Rootstock plays an important role in production of grafted watermelon plant. However, the sugars, organic acids, and internal bacterial communities associated with the grafted watermelon fruit are currently unknown. In this study, the effect of grafting with pumpkin and bottle gourd rootstocks on the quality and bacterial community structure shift in watermelon fruit were analyzed by using Illumina MiSeq sequencing. Results showed that grafting with pumpkin and bottle gourd rootstocks increased the nutrient uptake and positively affected the contents of soluble sugars and organic acids of the grafted watermelon fruit. Pumpkin and bottle gourd rootstocks have different effects on the transformation of sugarsand acids. Grafting with pumpkin and gourd rootstocks significantly (P < 0.05) increased the succinic, citric, shikimic, and tartaric acids in the fruit. Grafting with rootstocks was correlated with the shift of the bacterial community structures in the fruit. Further, nitrogen and phosphorus have greater effects on the bacterial community than potassium. The most predominant bacterial groups were Proteobacteria and Cyanobacteria. In addition sugar accumulation in the grafted watermelon fruit was correlated with its bacterial compositions. This study provides a new understanding of the fruit quality of grafted watermelon plant, and the findings are helpful in the breeding and application of suitable rootstocks.

Intracellular survival is a key virulence trait of Pseudomonas plecoglossicida, the agent of visceral granulomas disease in large yellow croaker (Larimichthys crocea). However, the bacterial determinants that support persistence within host phagocytes remain incompletely defined. Here, we investigated the contribution of the type VI secretion system-1 (T6SS-1) to macrophage infection using a ΔtssD-1 mutant and a head kidney-derived macrophage-like cell line (LYC-hk). Gentamicin protection assays showed that ΔtssD-1 bacteria entered macrophages at levels comparable to wild type at 0 h post-infection, but failed to undergo sustained intracellular replication at later time points, whereas a chromosomally complemented strain restored this phenotype. Differential permeabilization microscopy indicated that T6SS-1 is required for phagosomal rupture and exposure of bacteria to the host cytosol, and transmission electron microscopy confirmed cytosolic localization of wild-type and complemented bacteria but not the mutant. Time-resolved transcriptomic profiling revealed a shared early inflammatory signature in response to both strains, followed by divergence at 4-8 h post-infection. Wild-type infection was associated with distinct expression patterns, including strong induction of the osmotic stress marker lrrc8a, which was supported by RT-qPCR validation. Together, these results identify T6SS-1 as a key determinant of phagosomal rupture and cytosolic access in P. plecoglossicida macrophage infection and link this intracellular transition to distinct host transcriptional signatures.

A novel cell line (LYC-hK) was established from the head kidney of large yellow croaker (Larimichthys crocea), a major marine aquaculture fish species in China. The cell line has been continuously subcultured for over 100 passages and exhibits stable morphology, transitioning to fibroblast-like. Specifically, it exhibits a transition toward epithelium-like phenotype at high density. Karyotype analysis verified the stability of the diploid chromosome: approximately 50 % of the cells retained 48 chromosomes at the 120th passage. Functionally, LYC-hK displayed robust macrophage-like activity, including reactive oxygen species production upon stimulation with lipopolysaccharide (LPS) or phorbol myristate acetate (PMA), and efficient phagocytosis of Nocardia seriolae and Pseudomonas plecoglossicida, followed by time-dependent intracellular bacterial clearance. Gene expression profiling revealed high transcript abundance of macrophage-specific marker genes, including CD68, mpeg1, CD209/DC-SIGN, and CD302, alongside strong induction of the pro-inflammatory cytokines IL-1β and IL-8 upon bacterial challenge. Notably, high-level expression of the CD34 gene was also detected which implies that the LYC-hK might possess hematopoietic progenitor-like properties. Furthermore, successful transfection of the LYC-hK cells with the pEGFP-C2 plasmid confirmed the line's suitability for studies involving exogenous gene expression. Collectively, these findings indicate that LYC-hK is a stable macrophage-like cell line, providing a valuable in vitro model for studies of host-pathogen interactions and functional genomics in teleost fish.

A species of acanthocephalan collected from the hindgut of Larimichthys crocea was identified as Longicollum pagrosomi Yamaguti, 1935 based on morphological characteristics. The complete mitochondrial genome of this parasite was sequenced. The mitogenome exhibited a circular structure with a total length of 14 632 bp, containing 12 protein coding genes (PCGs), 2 ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs) and 2 major non-coding regions. The most frequently used start codon was GTG, and the most abundant amino acid was valine. The phylogenetic analyses of the mitogenome using Bayesian inference and maximum likelihood methods showed that the genus Longicollum formed a sister clade to the genus Pomphorhynchus, supporting the monophyly of Pomphorhynchus. This study reported a new host for L. pagrosomi and revealed the first complete mitogenome sequence of the genus Longicollum.

Nocardia seriolae and Pseudomonas plecoglossicida are two important pathogenic bacterial species in aquaculture, causing visceral granulomatous disease in large yellow croaker (Larimichthys crocea) and other economically important fish species. However, the difference of host-pathogen interactions between these two bacteria have not been well defined. In present study, ultrastructural changes, cell cytotoxicity, production of reactive oxygen species (ROS), apoptosis, ferroptosis, and transcriptomic profiles induced by P. plecoglossicida and N. seriolae were evaluated in a head kidney originating cell line from yellow large croaker (LYC-hK). Transmission electron microscope showed that both P. plecoglossicida and N. seriolae were able to invade into and multiply intracellularly, but display different intracellular ultrastructural changes upon bacterial infection. Lactate dehydrogenase assay showed that both two bacteria had low cell cytotoxicity toward LYC-hK cell, compared to the cytotoxic positive bacterium, Photobacterium damselae subsp. damselae. Flow cytometry detection showed that N. seriolae had significant stronger ability to trigger ROS production and apoptosis in LYC-hK than that of P. plecoglossicida. Intracellular reduced glutathione (GSH), total glutathione (GSH + GSSG) and Fe2+ measurements showed that N. seriolae infection led to an extremely low GSH or GSH + GSSG level and significant higher Fe2+ level in LYC-hK. Moreover, comparative transcriptomes by RNA-sequencing revealed some same and different cellular and molecular responses in LYC-hK after N. seriolae or P. plecoglossicida infection. These findings will provide new insight into understanding the pathogenesis of fish visceral granulomatous disease caused by these two bacterial pathogens.

Vibrio harveyi is a significant pathogen in marine aquaculture, causing vibriosis in various marine species. This study presents a comparative genomic analysis of two V. harveyi strains, N8T11 and 45T2, which exhibit differing virulence profiles. Virulence assays revealed that N8T11 caused 92% mortality in infected fish, while 45T2 resulted in 0% mortality. Whole-genome sequencing revealed that strain N8T11 harbors five plasmids (pN8T11a, pN8T11b, pN8T11c, pN8T11d and pN8T11e) absent in 45T2, encoding genes potentially linked to virulence, such as siderophore-mediated iron acquisition and stress response mechanisms. Pan-genome analysis highlighted substantial genomic plasticity within V. harveyi, with mobile genetic elements, including plasmids and prophages, contributing to horizontal gene transfer. Conjugation experiments demonstrated that all five N8T11 plasmids can transfer to 45T2 with efficiencies up to 87%, with pN8T11b remaining stable across multiple subcultures, enabling the dissemination of virulence-associated genes. These findings suggest that plasmid-mediated gene transfer plays a role in the virulence variability observed between V. harveyi strains. This study contributes to understanding the genomic factors underlying pathogenicity in V. harveyi and provides insights for future research aimed at controlling vibriosis in aquaculture.

The aim of this study was to clarify the taxonomic identification of a hemoflagellate and assess the effect of trypanosome infection on Larimichthys crocea. Giemsa staining showed the presence of three morphotypes of trypomastigotes. The trypanosomes had the following morphological characteristics: a slender body with a long flagellum at the front; body size 12.30–30.90 × 1.13–2.33 μm; elongated oval nucleus situated in the median region; kinetoplast small, oval, located at the posterior end. The parasite had significant morphological differences from Trypanosoma epinepheli Su, Feng, Jiang, Guo, Liu & Xu, 2014 and Trypanosoma carassii (Mitrofanov) Doflein, 1901. The 18S rDNA sequences of the trypanosome from L. crocea had the highest homology (98.4%) with T. carassii. Phylogenetic analysis indicated that the parasite clustered with freshwater fish trypanosomes. Based on the differences in morphological characteristics and molecular data, it is considered a new species, Trypanosoma larimichthysi n. sp. Trypanosome infection had no effect on the growth of L. crocea, but significantly increased the concentration of blood urea nitrogen (BUN), and induced pathological changes in the gills, liver, spleen and kidney. The pro-inflammatory immune genes, including TNF-α, IFN-γ, IL-1β, CXCL8 and iNOS, were significantly upregulated in the L. crocea infected with trypanosomes. These results suggest that the trypanosome has negative impacts on host health.

Large yellow croaker iridovirus (LYCIV) poses a growing threat to large yellow croaker (Larimichthys crocea) aquaculture, requiring early diagnosis for effective prevention and control. However, current detection methods are time-consuming, labor-intensive, or require specialized equipment, hindering timely diagnosis. To address this concern, we developed a novel platform combining CRISPR/Cas12a and recombinase polymerase amplification (RPA) for rapid and sensitive LYCIV detection. Our strategy targeted the conserved ATPase gene, utilizing specific crRNA and RPA primers for selective detection. Additionally, we employed a lateral flow strip technique for intuitive visual assessment. The performance evaluation demonstrated outstanding specificity and sensitivity, with a limit of detection as low as 5 x 103 copies / reaction of iridovirus. Validation on spiked and clinical fish samples confirmed the accuracy through PCR analysis. Overall, our developed platform offers a convenient, onsite, and user-friendly tool for early diagnosis, prevention, and control of LYCIV-related diseases in large yellow croaker aquaculture.

Pseudomonas plecoglossicida is a Gram-negative bacterium that causes chronic granulomatous diseases in fish. However, the molecular mechanisms underlying its pathogenicity are poorly understood. Here, we identified a novel two-component system (TCS) named PvgAS in P. plecoglossicida, which regulates the expression of genes including the type VI secretion system-1 (T6SS-1), a key virulence factor, as well as the type III secretion system (T3SS) gene cluster and several genes associated with metabolic pathways. We showed that PvgAS is essential for bacterial virulence in large yellow croaker (LYC), an important aquaculture species, by controlling T6SS-1 expression, which is crucial for tissue colonization and intracellular survival. In contrast, T3SS was dispensable for P. plecoglossicida virulence in this model. We also confirmed T6SS-1 gene regulation by PvgAS at the transcriptional and translational levels using RNA-seq, RT-qPCR, lacZ reporter, and western blot assays. Our study reveals a regulatory and functional role of PvgAS in P. plecoglossicida pathogenicity and suggests that PvgAS may be a potential target for new therapeutic interventions against this bacterium.

The Chinese soft-shelled turtle (CSST) (Pelodiscus sinensis) is the most farmed reptile in Asia because of its high nutritional and medicinal value. Increase in production due to intensified culture has led to increase in infectious diseases adversely impacting the CSST industry. To develop effective disease control strategies there is need for a comprehensive understanding of major diseases and predisposing factors leading to infections in CSSTs. Thus, this review puts together a summation of diseases adversely impacting the CSST industry. Major viral diseases include the red neck disease caused by the soft-shelled turtle iridovirus (STIV), soft-shelled turtle systemic septicemia (STSS) caused by STSS spherical virus (STSSSV) and trionyx sinensis hemorrhagic syndrome (TSHS) caused by TSHS virus (TSHSV). Bacteria cause the largest proportion of infectious diseases in CSSTs with the majority being caused by Aeromonas and Bacillus spp. They include the red neck, hemorrhagic septicemia, furunculosis, and red spot diseases caused by different Aeromonas spp. as well as craned neck, twisted necks and generalized edema caused by Bacillus spp. As for predisposing factors, the amphibious nature of CSSTs could be a disposure to waterborne pathogens like Aeromonas, Edwardsiella, Flavobacterium and Vibrio spp. during habitation in aquatic environments while exposure to saprophytic pathogens like Bacillus spp. could be linked to hibernation. Also, the omnivorous nature of CSSTs could be a disposure to ingestion of infected organisms while the presence of feral species can be a source of contact with infected feral species. Overall, this review identifies priority diseases that need vaccine development, and it also highlights the predisposing factors needed for the design of effective disease control measures.

The visceral white nodules disease in the internal organs of Larimichthys crocea has caused significant harm in the aquaculture of this species, with Pseudomonas plecoglossicida considered one of the core pathogens causing this disease. In this study, we designed three pairs of specific nested PCR primers targeting the sctU gene of P. plecoglossicida, a crucial component of the Type III secretion system (T3SS), which is instrumental in bacterial pathogenesis and virulence. Through the optimization of PCR reaction conditions, specificity testing, and sensitivity determination, a method was established for the accurate detection of P. plecoglossicida. This method yielded single amplification products, exhibited a false positive rate of zero for reference bacteria, and achieved a detection sensitivity of a minimum of 2.62 copies/reaction for the target sequence. Using the detection method, we conducted analyses on the diseased populations of L. crocea, involving a total of 64 screened fishes along the southeast coast of China from 2021 to 2023. The results revealed that the infection rate of P. plecoglossicida in diseased L. crocea exceeded over 90% in March and April, while in other months, the maximum recorded infection rate was merely 10%. The detection method developed in this study shows potential for early warning and routine monitoring of visceral white nodules disease in the internal organs of species such as L. crocea.

Pseudomonas plecoglossicida is a facultative Gram-negative bacteria that causes 'visceral granulomas disease' (VGD) in large yellow croaker (LYC; Larimichthys crocea) and several other fish species leading to high economic losses. Currently, there are no commercial vaccines against VGD. Thus, we evaluated the efficacy of a live attenuated vaccine (LAV) made by our laboratory in LYC administered orally in alginate-coated LAV-loaded feeds. To evaluate the tolerance of the LAV in the acidic environment of the stomach, we treated the alginatecoated LAV with a simulated gastric juice without pepsin. There was a 10-fold decrease in the number of viable LAV cells after treatment with the simulated gastric juice (pH 2.0). Despite so, we found a significant increase in LAV cells in the liver and hindgut within 4 h post first feeding of oral vaccine (hpff), which increased to high levels by 24 hpff indicating that LAV cells that survived the stomach acidic environment reached the hindgut for adsorption. Although there was a transient change in the composition of the gut microbiota at 24 hpff, analysis using the Shannon and beta-diversity indices showed a similarity in the bacterial composition found before and after vaccination indicating that the LAV had no long-term adverse effect on the gut microbiota. Systemic and mucosal humoral responses were characterized by high serum antibody levels and upregulation of the pig and sign while activation of cell-mediated immunity was characterized by upregulation of MHC-I alpha, MHCII beta, Prf1 and IFN gamma genes. The overall protection in vaccinated LYC was estimated at relative percent survival (RPS) of 60%. Altogether, this study has shown that LAVs can be used as oral vaccines to confer protective immunity without impacting adverse effects on the fish microbiota.

Pseudomonas plecoglossicida is a temperature-dependent opportunistic pathogen mediating visceral granulomas in many piscine species including the large yellow croaker (Larimichthys crocea) but the underlying mechanisms are unclear. RpoE is an alternative sigma (σ) factor involved in regulated intramembrane proteolytic (RIP) cascade, enabling bacterial pathogens to coordinate the expression of genetic traits associated with stress adaptation and virulence determinants in response to diverse stimuli in vitro and in vivo of the hosts. In this study, genes associated to RIP cascade in P. plecoglossicida were identified and characterized to show various sequence similarities to their counterparts in Escherichia coli and P. aeruginosa. The expression of P. plecoglossicida RIP locus was induced by higher temperatures. Moreover, RNA sequencing approach revealed that RpoE regulated the expression of ∼297 and ∼261 genes at virulent (18 °C) and non-virulent (28 °C) temperatures, respectively. RpoE regulon genes are involved in various processes associated with bacterial signal transduction, membrane homeostasis, energy metabolism and virulence. In particular, RpoE positively controlled expression of csrA encoding an RNA binding protein essential for central carbon metabolism. In addition, P. plecoglossicida RpoE was validated to regulate type VI secretion system (T6SS) expression, bacteria competition, biofilm formation and reproduction in macrophages. Collectively, RpoE-centered RIP cascade appeared to play important roles in control of the expression of genes involved in adaptation in vivo and in vitro niches by thermal sensing in P. plecoglossicida. These results facilitates to reveal the pathogenic mechanisms of P. plecoglossicida causing fish diseases and provides new perspectives to control bacterial infection.

Pseudomonas plecoglossicida is a facultative fish pathogen that possesses three distinct type VI secretion systems (named T6SS-1, T6SS-2, and T6SS-3). Our previous work indicated that only T6SS-2 of P. plecoglossicida mediates interbacterial competition. However, the antibacterial T6SS effectors and their functions are unclear. Here, we reported two T6SS effectors that mediate antibacterial activity. We first identified four putative antibacterial effectors (denoted as Txe1, Txe2, Txe3, and Txe4) and their cognate immunity proteins encoded in P. plecoglossicida strain XSDHY-P by analyzing the regions downstream of three vgrG genes. We showed that the growth of Escherichia coli cells expressing Txe1, Txe2, and Txe4 was inhibited, and these three effectors exhibited nuclease activity in vivo. The interbacterial competition assays with single- or multi-effector deletion mutants as attackers revealed that Txe1 was the predominant T6SS toxin of P. plecoglossicida strain XSDHY-P mediating the interbacterial killing. This work contributes to our understanding of bacterial effectors involved in the interbacterial competition.

Pseudomonas plecoglossicida is an important pathogenic bacterial species in aquaculture, causing visceral granulomas in large yellow croaker (Larimichthys crocea) and several other economically important fish species. The GacS/GacA is a two-component regulatory system (TCS) primarily found in Pseudomonads for its role in virulence, quorum sensing, and biofilm formation. To investigate the potential role of GacS/GacA system of P. plecoglossicida, a gacS deficient mutant (ΔgacS) was generated based on a clinical isolate XSDHY-P. Compared to the wild-type (WT) strain, the ΔgacS strain showed almost no growth defect in vitro but was impaired in swimming motility and biofilm formation. Furthermore, the ΔgacS strain exhibited significantly more sensitivity to the bactericidal action of normal fish serum mediated by the complement system. In a cell model originating from the head-kidney of large yellow croaker, the ΔgacS showed lower capacities of adhesion, invasion, and intracellular survival compared to the WT strain. In addition, the virulence of the ΔgacS and WT bacterial strains were evaluated in large yellow croaker. The results showed that the deficiency of gacS drastically decreased bacterial tissue loads and substantially attenuated P. plecoglossicida virulence in fish. Furthermore, a comparative transcriptome was performed between the ΔgacS and WT strains by RNA-sequencing (RNA-seq). Transcriptomic analysis results revealed 25 upregulated and 273 downregulated genes in the ΔgacS strain compared with the WT strain, and the downregulated genes were mainly involved in flagellar assembly, metabolism, bacterial chemotaxis, and biofilm formation, which are consistent with their respective phenotypic traits. These findings will provide functional insight into the role of GacS/GacA in P. plecoglossicida.

Pseudomonas plecoglossicida is an important pathogenic bacterium in aquaculture that causes visceral granulomas in large yellow croaker (Larimichthys crocea). Uridine diphosphate glucose phosphorylase encoded by galU plays a key role in biosynthesis of the bacterial envelope, particularly lipopolysaccharide and the capsule. In this study, we inactivated the galU gene in the P. plecoglossicida isolate XSDHY-P. The galU mutant strain showed impaired growth in the early exponential stage and lacked the O polysaccharide side chain in lipopolysaccharide, but almost no defect in biofilm formation was detected. The galU mutant strain also exhibited significantly more sensitivity to the bactericidal action of normal fish serum mediated by the complement system compared to the wild-type strain. In a cell model originating from the head kidney of large yellow croaker, the galU mutant strain showed lower capacities of adhesion, invasion, and intracellular survival compared to the wild-type strain. In addition, the deficiency of the galU mutant drastically decreased bacterial loads in tissues and attenuated P. plecoglossicida virulence in fish. These results suggest that the galU gene of P. plecoglossicida is required for in vivo survival in large yellow croaker.

In teleost, follicle atresia is a common degenerative process that can occur at different stages of ovarian development. In this study, we depicted the cellular morphology of silver pomfret (Pampus argenteus) follicular atresia in detail and divided it into four different stages from Aα to Aδ stages based on the main cellular characteristics. High-throughput RNA sequencing was used to profile follicle atresia from Aα to Aδ stages, and many stage-specific genes were identified. In early atretic ovary, a great number of genes in cytokine-cytokine receptor interaction were obviously downregulated, suggesting that somatic cells might directly induce the follicle atresia by disrupting the normal conservation with germ cells. Meanwhile, the regulatory network of immune cell–related pathways was discovered in the process of atresia. The genes enriched in Th cell differentiation, leukocyte transendothelial migration, cholesterol metabolism, and so on were abundantly expressed, indicating that the immune cells play key roles in the process of in follicle atresia. Moreover, a model was proposed to illustrate how somatic cells mediate the process of follicle atresia. The study provides important insights into the molecular networks underlying follicle atresia in teleost.

自我国实施了海洋伏季休渔制度以来,确定市售鱼类的死亡时间成了渔业执法领域的难题.本论文借鉴了法医学研究,探索死亡鱼类肌肉组织中18S核糖体RNA(18s)降解程度来推断死亡时间的可行性.先通过分析在(0～1℃)低温贮存条件下斑马鱼肌肉组织18s的降解动力学,结果表明肌肉组织内18s呈现区域偏好性的时序降解.在此基础上,建立了一种基于逆转录PCR检测多个特定片长18s降解物的技术,推测鱼类死亡时间的方法原型.虽然该方法仍存在局限性,但在1～7d的死亡后冷藏时间范围内具有可行性.

AIMS:Diseases of maricultured species caused by Vibrio harveyi are increasing in China and other regions. This study examined the genetic diversity, antimicrobial susceptibility, plasmid profiles and virulence potential of the V. harveyi isolated from marine organisms farmed in two provinces in eastern China between 2014 and 2019. METHODS AND RESULTS:A total of 54 V. harveyi were obtained from seven marine species. Enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting revealed substantial genetic heterogeneity among the V. harveyi isolates. There was no significant correlation between ERIC-PCR genotypes and host origins or fish farms. All the isolates were resistant to amoxicillin and ampicillin, and 79·6% to kanamycin. We found that 61·1% of the V. harveyi isolates had plasmid(s) and there were 14 different plasmid profiles. Most isolates from fish hosts (76·5%) contained plasmids; however, 75% of isolates from nonfish hosts lacked plasmids. Experimental infection results showed that isolates with plasmid(s) were more virulent to large yellow croaker than isolates lacking plasmids (P < 0·05). CONCLUSIONS:This study confirmed that V. harveyi isolates obtained from animals farmed in the coastal region of east China were genetically diverse. Our results suggest that the virulence of various V. harveyi strains to fish is associated with the plasmids they carry. SIGNIFICANCE AND IMPACT OF THE STUDY:More than 50% of the V. harveyi isolates carried one to 11 plasmids. The plasmid-borne traits of V. harveyi strains might be important for host adaptation and virulence, but they were not associated with susceptibility to the tested antibiotics.