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.

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.

Pseudomonas plecoglossicida is a Gram-negative fish pathogen that affects the large yellow croaker (LYC; Larimichthys crocea) and several other economically important fish species. The bacterium can cause systemic infections in LYC, resulting in visceral granulomas disease (VGD) with high mortality in cultured populations. Despite the severity of this disease, the LYC industry lacks an effective vaccine for VGD. We have constructed a highly attenuated P. plecoglossicida mutant by deleting a virulence gene (tssD-1) from the wild-type (WT) strain XSDHY-P. We then investigated the safety and efficacy of the mutant strain Delta tssD-1 in LYC juveniles as a live attenuated vaccine candidate. LYCs were vaccinated with Delta tssD-1 by two intraperitoneal injections per fish, and then challenged with P. plecoglossicida XSDHY-P by the immersion route. Vaccination of Delta tssD-1 induced a significantly higher level of P. plecoglossicida-specific serum IgM and increased expression of CD8 alpha and MHCI alpha, and resulted in a relative percentage survival of 86.3%. The safety of Delta tssD-1 was demonstrated because no mortality or clinical signs were found ina LYC group injected with a high dose (1.8 x 106 CFU per fish) of Delta tssD1. This is the first report of a defined live attenuated strain providing protective immunity in LYC against P. plecoglossicida.

Atmospheric transport could be a significant pathway for inland microplastics (MPs, with size＜5 mm) to the ocean in addition to catchment runoff and coastal discharge. However, atmospheric input of MPs to the ocean is rarely quantified. To address this issue, transport of atmospheric MPs from source to sink was studied in the Asia-Pacific region during nine cruises from October 2018 to September 2019. Both deposited atmospheric MPs (DAMPs) and suspended atmospheric MPs (SAMPs) were collected, ranging from 23.04 n/(m2·d) to 67.54 n/(m2·d), and 0 to 1.37 n/m3, respectively. Size composition revealed that atmospheric deposition of MPs originating in terrestrial regions seems inadequate and insufficient to quantify the atmospheric input to the ocean. In addition, combined with aerodynamic modelling, for the first time, we estimated that 7.64-33.76 t of fibrous atmospheric MPs was globally generated in 2018, which is 3 % and 31 % of riverine input MPs of The Yangtze River and The Pearl River in terms of mid-point mass, respectively. The increasing load of ingestible plastics from sea air could have a far-reaching impact on marine ecosystem.

It is documented that microplastics exist in coastal environments around the world.However, verified and standardized protocols for monitoring microplastics in intertidal sediments have not yet been developed.In this study, three quantitative methods, including direct floatation (method A), separatory funnel (method B) and volumetric flask (method C) were selected and optimized to isolate the microplastics in intertidal sediments with a sodium chloride solution as the flotation solution.At the same time, the effects of polymer type and sediment particle size on separation efficiency were assessed.Sediments containing microplastics were manually prepared to simulate field sediment samples and then microplastics extraction tests were conducted to determine the recovery efficiency of the three methods.The average recovery efficiency for high density polyethylene (HDPE), polyvinyl chloride (PVC), polypropylene (PP), polyamide (PA) and polystyrene (PS) of approximately 1 mm was as high as (91±7)%.Statistical analysis indicates that polymer type did not significantly (P>0.05) effect extraction efficiency, but the recovery ratio increased significantly (P<0.05) with sediment particle size: from clay (<0.004 mm) to silt (0.004 mm to 0.063 mm) and coarse sand (0.5-1 mm).The mean recovery ratio of method B was the highest and most consistent (95±3)%.In December 2015, method B was used to measure microplastics pollution in 27 sediment samples collected from nine randomly located sampling sites in Xiuzhen River Estuary, Ganyu harbor and Gaogongdao town.During ebb tide, 2-3 kg surface sediments (1-2 cm) were collected in the intertidal zone and sealed for further processing.Microplastic abundances in the intertidal sediment of Xiuzhen River Estuary, Ganyu harbor and Gaogongdao town were 34.97-144.33 n/kg, 43.70-119.9 n/kg and 372.98-1 103.02 n/kg, with corresponding concentrations of 2.56-10.70 mg/kg, 1.01-5.92 mg/kg and 3.36-8.48 mg/kg.All extracted microplastics were categorized into fiber, expanded foam, chip, fragment and film;fiber accounted for 47% of the 870 separate pieces of plastic, while fragments were the least abundant (5% of total).Our research suggests that microplastics pollution in the study area resulted from anthropogenic activity, including plastic items abandoned by tourists and fishermen.