UNASSIGNED: Due to the high-density farming of Larimichthys crocea over the years, diseases caused by pathogens such as bacteria, viruses, and parasites frequently occur in Ningbo, posing a huge threat and challenge to the sustainable and healthy development of the L. crocea\'s bay farming industry. In order to understand the diseases occurrence in L. crocea farming in Ningbo area, an epidemiological investigation of L. crocea diseases was carried out through regular sampling in 2023.
UNASSIGNED: From April to October 2023, routine sampling of L. crocea was conducted monthly in various farming areas in Ningbo. Each time, live or dying L. crocea with obvious clinical symptoms were sampled, with a total number of 55 L. crocea collected. The samples were preserved in ice bags and transported to the laboratory for pathogen detection(including bacterial isolation and identification,virus identification, and parasites detection).
UNASSIGNED: A total of fifty-five fish dying L. crocea with obvious clinical symptoms were collected in this study, of which 78.18% (43/55) were detected with symptoms caused by pathogenic infection, while 21.82% (12/55) did not have identified pathogens, which were presumed to be breeding abrasions, nutritional metabolic disorders, unconventional pathogens infection or other reasons. A total of twenty-five pathogenic bacteria strains were isolated, which mainly were Pseudomonas plecoglossicida and Vibrio harveyi, accounting for 52% (13/25) and 32% (8/25) of the pathogenic bacteria strains, respectively. Among them, both V. harveyi and Streptococcus. iniae co-infected one fish. Additionally, three other bacterial strains including Nocardia seriolae, Staphylococcus Saprophyticus, and Photobacterium damselae subsp.damselae were isolated. Microscopic examination mainly observed two parasites, Cryptocaryon irritans and Neobenedenia girellae. In virus detection, the red sea bream iridovirus (RSIV) was mainly detected in L. crocea. Statistical analysis showed that among the fish with detected pathogens, 55.81% (24/43) had bacterial infections, 37.21% (16/43) had parasitic infections, and 37.21% (16/43) had RSIV infections. Among them, five fish had mixed infections of bacteria and parasites, three had mixed infections of bacteria and viruses, three had mixed infections of parasites and viruses, and one L. crocea had mixed infections of viruses, bacteria, and parasites.
UNASSIGNED: These findings indicate that these three major types of diseases are very common in the L. crocea farming area in Ningbo, implying the complexity of mixed infections of multiple diseases.

The aim of this research was to estimate the immunopotentiation effect of brown algae Padina boergesenii water extract on Nile tilapia, Oreochromis niloticus through resistance to Pseudomonas putida infection. Gas Chromatography Mass Spectrometry was utilized to characterize the seaweed phytoconstituents. One hundred and twenty-six fish were divided in triplicates into two equal groups corresponding to two diet variants that used to feed Nile tilapia for 20 successive days: a basal (control), and P. boergesenii water extract supplemented group. Fish samples were collected at 10-days intervals throughout the experiment. Serum biochemical constituents, total antioxidant capacity (TAC), and some immune related genes expression of the spleen and intestinal tissues of experimental fish were studied, as well as histological examination of fish immune tissues. Moreover, following 20 days of feeding, the susceptibility of Nile tilapia to P. putida infection was evaluated to assess the protective effect of the used extract. The findings indicated that the studied parameters were significantly increased, and the best immune response profiles were observed in fish fed P. boergesenii water extract for 20 successive days. A bacterial challenge experiment using P. putida resulted in higher survival within the supplemented fish group than the control. Thus, the lowered post-challenge mortality of the fish may be related to the protection provided by the stimulation of the innate immune system, reduced oxidative stress by higher activity of TAC, and elevated levels of expression of iterleukin-1beta (IL-1β), beta-defensin (β-defensin), and natural killer-lysin (NKl). Moreover, the constituents of the extract used showed potential protective activity for histological features of the supplemented fish group when compared to the control. Collectively, this study presents a great insight on the protective role of P. boergesenii water extract as an additive in Nile tilapia feed which suggests its potential for improving the immune response against P. putida infection.

Myxidium rhodei Léger, 1905 (Cnidaria: Myxozoa) is a kidney-infecting myxosporean that was originally described from the European bitterling Rhodeus amarus. Subsequently, it has been documented based on spore morphology in more than 40 other cypriniform species, with the roach Rutilus rutilus being the most commonly reported host. This study introduces the first comprehensive data assessment of M. rhodei, conducted through morphological, ecological and molecular methods. The morphological and phylogenetic analyses of SSU rDNA sequences of Myxidium isolates obtained from European bitterling and roach did not support parasite conspecificity from these fish. In fact, the roach-infecting isolates represent three distinct parasite species. The first two, M. rutili n. sp. and M. rutilusi n. sp., are closely related cryptic species clustering with other myxosporeans in the freshwater urinary clade, sharing the same tissue tropism. The third one, M. batuevae n. sp., previously assigned to M. cf. rhodei, clustered in the hepatic biliary clade sister to bitterling-infecting M. rhodei. Our examination of diverse cypriniform fishes, coupled with molecular and morphological analyses, allowed us to untangle the cryptic species nature of M. rhodei and discover the existence of novel species. This underscores the largely undiscovered range of myxozoan diversity and highlights the need to incorporate sequence data in diagnosing novel species.
UNASSIGNED: Résoudre le casse-tête de Myxidium rhodei (Myxozoa) : aperçu de sa phylogénie et de sa spécificité d’hôte chez les Cypriniformes.
UNASSIGNED: Myxidium rhodei Léger, 1905 (Cnidaria : Myxozoa) est un Myxosporea infectant les reins qui a été décrit à l’origine chez la bouvière, Rhodeus amarus. Par la suite, il a été documenté, sur la base de la morphologie des spores, chez plus de 40 autres espèces de cypriniformes, le gardon Rutilus rutilus étant l’hôte le plus fréquemment signalé. Cette étude présente la première évaluation complète des données sur M. rhodei, réalisée par des méthodes morphologiques, écologiques et moléculaires. Les analyse morphologiques et phylogénétiques des séquences d’ADNr SSU des isolats de Myxidium obtenus à partir de bouvières et de gardons européens n’ont pas confirmé la conspécificité du parasite de ces poissons. En fait, les isolats infectant les gardons représentent trois espèces distinctes de parasites. Les deux premières, M. rutili n. sp. et M. rutilusi n. sp., sont des espèces cryptiques étroitement apparentées, regroupées avec d’autres Myxosporea du clade urinaire d’eau douce, partageant le même tropisme tissulaire. La troisième, M. batuevae n. sp., précédemment attribuée à M. cf. rhodei, appartient au clade biliaire hépatique, groupe-frère de M. rhodei infectant la bouvière. Notre examen de divers poissons cypriniformes, couplé à des analyses moléculaires et morphologiques, nous a permis de démêler la nature cryptique des espèces de M. rhodei et de découvrir l’existence de nouvelles espèces. Cela souligne la diversité largement méconnue des Myxozoaires et souligne la nécessité d’incorporer des données de séquence dans le diagnostic de nouvelles espèces.

UNASSIGNED: Red blood cells (RBCs), also known as erythrocytes, are underestimated in their role in the immune system. In mammals, erythrocytes undergo maturation that involves the loss of nuclei, resulting in limited transcription and protein synthesis capabilities. However, the nucleated nature of non-mammalian RBCs is challenging this conventional understanding of RBCs. Notably, in bony fishes, research indicates that RBCs are not only susceptible to pathogen attacks but express immune receptors and effector molecules. However, given the abundance of RBCs and their interaction with every physiological system, we postulate that they act in surveillance as sentinels, rapid responders, and messengers.
UNASSIGNED: We performed a series of in vitro experiments with Cyprinus carpio RBCs exposed to Aeromonas hydrophila, as well as in vivo laboratory infections using different concentrations of bacteria.
UNASSIGNED: qPCR revealed that RBCs express genes of several inflammatory cytokines. Using cyprinid-specific antibodies, we confirmed that RBCs secreted tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ). In contrast to these indirect immune mechanisms, we observed that RBCs produce reactive oxygen species and, through transmission electron and confocal microscopy, that RBCs can engulf particles. Finally, RBCs expressed and upregulated several putative toll-like receptors, including tlr4 and tlr9, in response to A. hydrophila infection in vivo.
UNASSIGNED: Overall, the RBC repertoire of pattern recognition receptors, their secretion of effector molecules, and their swift response make them immune sentinels capable of rapidly detecting and signaling the presence of foreign pathogens. By studying the interaction between a bacterium and erythrocytes, we provide novel insights into how the latter may contribute to overall innate and adaptive immune responses of teleost fishes.

UNASSIGNED: Oxygen deprivation (OD) is a critical condition that can lead to brain damage and even death. Current hypoxia management approaches are limited in effectiveness. Centella asiatica (CA), known for its neuroprotective properties, offers a potential alternative for OD treatment.
UNASSIGNED: This study aims to investigate the neuroprotective effects of CA on the expression of brain-derived neurotrophic factor (BDNF) and vesicular glutamate transporter 1 (VGLUT1) in zebrafish larvae under oxygen-deficient conditions.
UNASSIGNED: Zebrafish embryos were subjected to low oxygen levels (1.5 mg/l) 0-2 hours post-fertilization (hpf) until 3 days post-fertilization (dpf), simulating the early stages of OD. Subsequent treatment involved varying concentrations of CA (1.25-5 µg/ml) up to 9 days post-fertilization. The expression levels of BDNF and VGLUT1 were measured using PCR methods. Statistical analysis was conducted using a two-way analysis of variance to evaluate the impact of CA on the expression of BDNF and VGLUT1 in zebrafish larvae aged 3 and 9 dpf in oxygen-deprived conditions.
UNASSIGNED: CA significantly influenced the expression of BDNF and VGLUT1 under OD (p < 0.001). An increase in BDNF expression (p < 0.001) and a decrease in VGLUT1 (p < 0.01) were observed in zebrafish larvae experiencing OD and treated with CA. There was no significant difference in BDNF and VGLUT1 expression across age variations in zebrafish larvae at 3 dpf and 9 dpf in the treatment groups (p > 0.05). CA concentration of 2.5 µg/ml effectively enhanced BDNF and reduced VGLUT1 in 3-9 dpf zebrafish larvae.
UNASSIGNED: CA demonstrates potential as a neuroprotective agent, modulating increased BDNF expression and reduced VGLUT1 under OD conditions. These findings lay a foundation for further research in developing therapies for oxygen deficiency.

Nervous necrosis virus (NNV) is one of the greatest threats to Mediterranean aquaculture, infecting more than 170 fish species and causing mortalities up to 100% in larvae and juveniles of susceptible species. Intensive aquaculture implies stressed conditions that affect the welfare of fish and their ability to fight against infections. In fact, a higher susceptibility to NNV has been related to poor welfare conditions. In order to analyze the physiological link between stressed conditions and increased susceptibility to NNV, as well as its possible role in the pathogenesis of this disease, we reared shi drum (Umbrina cirrosa) juveniles (30.7 ± 3.10 g body weight), which are expected to be asymptomatic upon NNV infection, at three stocking densities (2, 15, and 30 kg/m3) for 27 days and subsequently challenged them with NNV. We firstly characterized the stressed conditions of the specimens before and after infection and recorded the mortalities, demonstrating that stressed specimens reared at 30 kg/m3 suffered mortalities. However, the viral loads in different tissues were similar in all experimental groups, allowing horizontal and vertical transmission of the virus from asymptomatic specimens. All of these data suggest that shi drum tolerates wide ranges of culture densities, although high densities might be a setback for controlling NNV outbreaks in this species. In an attempt to understand the molecular pathways orchestrating this susceptibility change in stressed conditions, we performed a transcriptomic analysis of four tissues under mock- and NNV-infected conditions. In addition to the modification of the exceptive pathways such as cell adhesion, leukocyte migration, cytokine interaction, cell proliferation and survival, and autophagy, we also observed a heavy alteration of the neuroactive ligand-receptor pathway in three of the four tissues analyzed. Our data also point to some of the receptors of this pathway as potential candidates for future pharmacological treatment to avoid the exacerbated immune response that could trigger fish mortalities upon NNV infection.

Infectious pancreatic necrosis virus (IPNV) causes economic losses with a highly variable mortality rate worldwide, especially in rainbow trout. The virus has a double-stranded bi-partite RNA genome designated segment A and B. New complete genome sequences of nine rainbow trout isolates from Turkey were determined and subjected to phylogenetic analysis, identifying all as genotype 5 (serotype Sp). A time-dependent change in the extended pathogenicity motif of VP2 from P217T221A247 (PTA) to PTE P217T221E247 over a period of 10 years was identified. A wider analysis of 99 IPNV sequences from Turkey and Iran revealed the emergence of the motif PTE from 2007 to 2017, inducing significant morbidity in fry by 2013. In fact, displacement of the PTA motif, by the PTE motif in IPNV isolates appeared to be connected to a production peak of rainbow trout in 2013. An additional CAI analysis provided more evidence, indicating that rainbow trout culture in Turkey has an influence on the evolution of IPNV.

The mode and outcome of fish-virus interactions are influenced by many abiotic factors, among which water temperature is especially important in poikilothermic fish. Rare minnow Gobiocypris rarus is a eurythermal small cyprinid fish that is sensitive to infection with genotype II grass carp reovirus (GCRV). HSP70, a conservative and key player in heat shock response, is previously identified as an induced pro-viral factor during GCRV infection in vitro. Here, rare minnow was subjected to heat shock treatment (HST), 1 h treatment at 32 °C followed by reverting to a normal temperature of 24 °C, and subsequently challenged with GCRV-II at a dosage of 1 × LD50. The effect of HST on GCRV virulence in vivo was evaluated by calculating virus-associated mortality and viral load in both dead and survival fish. The results revealed that HST enhanced the mortality of rare minnow infected with GCRV; the fact that viral loads in the tissue samples of HST-treated fish were significantly higher than those in samples of the control group at 6, 8 d p.i. reflected a faster infection process due to HST. Quantitative gene expression analysis was further employed to show that the expression levels of Hsp70 in intestine and liver tissues from the HST group declined faster than muscle tissue after HST. HST W/O GCRV challenge upregulated proinflammatory cytokines such as MyD88 and Nf-κB, which was in consistence with the inflammation observed in histopathological analysis. This study shed light on the complexity of the interaction between fish abiotic and biotic stress response, which suggested that HST, an abiotic stress, could enhance the virulence of GCRV in Gobiocypris rarus that involved modulating the gene expression of host heat shock, as well as a pro-inflammatory response.

The alewife (Alosa pseudoharengus) is an anadromous herring that inhabits waters of northeastern North America. This prey species is a critical forage for piscivorous birds, mammals, and fishes in estuarine and oceanic ecosystems. During a discovery project tailored to identify potentially emerging pathogens of this species, we obtained the full genome of a novel hepadnavirus (ApHBV) from clinically normal alewives collected from the Maurice River, Great Egg Harbor River, and Delaware River in New Jersey, USA during 2015-2018. This previously undescribed hepadnavirus contained a circular DNA genome of 3146 nucleotides. Phylogenetic analysis of the polymerase protein placed this virus in the clade of metahepadnaviruses (family: Hepadnaviridae; genus: Metahepadnavirus). There was no evidence of pathology in the internal organs of infected fish and virions were not observed in liver tissues by electron microscopy. We developed a Taqman-based quantitative (qPCR) assay and screened 182 individuals collected between 2015 and 2018 and detected additional qPCR positives (n = 6). An additional complete genome was obtained in 2018 and it has 99.4% genome nucleotide identity to the first virus. Single-nucleotide polymorphisms were observed between the two genomes, including 7/9 and 12/8 synonymous vs nonsynonymous mutations across the polymerase and surface proteins, respectively. While there was no evidence that this virus was associated with disease in this species, alewives are migratory interjurisdictional fishes of management concern. Identification of microbial agents using de novo sequencing and other advanced technologies is a critical aspect of understanding disease ecology for informed population management.

Grass Carp Reovirus (GCRV) and Aeromonas hydrophila (Ah) are the causative agents of haemorrhagic disease in grass carp. This study aimed to investigate the molecular mechanisms and immune responses at the miRNA, mRNA, and protein levels in grass carp kidney cells (CIK) infected by Grass Carp Reovirus (GCRV, NV) and Aeromonas hydrophilus (Bacteria, NB) to gain insight into their pathogenesis. Within 48 h of infection with Grass Carp Reovirus (GCRV), 99 differentially expressed microRNA (DEMs), 2132 differentially expressed genes (DEGs), and 627 differentially expressed proteins (DEPs) were identified by sequencing; a total of 92 DEMs, 3162 DEGs, and 712 DEPs were identified within 48 h of infection with Aeromonas hydrophila. It is worth noting that most of the DEGs in the NV group were primarily involved in cellular processes, while most of the DEGs in the NB group were associated with metabolic pathways based on KEGG enrichment analysis. This study revealed that the mechanism of a grass carp haemorrhage caused by GCRV infection differs from that caused by the Aeromonas hydrophila infection. An important miRNA-mRNA-protein regulatory network was established based on comprehensive transcriptome and proteome analysis. Furthermore, 14 DEGs and 6 DEMs were randomly selected for the verification of RNA/small RNA-seq data by RT-qPCR. Our study not only contributes to the understanding of the pathogenesis of grass carp CIK cells infected with GCRV and Aeromonas hydrophila, but also serves as a significant reference value for other aquatic animal haemorrhagic diseases.