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Metschnikowia bicuspidata causes a "milky disease" in Chinese mitten crab, Eriocheir sinensis, which inflicts significant damage on the breeding industry, but there are no effective drugs for this disease. Precise detection technologies and clarification of transmission routes are now essential to prevent its occurrence. A real-time fluorescent quantitative PCR (qPCR) detection method targeting the mitochondrial cytochrome c oxidase subunit VIA (COX6A) of M. bicuspidata was developed and its sensitivity, specificity, repeatability, and application effectiveness evaluated. There was a robust linear relationship between the qPCR threshold cycle value (Ct) and copy number of the standard with a wide dynamic range. The standard curve had a correlation coefficient (R2) of 0.996, amplification efficiency of 103.092%, and a lower limit of detection sensitivity of 7.6 × 101 copies/µL. The COX6A-qPCR method exhibited high specificity for the detection of M. bicuspidata, with no cross-reactivity. The intra- and inter-group variation coefficients were <1% and 2%, respectively. The qPCR exhibited superior sensitivity compared to existing detection methods, with a positivity rate of 76.67%. The M. bicuspidata content ranged from 1.0 × 101–2.7 × 106 copies/µL. The COX6A-qPCR detection technology exhibited high sensitivity, strong specificity, and excellent repeatability, enabling the accurate quantification of M. bicuspidata. [ABSTRACT FROM AUTHOR]

The "milky disease" of the Chinese mitten crab, Eriocheir sinensis , is a highly lethal fungal disease caused by Metschnikowia bicuspidata infection. To elucidate the immune responses of the hemolymph of E. sinensis to M. bicuspidata infection, a comparative analysis of the hemolymph of E. sinensis infected with M. bicuspidata and that treated with phosphate buffered saline was performed using label-free quantitative proteomics. A total of 429 proteins were identified. Using a 1.5-fold change in expression as a physiologically significant benchmark, 62 differentially expressed proteins were identified, of which 38 were significantly upregulated and 24 were significantly downregulated. The upregulated proteins mainly included cytoskeleton-related proteins (myosin regulatory light chain 2, myosin light chain alkali, tubulin α-2 chain, and tubulin β-1 chain), serine protease and serine protease inhibitor (clip domain-containing serine protease, leukocyte elastase inhibitor, serine protein inhibitor 42Dd), catalase, transferrin, and heat shock protein 70. Upregulation of these proteins indicated that phenoloxidase system, phagocytosis and the ROS systems were induced by M. bicuspidata. The downregulated proteins were mainly organ and tissue regeneration proteins (PDGF/VEGF-related factor protein, integrin-linked protein kinase homing pat-4 gene) and hemagglutination-associated proteins (hemolymph clottable protein, hemocyte protein-glutamine gamma-glutamyltransferase). Downregulation of these proteins indicated that M. bicuspidata inhibited hemocyte regeneration and hemolymph agglutination. Fifteen differentially expressed proteins related to immunity were verified using a parallel reaction monitoring method. The expression trend of these proteins was similar to that of the proteome. To the best of our knowledge, this is the first report on the proteome of E. sinensis in response to M. bicuspidata infection. These results not only provide new and important information on the immune response of crustaceans to yeast infection but also provide a basis for further understanding the molecular mechanism of complex host pathogen interactions between crustaceans and fungi. [ABSTRACT FROM AUTHOR]

In aquaculture, hypoxia is an important stressor that can inhibit the healthy development of Chinese mitten crab, Eriocheir sinensis. The aim of this work was to research on how hypoxia (1 mg/L) followed by reoxygenation affects the immune response and carbohydrate metabolism in the Chinese mitten crab. The activities of immune‐related enzymes including lysozyme (LZM), acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD) and phenoloxidase (PO) and concentration changes in haemolymph glucose, trehalose and hepatic glycogen were evaluated after 8 hr of hypoxia followed by 12 hr of reoxygenation. After 8 hr of hypoxia, the activities of LZM, PO, SOD and hepatic glycogen concentration significantly decreased to the minimum when compared to the control group, but the activities of ACP and AKP, and haemolymph glucose concentration significantly increased. After 12 hr of reoxygenation, the activities of LZM, ACP, AKP and SOD recovered to the normal level except SOD in plasma, while the PO, SOD in plasma and hepatic glycogen concentration were still significantly lower than the control level. No significant change was observed in trehalose concentration during hypoxia and reoxygenation periods. These results indicated that although the activities of ACP and AKP were likely increased to facilitate the hydrolysis of foreign matter, the overall strength of the immune response was decreased as LZM, PO and SOD activities were suppressed after exposure to hypoxic conditions. The energy stores of exposed crabs were reduced due to the expenditure of energy during exposure to hypoxic stress. In general, chronic hypoxia would impair the immunocompetence and increase the energy expenditure of Chinese mitten crab, further increased the risk of pathogen infection and feed coefficient, so the negative effects of hypoxia should not be ignored. [ABSTRACT FROM AUTHOR]

Chinese mitten crab, Eriocheir sinensis , often suffers from severe air exposure stress during transportation and culture; high mortality occurs due to desiccation. In this study, the effects of air exposure stress (0, 2, 4, 8, and 16 h) and reimmersion (2, 6, 12 h) on respiratory metabolism and antioxidant responses in Chinese mitten crabs were studied under laboratory conditions. The results showed that air exposure and reimmersion had a significant impact on the oxygen consumption rate (OCR), ammonia excretion rate (AER), oxygen to nitrogen ratio (O:N), superoxide dismutase (SOD), catalase (CAT), succinate dehydrogenase (SDH), and lactate dehydrogenase (LDH). Significant interaction between air exposure and reimmersion was observed for OCR, AER, O:N, SOD, CAT, SDH, and LDH in Chinese mitten crab. During the air exposure stage, SOD, CAT, and LDH activities in the gills and hepatopancreas first increased and then decreased as air exposure time increased. All of these parameters were significantly higher in the 4-h air exposure group than those in the control group. All the parameters were significantly lower in the 16-h air exposure group than those in the control group, except LDH in the hepatopancreas. However, SDH activity gradually decreased with increased air exposure time, and all the air exposure groups were markedly lower than those in the control group in the gills. During the reimmersion stage, OCR, AER, and O:N restored to normal levels after 12-h reimmersion, except in the 16-h air exposure group, where OCR and O:N were significantly higher than those in the control group and AER was significantly lower than that in the control group. The LDH activity in all groups restored to normal levels after 12-h reimmersion. The SDH, SOD, and CAT activities of the 2- and 4-h air-exposed groups returned to normal levels after 12-h reimmersion; however, these three parameters were still significantly higher in the 16-h air-exposed group than in the control group in the gills and hepatopancreas. Overall, Chinese mitten crabs reduce aerobic respiration and increase anaerobic respiration capacity during desiccation. Under air exposure stress, Chinese mitten crabs change their energy utilization mode to meet their energy demands and adjust their respiratory metabolism and antioxidant enzymes activities to adapt to adverse environments. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Eriocheir sinensis aquaculture has been significantly impeded by "milky disease," which is caused by Metschnikowia bicuspidata. This disease exhibits distinct seasonality and is closely associated with temperature fluctuations. The present study investigated the impact of temperature on the proliferation and pathogenicity of M. bicuspidata and the immune capacity of E. sinensis. We observed a significant temperature-dependent effect on the proliferation rate of M. bicuspidata in vitro and in vivo. Although low temperatures (4 °C) suppressed M. bicuspidata proliferation, different temperatures did not have a considerable influence on pathogenicity (P > 0.05). Cultivation at lower temperatures significantly reduced the proliferation rate of pathogens in infected E. sinensis and the time to death. Further, the bacteriostatic capacity of E. sinensis serum was significantly enhanced under high temperatures compared with that under low-temperature conditions (P < 0.01). Transcriptome analysis of hemocytes from E. sinensis at different breeding temperatures indicated that the host's metabolism and immunity were differentially affected at 4 °C compared to 32 °C, with pathways associated with growth and development primarily affected under low-temperature conditions and pathways related to energy supply predominantly influenced under high-temperature conditions. Notably, temperature also led to changes in the expression of genes closely linked to immunity and apoptosis, such as acid phosphatase, C-type lectin, glutathione peroxidase, caspase, and initiator caspase-8. The current findings have significant implications for understanding the effect of temperature on M. bicuspidata pathogenicity in E. sinensis and preventing milky disease infection during aquaculture. • Milky disease shows clear seasonality and is closely linked to temperature. • Low-temperature suppressed M. bicuspidata proliferation but not on pathogenicity. • High-temperature enhanced bacteriostatic capacity of crab serum. • Low-temperature downregulated the expression of immunity related genes. • These findings have significant implications for the control of milky disease. [ABSTRACT FROM AUTHOR]

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. • 253 proteins were differentially expressed after Polyascus gregaria infection. • Differentially expressed proteins are related to oxidative stress and apoptosis. • Polyascus gregaria infection results in hepatopancreatic tissue damage. • Infection affects hepatopancreatic metabolism and pathological phenotype. [ABSTRACT FROM AUTHOR]

The Chinese mitten crab, Eriocheir sinensis , is an economically important crustacean species in China. A recent epidemic of "milky disease" in Panjin, China resulted in a large loss of cultured E. sinensis. Clinical signs of the disease include weak response, swollen pereopod and claw joints, opaque or whitish muscles, and milky hemolymph, culminating in tissue or organ failure and death. Histological analysis of the crabs showed a marked disorder of fibers, discrete necrotic lesions, and high yeast infiltration in the muscles, heart, gills, and other organs. The yeast cells (LNES0119) isolated from the diseased crab tissues were subjected to polymerase chain reaction analysis. The resulting product, the D1/D2 domain of 26S rDNA of the LNES0119 yeast isolate, was used to identify the pathogen for milky disease, which was found to have 98% sequence identity with the sequence of Metschnikowia bicuspidata (GenBank accession nos. U44822 and U94944), a pathogenic yeast found in aquatic invertebrates. A laboratory challenge experiment showed that the pathogen was infectious and caused the same pathological alterations in crab hosts. The present findings demonstrated that the pathogen isolated from the Chinese mitten crab with milky disease was the yeast M. bicuspidata. This study is the first description of M. bicuspidata infection in the Chinese mitten crab and thus warrants further research on the effects of M. bicuspidata in crustaceans • We identified the pathogen of milky disease in crab Eriocheir sinensis as the yeast Metschnikowia bicuspidate. • The typical signs of milky disease of E. sinensis were opaque or whitish muscles, and milky hemolymph. • This study is the first description of a yeast M. bicuspidata infection in E. sinensis. [ABSTRACT FROM AUTHOR]