae, Picornavirales, and other bacteriophage families. The predominant viral families/order in Luzhou (LZ), Fuling (FL), and Wanzhou (WZ) were Caudovirales, Adinoviridae, and Microviridae, respectively. The virome from WZ had a unique community composition, with a high abundance of Picornavirales compared with LZ and FL. In LZ, the predominant double-stranded RNA virus family was Siphoviridae. Phylogenetic analyses showed that viruses presented high genetic diversity. Phylogenetically, Wenling pleuronectiformes picornavirus was close to the family Caliciviridae, which includes yellow catfish calicivirus (YcCV), responsible for the massive mortality of yellow catfish in 2020. This study provides insights into the viral community composition in yellow catfish in the upper reaches of the Yangtze River, revealing a diverse and unique river water virome and providing clues for future research on the origin of viral pathogens.

Aeromonas veronii, an opportunistic pathogen toward aquatic organisms, was identified as the causative pathogen (isolate WH10) in diseased bronze gudgeon via bacterial isolation, and morphological, physiological, biochemical, and molecular characterization. WH10 exerted its pathogenicity via five virulence genes, including those encoding cytotoxic enterotoxins (act and alt), lipase (lip), a quorum sensing-controlled virulence factor (LuxS), and a Type III secretion system inner membrane component (ascV). WH10 was shown to be sensitive to compound sulfamethoxazoles, cefothiophene, doxycycline, and sulfamethoxazole. Toward bronze gudgeon, WH10 had a median lethal dose (LD50) of 1.36 × 106 colony forming units/mL. Analysis of blood parameters of diseased fish revealed significant increases in monocytes and neutrophils, but decreased numbers of lymphocytes. Serum aspartate aminotransferase activity and triglyceride concentration were significantly higher in diseased fish than in healthy fish. The reverse was noted for alkaline phosphatase, total protein, albumin, total cholesterol, and glucose. Thus, Aeromonas veronii is implicated as the causative agent of the mass mortality observed in bronze gudgeon, warranting further investigations into the diagnosis, epidemiology, prevention, and treatment of this infectious disease.

Rhinogobio ventralis is a rare fish found in the Yangtze River in China and has significant ecological and economic value. In this study, a bacterial strain (RV-JZ01) was isolated from the livers of diseased R. ventralis. This isolate was identified as Aeromonas veronii based on its morphology, biochemical features and 16S rDNA phylogenetic analysis. The artificial infection of healthy R. ventralis (16 ± 2 cm) with RV-JZ01 resulted in the manifestation of clinical symptoms, in accordance with those of naturally infected fish. The 50% lethal dose (LD50) of RV-JZ01 for R. ventralis was 6.3 × 106 CFU/mL. Histopathological examination revealed various pathological changes in the diseased fish, including intestinal villus swelling and rupture, hepatocyte vacuolization, renal tubular cell nuclear enlargement and pyknosis, and myocardial fiber fracture and atrophy. RV-JZ01 infection significantly reduced the gut flora diversity of R. ventralis, with the relative abundances of Firmicutes and Fusobacteria increasing, and those of the Proteobacteria and Bacteroidetes decreasing. The abundance of Lactobacillus and Streptococcus dramatically increased, and the abundance of Clostridium and Escherichia reduced in the intestinal microbiota of R. ventralis infected with RV-JZ01. Antibiotic sensitivity testing revealed that RV-JZ01 was highly susceptible to 12 antimicrobials, including erythromycin, cefalexin, norfloxacin, furazolidone, sulfonamides, enrofloxacin, doxycycline, piperacillin, florfenicol, gentamicin, and lincomycin. These results contribute to the understanding of pathological alterations in R. ventralis following A. veronii infection, offering valuable data to support the implementation of disease treatment.

Objective: Non-alcoholic fatty liver disease (NAFLD) is strongly associated with hyperlipidemia, which is closely related to high levels of sugar and fat. β-sitosterol is a natural product with significant hypolipidemic and cholesterol-lowering effects. However, the underlying mechanism of its action on aquatic products is not completely understood. Methods: A high-fat diet (HFD)-induced NAFLD zebrafish model was successfully established, and the anti-hyperlipidemic effect and potential mechanism of β-sitosterol were studied using oil red O staining, filipin staining, and lipid metabolomics. Results: β-sitosterol significantly reduced the accumulation of triglyceride, glucose, and cholesterol in the zebrafish model. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that differential lipid molecules in β-sitosterol mainly regulated the lipid metabolism and signal transduction function of the zebrafish model. β-sitosterol mainly affected steroid biosynthesis and steroid hormone biosynthesis in the zebrafish model. Compared with the HFD group, the addition of 500 mg/100 g of β-sitosterol significantly inhibited the expression of Ppar-γ and Rxr-α in the zebrafish model by at least 50% and 25%, respectively. Conclusions: β-sitosterol can reduce lipid accumulation in the zebrafish model of NAFLD by regulating lipid metabolism and signal transduction and inhibiting adipogenesis and lipid storage.

The microbial community structure in aquaculture water plays an important role in the intestinal microbial diversity of aquatic animals. The Chinese soft-shelled turtle (SST) (Trionyx sinensis) is an important aquaculture species of high economic value in the Asia-Pacific region. An intuitive understanding of the microbial diversity and abundances of SST aquaculture is crucial for comprehending these ecosystems. Herein, the evolutionary characteristics of the bacterial communities in the SST and its aquaculture water systems were investigated using Illumina MiSeq sequencing. This experiment sampled nine SSTs from a pond outside a greenhouse and was repeated three times. The sequencing results revealed significant differences in the microflora composition at the phylum and genus levels in both the intestine and aquaculture water of the SSTs in the greenhouse and pond aquaculture environments. A total of 1039 genera belonging to 65 phyla were identified. At the phylum level, the relative abundances of Chloroflexi (24%), Acidobacteria (5%), and Nitrospira (3%) were higher in the greenhouse water than in the pond water. The relative abundances of Bacteroidetes (35%), Actinobacteria (8%), and Cyanobacteria (4%) were higher in the pond water than in the greenhouse water. The intestinal microorganisms in the SSTs experienced significant changes after the SSTs were transferred from a greenhouse culture to a pond culture environment for 28 days. After the SSTs were cultured in the ponds, we observed decreases in the relative abundances of Actinobacteria (39% to 25%), Cyanobacteria (24% to 0.8%), Chlorobacteria (9% to 3%), and Firmicutes (5.5% to 0.8%. However, we observed increases in the relative abundances of Bacteroidetes (2% to 35%) and Acidobacteria (0.3% to 25%). These results showed that the bacterial diversity and richness compositions in the intestinal tract and aquaculture water were the same. However, the relative abundances of bacterial communities varied. The results of this study are of great significance in understanding how the environment affects SST cultures. These data may provide valuable instructions for Chinese soft-shelled turtle aquaculture management.

Grass carp reovirus genotype Ⅱ (GCRV Ⅱ) causes a variety of fish hemorrhagic disease, which seriously affects the sustainable development of grass carp aquaculture in China. Rare minnow (Gobiocypris rarus) is an ideal model fish to study the pathogenesis of GCRV Ⅱ. To investigate the involved molecular responses against the GCRV Ⅱ infection, we performed comparative transcriptomic analysis in the spleen and liver of rare minnow injected with virulent strain DY197 and attenuated strain QJ205. Results showed that the virulent DY197 strain induced more differently expressed genes (DEGs) than the attenuated QJ205 strain, and tissue-specific responses were induced. In the spleen, the attenuated and virulent strains induced different DEGs; the attenuated QJ205 infection activated steroid synthesis pathway that involved in membrane formation; however, virulent DY197 infection activated innate immunity and apoptosis related pathways while suppressing cell proliferation and migration related pathways that are important for damage tissue repair, as well as hemorrhage related pathways. In the liver, the attenuated and virulent strains infection induced similar DEGs; both strains infection activated immunity and apoptosis related pathways but suppressed metabolism-related pathways; virulent DY197 infection especially activated protein digestion and absorption-related pathways and suppressed steroid synthesis pathway. To conclude, virulent strain infection especially induced tissue-specific alterations and caused severe suppression of hemorrhage-related pathways in spleen. Our findings will contribute to better understanding of the interactions between host and GCRV II.

Hybrid sturgeon is an important economic fish species in China. In 2021, a bacterium was isolated from the liver and kidneys of freshwater-farmed hybrid sturgeon in Yichang City, Hubei Province, causing a disease with high mortality and surface bleeding. Through morphological observation, 16S rDNA gene sequence analysis, pathogenicity, an antimicrobial sensitivity test, as well as serum physiological and biochemical analysis, it was identified as Staphylococcus saprophyticus and named E702. The 16S rDNA gene sequence of E702 is highly homologous to S. saprophyticus in GenBank. Phylogenetic analysis showed that E702 and S. saprophyticus clustered into one clade. The 50% lethal dose of E702 was 2.14 × 105 CFU/g. The percentages of monocytes and eosinophils were markedly increased in the diseased sturgeon’s blood, whereas the percentages of platelets and lymphocytes were decreased. The activity levels of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase in the diseased fish were significantly increased. The diseased fish suffered obvious damage to many tissues and organs, especially the liver and kidney, showing swelling, hyperemic and inflammatory cell infiltration. E702 was sensitive to antibiotics such as neomycin, cefazolin, norfloxacin, carbenicillin, gentamicin and ciprofloxacin. The study not only proved that S. saprophyticus was responsible for a great deal of hybrid sturgeon deaths, but also shed light on its potential risks in hybrid sturgeon farming. The research results provided the theoretical basis for the diagnosis as well as prevention of this disease.

Aeromonas veronii is widespread in aquatic environments and is capable of infecting various aquatic organisms. A. veronii infection is lethal for Chinese soft-shelled turtles (Trionyx sinensis, CSST). We isolated a gram-negative bacterium from the liver of diseased CSSTs, which was named XC-1908. This isolate was identified as A. veronii based on its morphological and biochemical characteristics, and 16S rRNA gene sequence analysis. A. veronii was pathogenic for CSSTs with an LD50 of 4.17 × 105 CFU/g. The symptoms of CSSTs artificially infected with isolate XC-1908 were consistent with those of the naturally infected CSSTs. The levels of total protein, albumin, and white globule in the serum samples of the diseased turtles were decreased, whereas those of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase were elevated. Moreover, the diseased CSSTs exhibited the following histopathological changes: the liver contained numerous melanomacrophage centers, renal glomerulus were edematous, intestinal villi were shed, and in oocytes, the number of vacuoles increased and red-rounded particles were observed. Antibiotic sensitivity tests revealed that the bacterium was sensitive to ceftriaxone, doxycycline, florfenicol, cefradine, and gentamicin, and resistant to sulfanilamide, carbenicillin, benzathine, clindamycin, erythromycin, and streptomycin. This study provides control strategies to prevent outbreaks of A. veronii infection in CSSTs.

Largemouth bass ranavirus (LMBV) infects largemouth bass, leading to significant mortality and economic losses. There are no safe and effective drugs against this disease. Oral vaccines that directly target the intestinal mucosal immune system play an important role in resisting pathogens. Herein, the B subunit of Escherichia coli heat-labile enterotoxin (LTB, a mucosal immune adjuvant) and the LMBV main capsid protein (MCP) were expressed using Saccharomyces cerevisiae surface display technology. The yeast-prepared oral vaccines were named EBY100-OMCP and EBY100-LTB-OMCP. The candidate vaccines could resist the acidic intestinal environment. After 7 days of continuous oral immunization, indicators of innate and adaptive immunity were measured on days 1, 7, 14, 21, 28, 35, and 42. High activities of immune enzymes (T-SOD, AKP, ACP, and LZM) in serum and intestinal mucus were detected. IgM in the head kidney was significantly upregulated (EBY100-OMCP group: 3.8-fold; BY100-LTB-OMCP group: 4.3-fold). IgT was upregulated in the intestines (EBY100-OMCP group: 5.6-fold; EBY100-LTB-OMCP group: 6.7-fold). Serum neutralizing antibody titers of the two groups reached 1:85. Oral vaccination protected against LMBV infection. The relative percent survival was 52.1% (EBY100-OMCP) and 66.7% (EBY100-LTB-OMCP). Thus, EBY100-OMCP and EBY100-LTB-OMCP are promising and effective candidate vaccines against LMBV infection.

β-Sitosterol, which is used extensively in pharmaceuticals, nutraceuticals, and cosmetics, has high nutritional value along with immunomodulatory and anti-inflammatory properties. In this study, we investigated the antioxidant and anti-inflammatory effects of β-sitosterol in zebrafish and explored the associated molecular mechanisms. In an in vivo antioxidant experiment, zebrafish (Danio rerio) larvae were treated with different concentrations of β-sitosterol and then exposed to a nonlethal concentration of CuSO4 to induce oxidative stress. Treatment with β-sitosterol at 70 or 100 μg/mL significantly reduced CuSO4-induced oxidative stress in the zebrafish, demonstrating the strong antioxidant activity of β-sitosterol. Treatment with β-sitosterol protected zebrafish larvae against oxidative damage from CuSO4 by upregulating the expressions of sod and gpx4b. In a zebrafish model of inflammation, pretreatment with β-sitosterol before CuSO4 exposure inhibited neutrophil recruitment and damage to lateral line neuromasts, indicating a potent anti-inflammatory effect derived from reductions in the expressions of il-8 and myd88. The results demonstrate the antioxidative and anti-inflammatory activities of β-sitosterol and suggest that β-sitosterol may be useful for the treatment of various inflammatory diseases.

Aflatoxin contamination of food and water is a serious problem worldwide. This study investigated the defensive ability of gibel carp exposed to aflatoxin B1 (AFB1) by challenging it with cyprinid herpesvirus 2 (CyHV-2) infection. The data showed that AFB1 exposure significantly increased the mortality of CyHV-2-infected gibel carp, and enhanced the viral load in the fish liver, kidney, and spleen. The oxidative-antioxidant balance suggested that AFB1 induced severe oxidative stress, including increased reactive oxygen species (ROS) and malondialdehyde (MDA) levels in the AFB1 exposed group, and the reduced activity of superoxide dismutase (SOD), glutathione-S-transferase (GST) and catalase (CAT) in the AFB1 exposed group. Meanwhile, the related expression of nuclear factor erythroid 2-related factor 2 (Nrf2), interferon regulatory factor 3 (IRF3) and the type 1 interferon (IFN1) were noticeably down-regulated, but caspase-1 was up-regulated, after exposure to AFB1, demonstrating that fish are unable to avoid the virus infection. It should be noted that the intestinal microbiota diversity and richness were lower in the AFB1 exposed group, and the composition of intestinal microbiota was affected by AFB1, resulting in the higher abundance of bacteria (such as Aeromonas and Bacteroides) and the lower abundance of potentially beneficial bacteria (such as Cetobacterium and Clostridium) in the AFB1 exposed group. This research provides insight into the possibility that AFB1 may increase the susceptibility of C. gibelio to CyHV-2 infection, and thus amplify the viral outbreak to endanger ecological safety in aquatic environment.

Molecular diagnostic testing for viral pathogens is crucial in aquaculture. The efficient and convenient preparation of pathogenic microbial nucleic acids is the basis of molecular diagnosis. Here, we developed a simplified deoxyribonucleic acid (DNA) extraction method from aquatic animal DNA viruses using the Chelex 100 resin. The nucleic acid was extracted from infected tissues and cell culture for the detection of three common aquatic viral pathogens (CEV, CyHV-2, and GSIV). We compared the extraction effects of a current commercial kit extraction method and the Chelex 100 resin extraction method according to nucleic acid concentration, conventional polymerase chain reaction (PCR), and digital droplet PCR (ddPCR). The results indicated that both extraction procedures could obtain high-quality nucleotide samples. Extracting DNA using the Chelex 100 resin led to better detective efficiency for ddPCR molecular diagnostic testing. The whole process took less than 20 min, and only Chelex 100 resin solution was added to the tissues or cells without multiple tubes being transferred several times. The extracted DNA concentration and the detection sensitivity were high. These results indicated that the Chelex 100 resin solution has the advantages of speed, efficiency, and economy compared to the commercial kit. In addition, the higher pH value (10–11) of the Chelex 100 resin solution markedly improved the detection sensitivity compared to a lower pH value (9–10). In conclusion, the comparison of the Chelex 100 Resin and commercial viral DNA extraction kits revealed the good performance of the Chelex 100 resin solution at pH 10–11 in DNA extraction for PCR amplification from aquatic animal viral samples of tissues and cells in molecular diagnostic testing. It is both rapid and cost-effective.

Amphibians, including Andrias davidianus, are declining worldwide partly due to infectious diseases. The Myxovirus resistance (Mx) gene is a typical interferon (IFN)-stimulated gene (ISG) involved in the antiviral immunity. Therefore, knowledge regarding the antiviral immunity of A. davidianus can be used for improved reproduction in captivity and protection in the wild. In this study, we amplified and characterized four different A. davidianus Mx genes (adMx) and generated temporal mRNA expression profiles in healthy and Chinese giant salamander iridovirus (GSIV) infected A. davidianus by qualitative real-time PCR (qPCR). The four adMx genes ranged in length from 2008 to 2840 bp. The sequences revealed conserved protein domains including the dynamin superfamily signature motif and the tripartite guanosine-5-triphosphate (GTP)-binding motif. Gene and deduced amino acid sequence alignment revealed relatively high sequence identity with the Mx genes and proteins of other vertebrates. In phylogenetic analysis, the adMx genes clustered together, but also clustered closely with those of fish species. The four adMx genes were broadly expressed in healthy A. davidianus, but were differentially expressed in the spleen during the GSIV infection. Our results show that the adMx genes share major structural features with their homologs, suggesting similar functions to those in other species.

The largemouth bass (Micropterus salmoides) is one of the most economically valuable fish species in China. In this study, a bacterial pathogen was isolated from the internal organs of diseased M. salmoides, and the strain was named WH21406. This isolate was identified as Aeromonas caviae on the basis of its morphology, biochemical features and 16S rDNA phylogenetic analysis. Four virulence genes related to pathogenicity, namely, flagella (fla), elastase (ela), haemolysin (hly) and aerolysin (aer), were detected in this isolate. The median lethal dosage (LD50) of A. caviae WH21406 for M. salmoides was calculated to be 3.46 × 105 CFU mL−1. The histopathological analysis showed obvious tissue damage in the gill, liver, kidney, spleen and gut of the diseased fish. The antibiotic susceptibility test demonstrated that strain WH21406 was highly sensitive to enrofloxacin, norfloxacin, streptomycin and amikacin. The results of this study provide a foundation for the diagnosis, prevention and treatment of A. caviae infection in M. salmoides.

In July 2021, a disease with a high mortality rate broke out in freshwater cultured hybrid sturgeon in Zhengzhou, Henan Province. A dominant strain, H-701, was isolated from diseased fish; physiological changes in diseased fish were investigated and molecular identification, biochemical characterization, and pathogenicity and drug sensitivity tests of H-701 were performed. The 16S rRNA gene sequence of H-701 was 99.86% homologous with that of Vibrio metschnikovii in GenBank. The 50% lethal dose of H-701 was 3.72 ± 0.929 × 104 CFU/g fish weight. The proportion of monocytes, neutrophils, and eosinophils in the blood of diseased sturgeon increased significantly, whereas the proportion of lymphocytes decreased. In diseased fish, the serum levels of total protein, albumin, globulin, and alkaline phosphatase decreased significantly, and those of aspartate aminotransferase, alanine aminotransferase, and complement C3 increased significantly. There were obvious pathological changes in several tissues of the diseased fish. H-701 was sensitive to antibiotics such as florfenicol, enrofloxacin, and doxycycline. This study not only demonstrated that V. metschnikovii was the cause of death of a large number of hybrid sturgeon but also revealed its potential risk in hybrid sturgeon aquaculture. The results provide a basis for the diagnosis and prevention of this disease.

Diseases of crucian carp (Carassius auratus) are closely related to intestinal parameters. Enterococcus faecalis has strong colonization ability in the intestinal tract, and produces natural antibiotics, bacteriocin, and other bacteriostatic substances, which can effectively inhibit some pathogenic bacteria and improve the intestinal microenvironment. This study aimed to assess the effects of E. faecalis YFI-G720 which was isolated from the intestinal of crucian carp on the growth, immunity, intestinal health, and disease resistance of crucian carp. Fish (48.16 ± 0.55 g) were fed four diets, commercial diet or diet containing E. faecalis at 105 CFU/g (EF1), 106 CFU/g (EF2), or 107 CFU/g (EF3) for 28 days. The results showed that supplementation of E. faecalis significantly improved the weight gain ratio (WGR) and the specific growth rate (SGR) compared with control group (p < 0.05). Intestinal mucosal epithelial cells in EF2 were intact and normal, but there was obvious vacuolation in CG. Compared with CG, serum C3 and IgM in EF2 were significantly increased at the end of the experiment (p < 0.05), and serum alkaline phosphatase was significantly higher in all experimental groups (p < 0.05). Among studied immune-related genes, expression was detected by qPCR, C3, IgM, and IL-1βwere upregulated in all experimental groups to varying degrees from 14 days, with highest expression in EF2 at 28 days. Intestinal microbiota structure analyzed through high-throughput sequencing, and the results showed that the relative abundance of Aeromonas and Acinetobacter decreased while Cetobacterium increased in all experimental groups, with the greatest changes in EF2. Challenge tests showed that fish fed E. faecalis were more resistant to Aeromonas veronii (p < 0.05). In conclusion, dietary E. faecalis YFI-G720 at 106 CFU/g can improve the health status, immune parameters, intestinal microbiota composition, and disease resistance of crucian carp.

Chinese giant salamander iridovirus (GSIV) infection could lead to mitochondrial apoptosis in this animal, a process that involves B-cell lymphoma-2 (BCL-2) superfamily molecules. The mRNA expression level of Bcl-xL, a crucial antiapoptotic molecule in the BCL-2 family, was reduced in early infection and increased in late infection. However, the molecular mechanism remains unknown. In this study, the function and regulatory mechanisms of Chinese giant salamander (Andrias davidianus) Bcl-xL (AdBcl-xL) during GSIV infection were investigated. Western blotting assays revealed that the level of Bcl-xL protein was downregulated markedly as the infection progressed. Plasmids expressing AdBcl-xL or AdBcl-xL short interfering RNAs were separately constructed and transfected into Chinese giant salamander muscle cells. Confocal microscopy showed that overexpressed AdBcl-xL was translocated to the mitochondria after infection with GSIV. Additionally, flow cytometry analysis demonstrated that apoptotic progress was reduced in both AdBcl-xL-overexpressing cells compared with those in the control, while apoptotic progress was enhanced in cells silenced for AdBcl-xL. A lower number of copies of virus major capsid protein genes and a reduced protein synthesis were confirmed in AdBcl-xL-overexpressing cells. Moreover, AdBcl-xL could bind directly to the proapoptotic molecule AdBak with or without GSIV infection. In addition, the p53 level was inhibited and the mRNA expression levels of crucial regulatory molecules in the p53 pathway were regulated in AdBcl-xL-overexpressing cells during GSIV infection. These results suggest that AdBcl-xL plays negative roles in GSIV-induced mitochondrial apoptosis and virus replication by binding to AdBak and inhibiting p53 activation.

The present study aimed to evaluate the effect of the dietary supplementation of Saccharomyces cerevisiae YFI-SC2 on the growth performance, intestinal morphology, immune parameters, intestinal microbiota, and disease resistance of crayfish (Procambarus clarkia). Crayfish were randomly assigned to six different boxes and two different groups in triplicate. The control group received a basal diet and the treatment group received a diet containing S. cerevisiae at 107 CFU/g. After feeding for 28 days, crayfish of the treatment group exhibited a significantly better weight gain ratio (WGR) and a specific growth rate (SGR) (p < 0.05) than crayfish of the control group. Compared to the treatment group, the control group intestines showed an oedema connective tissue layer and a weak muscle layer. For immune-related genes, Crustin2 expression was similar between the groups, whereas Lysozyme and prophenoloxidase from treatment group expression levels were upregulated significantly (p < 0.05) after 14 and 28 days of feeding. Prophenoloxidase showed the highest expression, with 10.5- and 8.2-fold higher expression than in the control group at 14 and 28 days, respectively. The intestinal microbiota community structure was markedly different between the two groups. After 14 and 28 days of feeding, the relative abundance of Cetobacterium and Lactobacillus increased, whereas Citrobacter and Bacteroides decreased in the treatment group compared with that of the control group. The challenge test showed that crayfish of the treatment group had a significantly enhanced resistance against Citrobacter freundii (p < 0.05). Our results suggest that a S. cerevisiae-containing diet positively influenced the health status, immune parameters, intestinal microbiota composition, and disease resistance of crayfish.

The grass carp reovirus (GCRV) causes severe hemorrhagic disease with high mortality and leads to serious economic losses in the grass carp (Ctenopharyngodon idella) industry in China. Oral vaccine has been proven to be an effective method to provide protection against fish viruses. In this study, a recombinant baculovirus BmNPV-VP35-VP4 was generated to express VP35 and VP4 proteins from GCRV type Ⅱ via Bac-to-Bac baculovirus expression system. The expression of recombinant VP35-VP4 protein (rVP35-VP4) in Bombyx mori embryo cells (BmE) and silkworm pupae was confirmed by Western blotting and immunofluorescence assay (IFA) after infection with BmNPV-VP35-VP4. To vaccinate the grass carp by oral route, the silkworm pupae expressing the rVP35-VP4 proteins were converted into a powder after freeze-drying, added to artificial feed at 5% and fed to grass carp (18 ± 1.5 g) for six weeks, and the immune response and protective efficacy in grass carp after oral vaccination trial was thoroughly investigated. This included blood cell counting and classification, serum antibody titer detection, immune-related gene expression and the relative percent survival rate in immunized grass carp. The results of blood cell counts show that the number of white blood cells in the peripheral blood of immunized grass carp increased significantly from 14 to 28 days post-immunization (dpi). The differential leukocyte count of neutrophils and monocytes were significantly higher than those in the control group at 14 dpi. Additionally, the number of lymphocytes increased significantly and reached a peak at 28 dpi. The serum antibody levels were significantly increased at Day 14 and continued until 42 days post-vaccination. The mRNA expression levels of immune-related genes (IFN-1, TLR22, IL-1β, MHC I, Mx and IgM) were significantly upregulated in liver, spleen, kidney and hindgut after immunization. Four weeks post-immunization, fish were challenged with virulent GCRV by intraperitoneal injection. The results of this challenge study show that orally immunized group exhibited a survival rate of 60% and relative percent survival (RPS) of 56%, whereas the control group had a survival rate of 13% and RPS of 4%. Taken together, our results demonstrate that the silkworm pupae powder containing baculovirus-expressed VP35-VP4 proteins could induce both non-specific and specific immune responses and protect grass carp against GCRV infection, suggesting it could be used as an oral vaccine.

The grass carp hemorrhagic disease, caused by the grass carp reovirus (GCRV), has resulted in severe economic losses in the aquaculture industry in China. VP4 and VP35 are outer capsid proteins of GCRV and can induce an immune response in the host. Here, three recombinant baculoviruses, AcMNPV-VP35, AcMNPV-VP4, and AcMNPV-VP35-VP4, were generated to express recombinant VP4 and VP35 proteins from GCRV type II in insect cells by using the Bac-to-Bac baculovirus expression system to create a novel subunit vaccine. The expression of recombinant VP35, VP4, and VP35-VP4 proteins in Sf-9 cells were confirmed by Western blotting and immunofluorescence. Recombinant VP35, VP4, and VP35-VP4 were purified from baculovirus-infected cell lysates and injected intraperitoneally (3 μg/fish) into the model rare minnow, Gobiocypris rarus. After 21 days, the immunized fish were challenged with virulent GCRV. Liver, spleen, and kidney samples were collected at different time intervals to evaluate the protective efficacy of the subunit vaccines. The mRNA expression levels of some immune-related genes detected by using quantitative real-time PCR (qRT-PCR) were significantly upregulated in the liver, spleen, and kidney, with higher expression levels in the VP35-VP4 group. The nonvaccinated fish group showed 100% mortality, whereas the VP35-VP4, VP4, and VP35 groups exhibited 67%, 60%, and 33% survival, respectively. In conclusion, our results revealed that recombinant VP35 and VP4 can induce immunity and protect against GCRV infection, with their combined use providing the best effect. Therefore, VP35 and VP4 proteins can be used as a novel subunit vaccine against GCRV infection.