No abstract

Chinese grass shrimp (Palaemonetes sinensis) is an economically important crustacean in Chinese aquaculture. Recently, we found that shrimp in Panjin city were infected with microsporidia, a group of fungi. The hepatopancreas of several infected shrimp showed white turbidity and pathological changes that negatively affected the health and appearance of the shrimp. Histopathology and transmission electron microscopy were used to examine the development of the parasite within its parasitophorous vacuole. Our results indicated that microsporidia developed asynchronously within the same parasitophorous vacuole. The spores were predominantly small, and rod or oval-shaped. The sizes of fresh spores were approximately 3.1 × 2.4 μm and fixed spores were 1.9 × 1.1 μm. The polar filament was isofilar with 5–6 coils and the thickness was 103.2 nm. Merogonial divisions occurred by binary fission and sporogonial division occurred by plasmotomy. The small subunit ribosomal DNA sequence (1295 bp) from the parasite was highly similar to the previously reported parasite Enterocytospora artemiae (99% nucleotide identity, JX915760). Using maximum likelihood to analyze the phylogenetic relationships, we found that this microsporidian should be grouped within Clade IV, an Enterocytospora-like clade, of the Microsporidia phylum. Based on this parasite’s life cycle characteristics, morphology, and small subunit ribosomal DNA sequence, the parasite described here is likely E. artemiae, which has previously only been described in Europe and North America. Thus, this is the first report of E. artemiae both in Asia and economically important shrimp.

No abstract

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.

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.

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.

Milky disease caused by Metschnikowia bicuspidata fungus has significantly harmed the Chinese mitten crab Eriocheir sinensis aquaculture industry. However, the effect of M. bicuspidata infection on the metabolism and intestinal flora of the crab remains unclear. In this study, we aimed to explore the changes in the metabolism and intestinal flora E. sinensis after 48 h of infection with M. bicuspidata, using metabolomic and metagenomic analyses. Metabolomic analysis results revealed 420 significantly different metabolites between the infected and control groups, and these metabolites were enriched in 58 metabolic pathways. M. bicuspidata infection decreased the levels of metabolites related to amino acid biosynthesis, the tricarboxylic acid cycle, as well as lysine, histidine, linolenic, arachidonic, and linoleic acid metabolism. These results indicated that M. bicuspidata infection significantly affected the energy metabolism, growth, and immunity of E. sinensis. The results of metagenomic analysis showed that the anaerobes and ascomycetes populations significantly increased and decreased, respectively, after M. bicuspidata infection. These changes in intestinal flora significantly upregulated metabolic and synthetic pathways while downregulating immunity-related pathways. The results of integrated metabolomic and metagenomic analyses showed that 55 differentially expressed genes and 28 operational taxonomic units were correlated with 420 differential metabolites. Thus, the intestinal flora changes caused by M. bicuspidata infection also affected the metabolites. This study provides novel insights into the metabolic-and intestinal microflora-based effects of M. bicuspidata infection in E. sinensis, as well as a theoretical basis for the interaction between fungi and crustaceans.

Trace metal contamination in the aquatic ecosystem occurs worldwide: although copper is an essential trace metal, it is considered as a pollutant at certain levels in China. Freshwater crayfish Cambaroides dauricus is a commercially important wild species in northeastern China, in which is an important heavy industry area. The population of C. dauricus was decreasing sharply due to the environmental pollution and human intervention over the past 20 years. However, nothing is known regarding the responses of this species to trace metal toxicants. This study aimed to determine the acute and chronic toxicity of Cu and its toxicological effects on respiratory metabolism, as well as Cu accumulation in C. dauricus. The acute (96 h) median lethal concentration (LC50) value of 32.5 mg/L was detected in C. dauricus. Then, acute (96 h; 8.24, 16.48 mg/L) and sub-chronic (14 days; 2.06, 4.12 mg/L) exposure in Cu was investigated by estimating the oxygen consumption rate, ammonium excretion rate, and Cu accumulation. Both acute and sub-chronic Cu exposure induced an inhibition of the oxygen consumption rate and ammonium excretion rate, and thereby, an increased O:N ratio. The shift in O:N ratio indicated a metabolic substrate shift towards lipid and carbohydrate metabolism under Cu stress. Cu accumulation in the hepatopancreas and muscles throughout the study was found to be time-dependent and concentration-dependent. The maximum accumulation in the hepatopancreas and muscle were almost 31.6 folds of the control after 14 days’ exposure to 4.12 mg/L concentration. Based on the present work, we suggest that crayfish be considered a potential bioindicator of environmental pollution in freshwater systems. The study provides basic information for further understanding of the toxicological responses of this species to trace metals.