In this study, antifreeze peptides extracted from crayfish shells (CSPs) were glycosylated through dextran using the Maillard reaction (MR), successfully producing antifreeze glycopeptides (GCSPs) with excellent antifreeze activity and satisfying flavor. Infrared and fluorescence spectroscopies indicated that CSPs and dextran mainly formed covalent C–N and C–O bonds to generate GCSPs. Compared with CSPs, GCSPs formed more O–H and C=O bonds, resulting in a more compact microstructure and a higher molecular weight. With a thermal hysteresis activity of 1.23°C and a glass transition temperature of –19.5°C, as determined using differential scanning calorimetry, GCSPs also had better frost resistance than CSPs. Thermogravimetric analysis showed that glycosylation significantly enhanced the thermal stability of CSPs. Additionally, GCSPs generated pyran derivatives, terpenoids, and aldehydes with special aromas through the MR, substantially diminishing the fishy odor of CSPs. In the preservation of frozen pork, GCSPs considerably reduced water loss, improved the water-holding capacity, and enhanced its firmness, resilience, and chewiness. Low-field nuclear magnetic resonance spectroscopy also confirmed that GCSPs effectively reduced the loss of free water during thawing and maintained moisture stability. Overall, GCSPs have excellent antifreeze and water-retention properties and the potential to be widely used to preserve frozen foods.

Viral nervous necrosis (VNN), caused by nervous necrosis virus (NNV), is a devastating viral disease affecting marine fish aquaculture worldwide, particularly during larval and juvenile stages. The virus primarily targets the central nervous system and retina, often resulting in severe neurological symptoms and high mortality. To clarify the relationship between greenfin horse-faced filefish (Thamnaconus septentrionalis) and NNV, this study investigated an outbreak of abnormal mortality in juvenile fish from a marine aquaculture farm in Rizhao, Shandong Province, China. A comprehensive etiological diagnosis was conducted using bacterial isolation and identification, real-time quantitative RT-PCR (RT-qPCR), histopathological analysis, transmission electron microscopy (TEM), and phylogenetic analysis. Diseased fish exhibited typical neurological signs, including spiral swimming, anorexia, and body darkening. Several opportunistic bacteria were isolated from affected fish; however, they were not considered the primary cause of mortality. RT-qPCR results demonstrated that all diseased samples were positive for NNV nucleic acid, with high viral loads detected in neural tissues. Histopathological examination revealed pronounced vacuolation and neuronal nuclear pyknosis in the brain and retinal tissues, while TEM identified abundant non-enveloped, icosahedral viral particles approximately 25–26 nm in diameter, consistent with the ultrastructural characteristics of NNV. Phylogenetic analysis based on the RNA2 capsid protein gene revealed that the NNV isolate clustered within the red-spotted grouper nervous necrosis virus (RGNNV) genotype and showed a close evolutionary relationship with RGNNV strains previously reported from groupers and barramundi. Based on clinical manifestations, molecular detection, histopathological lesions, ultrastructural evidence, and phylogenetic characterization, and in accordance with the criteria of the World Organisation for Animal Health (WOAH) for defining susceptible hosts, this study confirms that T. septentrionalis can be naturally infected by NNV and represents a newly confirmed susceptible host. These findings expand the known host range of NNV and provide essential baseline information for disease surveillance, molecular epidemiology, and biosecurity management in filefish aquaculture. Moreover, the addition of T. septentrionalis to the susceptible species allows further inference of the susceptibility of 109 species in family Monacanthidae to infection with NNV, according to the WOAH standard.

ABSTRACT Due to its advantages of a short production cycle, wide market demand, and profitability, decapod crustacean aquaculture has become one of the fastest‐growing food production systems. However, disease outbreaks have become one of the most important factors limiting the sustainability of decapod crustaceans. This review synthesizes current advancements in the understanding of emerging viral pathogens in decapod crustacean aquaculture, with emphasis on their genomic‐based taxonomical classification, susceptible hosts, global distribution, clinical manifestations, pathogenic mechanisms, diagnostic developments, and management strategies. This paper summarizes details of emerging viruses with confirmed pathogenicity (such as decapod iridescent virus 1), as well as viruses with uncertain or yet‐unconfirmed pathogenic roles. In addition, we highlight the role of next‐generation sequencing in virus discovery and examine the challenges posed by coinfection with other microorganisms. This review provides information to help the decapod crustacean aquaculture industry in setting up early warning systems to prevent the introduction of emerging pathogens into the production systems.

Decapod iridescent virus 1 (DIV1) poses a substantial threat to global crustacean aquaculture. The Chinese mitten crab, Eriocheir sinensis, a species of immense economic importance, has an unverified susceptibility status under the stringent criteria of the World Organization for Animal Health (WOAH), as prior investigations predominantly relied on invasive injection challenges. This study rigorously evaluated the susceptibility of E. sinensis to DIV1 by employing both intramuscular (IM) injection and a non-invasive per os (PO) challenge, the latter simulating a natural infection pathway. A multi-faceted diagnostic approach, integrating clinical monitoring, quantitative PCR (qPCR), in situ DIG-labeling loop-mediated isothermal amplification (ISDL), histopathology, and transmission electron microscopy (TEM), was utilized to confirm productive infection. The IM challenge induced acute disease progression, resulting in 100 % cumulative mortality by 7 days post-infection (dpi). In contrast, the PO route produced a protracted disease course with 53.3 % survival at 15 dpi, yet successfully established persistent systemic infection. Quantitative PCR revealed widespread viral dissemination in both groups, although viral loads were several orders of magnitude higher in IM-challenged crabs. The hepatopancreas, gills, and intestine were identified as primary sites of viral accumulation. Histopathological and ultrastructural analyses provided compelling evidence of DIV1-induced cytopathology. A novel and critical finding was the extensive targeting and destruction of circulating hemocytes, evidenced by nuclear pyknosis, cytoplasmic vacuolization, and advanced necrosis, confirmed by both ISDL and TEM. This targeted immunopathology was observed in both infection routes, suggesting a primary mechanism for viral dissemination and immunosuppression. Collectively, these data provide the first definitive, WOAH-compliant evidence of E. sinensis susceptibility to DIV1. The findings establish this commercially vital species as a potential reservoir capable of amplifying and transmitting the virus, posing a significant epidemiological risk to polyculture systems. This underscores the urgent need for revised biosecurity protocols and targeted surveillance to mitigate the spread of DIV1 in global aquaculture.

Decapod iridescent virus 1 (DIV1) is a newly recognized pathogen that causes severe disease in farmed crustaceans, leading to substantial economic losses in the shrimp and crab aquaculture industries. This study investigates the thermal persistence and heat inactivation conditions of DIV1, aiming to establish effective heat treatment protocols for seafood safety in international trade and aquaculture waste management. DIV1 was treated at different temperatures (56 degrees C, 60 degrees C, and 70 degrees C) for varying durations, and infectivity was assessed through experimental infection of Penaeus vannamei. Results indicated that treatment at 56 degrees C for 30 min, 60 degrees C for 15 min, or 70 degrees C for 1 min effectively inactivated DIV1, with these parameters being less stringent than the current WOAH recommendation of 80 degrees C for 30 min. Additionally, this study revealed that DIV1 could remain infectious at room temperature for more than 168 h and at ultralow temperatures (-80 degrees C) for more than 8 years. These findings provide critical data for optimizing heat treatment strategies to mitigate pathogen transmission risks in aquaculture and seafood products, ensuring biosecurity and safety in international markets.

Mechanical vibration is unavoidable during waterless live-fish transport, yet the chemical mechanisms driving muscle quality deterioration remain unclear. This study examined the effects of vibration frequency on muscle quality in yellow catfish Pelteobagrus fulvidraco during simulated waterless transport by integrating physicochemical traits, histology, low-field nuclear magnetic resonance, serum redox indices, and untargeted metabolomics. Higher vibration frequencies exacerbated water loss and texture deterioration, accompanied by progressive microstructural disruption and enhanced water mobility. Serum biomarkers indicated intensified oxidative stress, evidenced by increased malondialdehyde and glutathione depletion. Untargeted metabolomics annotated 1117 metabolites and revealed frequency-dependent metabolic remodeling, with pronounced disturbances in glycerophospholipid metabolism and amino sugar and nucleotide sugar metabolism. Key membrane phospholipids and glycosyl donors were depleted under high-frequency vibration, suggesting impaired membrane stability and structural maintenance. Results demonstrate that transportation vibration stress impairs muscle quality via regulating energy metabolism, membrane lipid stability, glycosylation processes, and cell apoptosis, thus providing a theoretical basis to optimize aquaculture transport and improve the quality of flesh quality resilience during waterless transport.

Effective transport strategies are critical for the survival and welfare of juvenile Ictalurus punctatus, but the effects of pre-transport salt bath treatments remain uncertain. In this study, we systematically evaluated the effects of pre-transport salt bath acclimation at 0‰ (S1), 1‰ (S2), 5‰ (S3), and 9‰ (S4) salinity for 30 min on stress resilience and recovery in fingerlings during 12 h of simulated transport and 24 h of recovery. All fish survived, but total ammonia nitrogen (TAN) increased, and pH decreased in all groups, except S3, which showed significantly lower TAN and higher pH (p < 0.05). The S3 and S4 groups showed attenuated increases in serum cortisol and glucose, with S3 exhibiting the fastest return to baseline levels and stable serum sodium and potassium levels. Liver antioxidant enzyme activities in group S3 remained stable, with the lowest malondialdehyde (MDA) accumulation. Integrated biomarker response (IBR) and histological analyses demonstrated that S3 had the lowest systemic stress and tissue damage, whereas S1 and S4 displayed marked cellular disruption. These results indicate that a 5‰ salt bath applied prior to transport may improve water quality, mitigate stress responses, and preserve tissue integrity in juvenile channel catfish. Further studies are needed to confirm these findings in other species and under commercial transport conditions.

This study presents a strategy to develop crayfish shell peptides with enhanced antioxidant and angiotensin-I-converting enzyme (ACE) inhibitory properties. Crayfish shell protein hydrolysates (CSPH1–3) with different molecular weights were analyzed. CSPH2 (3–5 kDa) exhibited the strongest antioxidant activities, which could scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the 2,2′-azobis(3-ethylbenzothiazoline-6-sulfonic acid) sodium salt (ABTS) radical by (77.40 ± 4.54)% and (91.59 ± 0.30)%, respectively, and ACE inhibition activity of (64.74 ± 0.64)%. CSPH2 was further separated into three fractions, and CSPHF2 showed the maximum biological activity. The sequences of the purified antioxidant peptide (APAPLPPPAP) and ACE inhibitory peptide (QGPDDPLIPIM) were identified by liquid chromatography–tandem mass spectrometry (LC-MS/MS) in CSPHF2. These peptides increased the nitric oxide (NO) concentration and decreased the endothelin-1 (ET-1) content in human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner, while also inhibiting reactive oxygen species (ROS). In addition, CSPH showed protective effects in terms of oxidative damage to HepG2 cells induced by H2O2. These findings suggest that crayfish shell peptides have potential applications as ingredients in antihypertensive agents and antioxidants, offering significant health benefits when consumed.

This study examined the effects of bioactive carbohydrate additives on yellow catfish juveniles following transport. Three additives-Astragalus polysaccharides (APS), chitosan (COS), and xylooligosaccharides (XOS)-were evaluated. The results revealed that, after the holding and transport period, cortisol levels were significantly elevated in the APS group (P < 0.05), while they decreased in the COS group. Both APS and XOS were found to increase the activity levels of immunoglobulin M, component 3, and lysozyme in the fish. Additionally, the APS and COS groups exhibited reduced activity levels of superoxide dismutase and catalase, indicating a mitigation of transport-induced stress. Reduced activity levels of Caspase-3 and Caspase-9 were observed in the intestinal tract of fish from the APS and COS groups. APS also increased the number of goblet cells and the height of intestinal villi, thereby improving intestinal structure integrity. Following transport, the relative abundance of Proteobacteria, which includes pathogenic bacteria, was significantly lower in the APS group, while the abundance of the potentially beneficial genus Cetobacterium increased substantially, resulting in improved microbiota environment compared to the other groups. In conclusion, the addition APS to the water during the holding period followed by transport significantly impacted the health and immune function of yellow catfish juveniles.

This study delves into the susceptibility of Chinese shrimp, Penaeus chinensis, to Decapod iridescent virus 1 (DIV1), a pathogen of significant concern in crustacean aquaculture. Our research meticulously evaluated the infection potential of DIV1 through both invasive (intramuscular injection) and non-invasive (per os) experimental methodologies. The outcomes revealed that intramuscular injection effectively induced infection in P. chinensis, manifesting clinical signs and resulting in 100 % mortality within five days post-infection (dpi). In contrast, per os challenge resulted in a low viral load infection at 3 day post-infection (dpi), which did not persist beyond the 9 dpi, showing a late-stage clearance of iDIV1. These results were corroborated through molecular detection, histopathological examination, in situ DIG-labeling loop-mediated isothermal amplification (ISDL), and transmission electron microscopy (TEM). The study concludes that, while P. chinensis is not considered a susceptible host for DIV1, its susceptibility is nearing a critical threshold. These findings underscore the imperative for continuous virus surveillance and research into host susceptibility to preempt potential economic repercussions for the aquaculture industry.

Decapod iridescent virus 1 (DIV1) is a newly identified pathogen responsible for significant disease outbreaks and high mortality rates in farmed crustaceans. While previous studies have reported the susceptibility of mud crab Scylla paramamosain, a species of considerable commercial importance in Asia, to DIV1, detailed investigations into its infection by DIV1, particularly in situ confirmation evidence, are still lacking. This study experimentally confirms that S. paramamosain is susceptible to DIV1, demonstrating that the virus can infect and induce disease through both intramuscular injection and oral routes. The infection resulted in high mortality, especially in the high-dose injection group, while oral exposure led to slower disease progression. Quantitative PCR analysis revealed high viral loads in hepatopancreas, gonads, and gills. Histopathological examination identified typical signs of infection, including eosinophilic inclusions and nuclear pyknosis. Furthermore, in situ DIG-labeling loop-mediated isothermal amplification (ISDL) provided direct evidence of the widespread distribution of DIV1 across various tissues, including the gonads, suggesting potential implications for reproduction. Transmission electron microscopy (TEM) revealed the presence of icosahedral viral particles with typical iridovirus morphology in infected cells, further confirming DIV1 replication in S. paramamosain. The study underscores the importance of the route of exposure in determining disease progression and mortality, with oral exposure resulting in lower mortality compared to injection. These findings establish S. paramamosain as a susceptible species for DIV1, expanding the known host range of the virus and offering valuable insights into its pathogenesis, tissue tropism, and potential impacts on aquaculture.

Transport procedures usually cause fish stress, especially at high densities, which might cause considerable stress and compromise product quality for adult fish. However, many previous studies of density on sturgeon have hindered understanding of its roles in the physiological change during and after the transportation of sturgeon and no study explored high density as fish-to-water ratios up to 1:1. The objective of this study was to investigate the effects of transport density and duration on antioxidant indices, immune response, and post-transported recovery time of adult sturgeon (758.30 ± 42.8 g, one and a half years old) during long-distance and high-density transportation. Sturgeon were anesthetized with MS-222 under different fish-to-water ratios of 1:1 (D1), 1:2 (D2), 1:3 (D3) and 1:4 (D4), and simulated transport was carried out for 0 h, 1 h, 6 h, 24 h, 48 h and 72 h, and resuscitation was carried out for 6 h, 24 h and 48 h after transport. The relevant measurements were made on the water and serum samples. The findings demonstrated that after 72 h of transportation at various densities, the survival rate was 100%. Total ammonium nitrogen concentrations in the water increased in parallel with the stocking density after 48 h. After 72 h of transport, blood levels of malondialdehyde (MDA), glutathione peroxidase (GSH-PX), catalase (CAT), glutamate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and total protein (TP) were substantially higher in group D1 than in group D4 (P < 0.05), although serum levels of MDA, cortisol (COR), TP, and immunoglobulin M (IGM) in group D2 were not statistically different from those in group D3 (P > 0.05). To cope with the pressure imposed by high-density transport, the antioxidant, metabolic, and immunological indexes of sturgeon in the D1 and D2 were significantly greater than those of sturgeon in D4 group during transport. The D1 group had the most severe damage to antioxidant, metabolic, and immune abilities after resuscitation, and at 48 h after resuscitation, the levels of MDA, AST, and IGM were still significantly higher than those in the control group (P<0.05), whereas the levels of IGM in the D2 group were not significantly different (P > 0.05). Sturgeon under anesthesia should not be transported for longer than 72 h at a time with a fish-to-water ratio of 1:1 because doing so resulted in irreparable harm to all fish indicators. To better comprehend the circumstances necessary for efficient transportation, the study may contribute to evaluate the changes in physiological and biochemical characteristics of sturgeon.

Live fish transport is an important aspect of aquaculture, especially at high densities, which might cause considerable stress and compromise product quality. This study investigated the effects of transport density and time on stress responses, innate immunity, and immune gene expression in juvenile yellow catfish. The fish were designated to five density groups: D11, D12, D13, D14, and D15 (500, 333, 250, 200, and 167 kg/m3). After 36 h of transport, only the D15 group had a 100% survival rate, followed by D14 with 83% survival, and 0% in the remaining three density groups. The damage to gill filaments and the liver worsened with increasing environmental stress severity. The results showed that with the prolonged transport time, plasma cortisol (COR), and catalase (CAT), malonaldehyde (MDA) and lysozyme (LZM) levels in the livers first increased and later gradually decreased after 16 h of transport, while the activity of superoxide dismutase (SOD) continued to rise. The activities of CAT and MDA content increased as the density increased. The expression of heat shock protein (HSP) 70 and 90, tumor necrosis factor α (TNF-α), and interleukin-1β (IL-1β) in juvenile livers in D11 group was significantly higher than the control group (p < 0.05), confirming that density imposed stress on fish during transport. In conclusion, we demonstrated that increased density and time during live transport induced significant physiological stress responses in juvenile P. fulvidraco. This study provides insights into the transport of yellow catfish to improve transport measures and reduce economic losses to the aquaculture industry.

DIV1 has the characteristics of fast transmission and a broad host range. Its infection leads to a high mortality rate, posing a serious threat to the global crustacean aquaculture industry. In order to increase the accuracy of DIV1 detection and reduce the difficulty of result interpretation, this study modified the original nested PCR method targeting the DIV1 ATPase gene. The internal primers for the nested PCR were redesigned to produce a 338 bp amplification product in the second step PCR, effectively distinguishing the target band from primer dimers. The newly established nested PCR method exhibits strong specificity and high sensitivity, with a detection limit as low as 1.37 × 101 copies/reaction. The developed nested PCR assay provides new technical support for the accurate detection of DIV1 in global crustacean aquaculture.

Perinereis species are essential benthonic animals in coastal ecosystems and have significant roles as live feed in aquaculture, owing to their high-protein and low-fat nutritional profile. Despite their ecological importance, the viral communities associated with these organisms need to be better understood. In this study, we generated 2.6 × 108 reads using meta-transcriptomic sequencing and de novo assembled 5.3 × 103 virus-associated contigs. We identified 12 novel RNA viruses from two species, Perinereis aibuhitensis and P. wilsoni, which were classified into four major viral groups: Picobirnaviridae, Marnaviridae, unclassified Picornavirales, and unclassified Bunyavirales. Our findings revealed the hidden diversity of viruses and genome structures in Perinereis, enriching the RNA virosphere and expanding the host range of Picobirnaviridae, Marnaviridae, and Bunyavirales. This study also highlighted the potential biosecurity risk of the novel viruses carried by Perinereis to aquaculture.

All authors declare that there is no conflict of interest or financial interest. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

Although thermosonication (TS) treatment has been widely used in food sterilization, the viable but non‐culturable (VBNC) of bacteria with TS treatment has still concerned potential food safety and public health. The molecular mechanism of VBNC status of bacteria with TS treatment is not clearly known. Therefore, in this study, we used Shewanella putrefaciens, which was a common putrefactive bacteria in aquatic products, to study the VBNC state of bacteria with TS treatment. Firstly, our results revealed that S. putrefaciens still could enter the VBNC state after TS treatments: 50 kHz, 300 W, 30 min ultrasonic treatment and 70 °C heating; Subsequently, we found the VBNC state of S. putrefaciens can resist the damage of TS treatment, such as cell wall break, DNA degradation, etc; Finally, four-dimensional data-independent acquisition-based proteomics showed that under VBNC state, S. putrefaciens upregulated functional proteins to resist TS treatment, such as: ribosomal proteins to accelerate the synthesis of stress proteins to counteract TS treatments, ornithine decarboxylase SpeF and MraY to repair TS treatment-induced damage, etc. Meanwhile, S. putrefaciens downregulates metabolic and transport functional proteins such as dehydrogenase to reduce the metabolism. Importantly, among those proteins, the ribosomal transcriptional regulatory protein family, such as rpsB, etc, may be the key proteins for S. putrefaciens entering VBNC state. This finding can provide some new strategies for preventing VBNC status of bacteria with TS treatment, such as: inhibition of key proteins, etc.

In this study, a novel antifreeze peptide was isolated from crayfish shells and its antifreeze mechanism and activity were investigated. Firstly, the crayfish shells peptides (CSPs) were prepared by Enzymatic Hydrolysis Technology (alkaline protease and trypsin) and the CSPs could significantly enhance the survival rate of Saccharomyces cerevisiae from 12.5% to 88.5% after 24h freeze-thaw cycle. Then, a total 1004 peptides were identified from CSPs by liquid chromatography/tandem mass spectrometry (LC-MS/MS), among those peptides, two peptide sequences YWDHPIRDGFAPH (AFP1) and GPPGKPGIPDIVDW (AFP1) were selected as potential antifreeze polypeptides (AFPs) according the physicochemical properties: instability index, hydrophilicity, and confidence level. Furthermore, the antifreeze activity and mechanism of AFP1 and AFP2 were studied through molecular dynamics simulation, revealing that the AFP1 and AFP2 could adsorb to ice surface with 7 and 14 hydrogen bonds, respectively. Moreover, AFP2 has a more stable binding capacity and could interact with 33 water molecules. At last, the results of differential scanning calorimetry (DSC) and cold-stage polarization microscope indicated that the thermal hysteresis (TH) values of AFP2 were 1.62 - 2.09 degrees C and could inhibit ice recrystallization. Therefore, this study provides a theoretical basis for developing safe peptide-based cryoprotectants which will improve the quality of frozen foods.

Viral diseases have become a significant impediment to the sustainable development of the global shrimp aquaculture industry. Decapod iridescent virus 1 (DIV1) is an emerging shrimp virus that has affected shrimp in China recent years. Rapid detection of DIV1 could improve enhance the effectiveness of prevention, control and treatment in the absence of good prevention and control measures. This study established loop-mediated isothermal amplification (LAMP) along with two visual interpretation methods, LAMP-dye and LAMP-LFD, to detect DIV1. The newly developed method would not cause cross-reactions with other shrimp pathogens such as white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), Enterocytozoon hepatopenaei (EHP), and Vibrio parahaemolyticus acute hepatopancreatic necrosis disease (VpAHPND). The detection limit of DIV1 LAMP was as low as 103 copies of DIV1 per reaction, with a reaction time of less than 40 min. The diagnostic sensitivity and diagnostic specificity of this method were determined to be 88% and 100%, respectively, when compared with the conventional PCR. Both of the LAMP-dye and LAMP-LFD methods are cost-effective and do not require expensive amplification equipment. They can be combined with LAMP and other temperature amplification methods for rapid on-site detection, effectively prevent aerosol contamination, and which are convenient and suitable for field testing or preliminary infection rish prediction experiments to predict the risk of infection.