First report of Metschnikowia bicuspidata infection in the oriental river prawn (Macrobrachium nipponense, de Haan) in China.

During breeding, some oriental river prawns (Macrobrachium nipponense, de Haan), an important aquaculture species in China, exhibit yellowish-brown body colouration, reduced appetite, and vitality. Diseased prawns revealed characteristic emulsifying disease signs, including whitened musculature, hepatopancreatic tissues, milky haemolymph, and non-coagulation. The present study investigated the causative agent of M. nipponense infection through isolation, histopathology, molecular sequencing, and infection experiments. The pathogenic strain exhibited distinctive white colonies on Bengal red medium, with microscopic examination confirming the presence of yeast cells. Histopathological analysis revealed prominent pathological alterations and yeast cell infiltration in muscles, hepatopancreas and gills. Additionally, 26S rDNA sequencing of the isolated yeast strain LNMN2022 revealed Metschnikowia bicuspidata (GenBank: OR518659) as the causative agent. This strain exhibited a 98.28% sequence homology with M. bicuspidata LNMB2021 (GenBank: OK094821) and 96.62% with M. bicuspidata LNES0119 (GenBank: OK073903). The pathogenicity test confirmed that M. bicuspidata elicited clinical signs in M. nipponense consistent with those observed in natural populations, and the median lethal concentration was determined to be 3.3 × 105 cfu/mL. This study establishes a foundation for further investigations into the host range and epidemiological characteristics of the pathogen M. bicuspidata in aquatic animals and provides an empirical basis for disease management in M. nipponense.

Multi-omics analysis of oxidative stress and apoptosis in hepatopancreas cells induced by Polyascus gregaria parasitizing the Eriocheir sinensis.

Polyascus gregaria, a parasitic barnacle, poses a significant threat to Eriocheir sinensis farms by inhibiting crab growth. However, the molecular and pathological mechanisms behind P. gregaria infection in the hepatopancreas of E. sinensis remain unclear. In this study, we investigated the impact and underlying mechanisms of P. gregaria infection on E. sinensis through analyzing the infected hepatopancreatic tissues by tandem mass tag technology and RNA-Seq high-throughput sequencing. Among the identified 10,693 differentially expressed genes, 294 genes were significantly altered following P. gregaria infection, including 92 upregulated and 202 downregulated genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses further revealed the involvement of these genes in oxidative decomposition, lipid metabolism, inflammation, and hepatopancreas metabolism. Meanwhile, the identified 253 differentially expressed proteins, including 143 upregulated and 110 downregulated proteins, are mainly related to cellular and metabolic processes, catalytic activity, and cell components. The pathway analysis indicated their enrichment in glycolysis/gluconeogenesis, oxidative phosphorylation, endoplasmic reticulum protein processing, and actin cytoskeleton regulation. The involvement of these differentially expressed genes and proteins in the peroxisome proliferator-activated receptors pathway during host immune responses against P. gregaria infection has been highlighted. Furthermore, pathological examinations and biochemical indicators jointly demonstrated the hepatopancreatic damage and increased oxidative stress and apoptosis in the infected E. sinensis. Collectively, our study provides crucial insights into the mechanisms underlying the E. sinensis-P. gregaria interactions, and may contribute to the development of novel strategies for parasite control and reducing economic losses in aquaculture.

Comparison of the gut microbiota and untargeted gut tissue metabolome of Chinese mitten crabs (Eriocheir sinensis) with different shell colors.

Introduction: The Chinese mitten crab (Eriocheir sinensis) is a highly valued freshwater crustacean in China. While the natural shell color of E. sinensis is greenish brown (GH), we found a variety with a brownish-orange shell color (RH). Although RH is more expensive, it exhibits a lower molting frequency and growth rate compared with GH, which significantly reduces its yield and hinders large-scale farming. The growth and development of animals are closely related to their gut microbiota and gut tissue metabolic profiles. Methods: In this study, we compared the gut microbiome communities and metabolic profiles of juvenile RH and GH crabs using 16S rRNA gene sequencing and liquid chromatography-mass spectrometry (LC-MS), respectively. Results: Our findings indicated that the intestinal microbial composition and metabolic characteristics of E. sinensis differed significantly between RH and GH. At the operational taxonomic unit (OTU) level, the α-diversity of the gut microbiota did not differ significantly between RH and GH, while the ß-diversity of the RH gut microbiota was higher than that of the GH gut microbiota. At the species level, the richness of unclassified_c_Alphaproteobacteria was significantly higher in the GH group, while the RH group had a significantly higher richness of three low-abundance species, Flavobacteria bacterium BAL38, Paraburkholderia ferrariae, and uncultured_bacterium_g__Legionella. In the current study, 598 gut tissue metabolites were identified, and 159 metabolites were significantly different between GH and RH. The metabolite profile of RH was characteristic of a low level of most amino acids and lipid metabolites and a high level of several pigments compared with that of GH. These metabolites were enriched in 102 KEGG pathways. Four pathways, including (1) Central carbon metabolism in cancer, (2) protein digestion and absorption, (3) alanine, aspartate and glutamate metabolism, and (4) aminoacyl-tRNA biosynthesis, were significantly enriched. The correlation analysis between metabolites and microbiotas indicated that most key differential metabolites were positively correlated with the abundance of Shewanella_sp_MR-7. Discussion: This research provided a greater understanding of the physiological conditions of E. sinensis varieties with different shell colors by comparing the gut microbiota and gut tissue metabolome.

The Complete Mitochondrial Genome of Torix tukubana (Annelida: Hirudinea: Glossiphoniidae).

Torix tukubana is a poorly understood proboscidate leech species, generally an ectoparasite on amphibian species. In this study, the complete mitochondrial genome (mitogenome) of T. tukubana was sequenced using next-generation sequencing (NGS), and the essential characteristics, gene arrangement, and phylogenetic relationship were analyzed. The results showed that the T. tukubana mitogenome was 14,814 bp in length, consisting of 13 protein-coding genes (PCGs), 22 tRNAs, 2 rRNAs, and 1 control region (CR). The mitogenome composition presented a strong A + T bias (73.6%). All tRNAs had the typical clover structure except the trnS1 (TCT), whose dihydrouridine (DHU) arm was short, having only one complementary base pair. Additionally, 8 gene order patterns were identified among 25 known Hirudinea species, and T. tukubana was identical to the Hirudinea ground pattern. A phylogenetic analysis based on 13 PCGs indicated that all the studied species clustered into three main clades. The relationships among Hirudinea species were basically consistent with their gene arrangement results, but different from their morphological taxonomy. T. tukubana was in the monophyletic group of Glossiphoniidae, a finding consistent with previous research. Our results provided the essential characteristics of the T. tukubana mitogenome. As the first complete mitogenome of Torix, it could offer valuable information for a systematic understanding of the Hirudinea species.

Transcriptome analysis of hepatopancreas of Chinese grass shrimp, Palaemonetes sinensis, infected by Enterocytospora artemiae.

The emergence of the microsporidian, Enterocytospora artemiae, has caused serious economic losses to the aquaculture industry of Palaemonetes sinensis. The hepatopancreas is the main digestive and immune organ of P. sinensis, and the main site of E. artemiae infection. We used next-generation sequencing to determine the effects of E. artemiae parasitism on the hepatopancreas of P. sinensis at the transcriptome level. The hepatopancreas of P. sinensis was parasitized by E. artemiae, and 881 differentially expressed genes (DEGs) were obtained, of which 643 were upregulated and 238 were downregulated. These DEGs are mainly involved in DNA replication, transcription, translation, immunity, and metabolism. Among them, the cellular processes of DNA replication, transcription and translation are significantly strengthened, which may be related to the use of host ATP and nucleic acid by E. artemiae to achieve proliferation and damage to host cells to enhance DNA replication and repair. Moreover, to defend against E. artemiae, some immune genes related to antioxidation, such as glutathione metabolism, seleno compound metabolism, and cytochrome p450 2L1, were significantly upregulated, but simultaneously, tumor necrosis factor, NF-κB inhibitor α, and other immune-related genes were significantly down regulated, indicating that the parasitism of E. artemiae led to a significant decline in the immune defense ability of P. sinensis. From the perspective of metabolism, the metabolism-related DEGs of retinol, glycine, serine, and threonine metabolism, were significantly downregulated, resulting in insufficient nutrient absorption and decreased energy supply of the P. sinensis, which in turn affected their growth. The differential genes and pathways identified in this study can provide a reference basis to further elucidate the pathogenic mechanism of P. sinensis infected with E. artemiae and the prevention and control of microsporidia disease.

Development of a nested PCR assay for specific detection of Metschnikowia bicuspidata infecting Eriocheir sinensis.

In recent years, the "milky disease" caused by Metschnikowia bicuspidata has seriously affected the Eriocheir sinensis culture industry. Discovering and blocking the transmission route has become the key to controlling this disease. The existing polymerase chain reaction (PCR) detection technology for M. bicuspidata uses the ribosomal DNA (rDNA) sequence, but low sensitivity and specificity lead to frequent false detections. We developed a highly specific and sensitive nested PCR method to detect M. bicuspidata, by targeting the hyphally regulated cell wall protein (HYR) gene. This nested HYR-PCR produced a single clear band, showed no cross-reaction with other pathogens, and was superior to rDNA-PCR in specificity and sensitivity. The sensitivity of nested HYR-PCR (6.10 × 101 copies/µL) was greater than those of the large subunit ribosomal RNA gene (LSU rRNA; 6.03 × 104 copies/µL) and internal transcribed spacer (ITS; 6.74 × 105 copies/µL) PCRs. The nested HYR-PCR also showed a higher positivity rate (71.1%) than those obtained with LSU rRNA (16.7%) and ITS rDNA (24.4%). In conclusion, we developed a new nested HYR-PCR method for the specific and sensitive detection of M. bicuspidata infection. This will help to elucidate the transmission route of M. bicuspidata and to design effective management and control measures for M. bicuspidata disease.

Experimental Transmission of the Yeast, Metschnikowia bicuspidata, in the Chinese Mitten Crab, Eriocheir sinensis.

The Chinese mitten crab, Eriocheirsinensis, is an important farmed crustacean species in China, outranking other farmed crabs in yield and economic importance. An infection called "milky disease", caused by the yeast, Metschnikowiabicuspidata, has emerged in E. sinensis farms in northeast China and has caused progressive economic losses. The diseased crabs present with opaque, whitish muscles and milky hemolymph. Currently, there are no effective drugs to treat the infection. Clarifying the transmission route of M. bicuspidata would help to treat and prevent the disease. We investigated the effects of three different M. bicuspidata infection methods (feeding, immersion, and cohabitation) on E. sinensis. All three infection methods led to a high infection rate in healthy crabs. After 35 d, the infection rate was 76.7%, 66.7%, and 53.3% in the feeding, immersion, and cohabitation groups, respectively. Diseased crabs exhibited the typical symptom of hemolymph emulsification, with a high pathogen load of M. bicuspidata. The yeast was not detected in the oocytes of infected crabs. Fertilized embryos, zoea larvae, and megalopae of infected ovigerous crabs tested negative for yeast, indicating that direct transmission from mother to offspring does not occur. Our results highlight avenues for the prevention and control of this yeast.

Freshwater crabs could act as vehicles of spreading avian influenza virus.

Avian influenza virus (AIV) possessed significant risk to various animals and human health. Wild birds, especially waterfowls are considered to be the natural reservoir of AIVs. The ecology of AIV is still far from being fully understood. Freshwater crabs are nonnegligible biotic factor in AIV ecosystem. We analyzed the ability of freshwater crabs accumulate and spread AIV. We found that AIV remain infectious in water only for 36 h but persist in crabs for 48 h. Crabs could accumulate AIV in their gills and gastrointestinal tracts. The AIV titers in crabs were higher than the surrounding contaminated water. Crabs could accumulate AIV from contaminated water, carry the virus and spread to naïve crabs via surrounding water. Our study identified freshwater crab as a novel transmission vehicle in AIV ecosystem.

Impact of Dietary Protein Content on Soil Bacterial and Fungal Communities in a Rice-Crab Co-culture System.

Although co-culture of paddy fields with aquatic animals is lucrative, the ecological impacts of high-protein content entering the agricultural soil via animal pellet feed and feces have not been well studied. Moreover, the effects of dietary protein on soils and soil microbes remain unclear. To elucidate this, we examined soil bacterial and fungal community composition and temporal changes in paddy fields subjected to different protein-content diets via 16S/18S rRNA gene amplicon sequencing analysis with a high-throughput next-generation sequencer. MiSeq sequencing revealed that protein content significantly impacted fungal community structure. High-protein diets reduced bacterial community diversity and relative abundance in both July and October. The phylum-level bacterial taxonomic composition was not affected by diet treatment, while in fungi, a major phylum-level shift was evident. Hierarchically clustered analysis showed that high-protein diets significantly reduced the relative abundance of Brevundimonas in both July and October. Saprotrophic macrofungal diversity was negatively related to dietary protein content. Considering microbial community structure and environmental factors, ca. 15% protein content is appropriate for the rice-crab co-culture system that we studied.