Study on the pharmacokinetics of enrofloxacin in the Chinese mitten-handed crab, Eriocheir sinensis, after different administration regimes

A pharmacokinetic study on enrofloxacin (ENR) following different administration regimes (i.e. intramuscular, oral and bath administrations) was carried out for the Chinese mitten-handed crab ( Eriocheir sinensis ) at 17 °C water temperature. Muscle and hepatopancreas samples were collected at each sampling time of 1 and 10 min and 0.5, 1, 2, 8, 16, 24, 48, 72 and 168 h for intramuscular administration and 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72 and 168 h for oral and bath administration. The levels of ENR and ciprofloxacin (CIP) were simultaneously determined ( n =5) by high-performance liquid chromatography. The results showed that similar absorption and elimination patterns of ENR level in muscle and hepatopancreas were observed for oral administration and bath treatment; however, the trend in ENR level changes in muscle and hepatopancreas after intramuscular administration was quite different from oral and bath treatments. Meanwhile, the level of ENR in hepatopancreas was much higher than that in muscle for the same administration regimes. The concentration–time data were analysed with the Practical Pharmacokinetics Program (3P97), and the kinetic profiles of ENR in the hepatopancreas and muscle conformed to two-compartment models. The absorption and elimination of ENR in hepatopancreas for bath treatment were faster than those for oral and intramuscular administrations. However, the pattern was quite different in muscle. The fastest absorption and elimination of ENR in muscle were seen for intramuscular administration.     

Screening and identification of microRNAs during larval metamorphic development of Chinese mitten crab Eriocheir sinensis

MicroRNAs (miRNAs) represent a class of endogenous small non‐coding RNAs with 18–25 nucleotides that are involved in diverse biological processes. In the present study, differentially expressed miRNAs in four different metamorphic stages of Eriocheir sinensis were comprehensively detected and validated from high‐throughput sequencing miRNA data sets. We identified and characterized 597 known miRNAs and 901 novel miRNAs from the four small RNA libraries. We also demonstrated the evolutionary conservation of miRNAs from Chinese mitten crab with those from other species. Analyses of miRNA expression profiles during brachyurization development of the Chinese mitten crab revealed that 86 mature miRNAs were dysregulated, 47 of which were significantly differentially expressed. The results of real‐time quantitative PCR analyses for a few selected miRNAs were similar to those of deep sequencing analyses. Intriguingly, the results of Gene Ontology annotation and KEGG pathway analyses indicated that miRNA gene clusters and families were consistently dysregulated at four different metamorphic stages, although they were enriched at various levels. Functional enrichment analysis revealed the versatile physiological roles (over six signal pathways) of these miRNAs. The results suggest that these miRNAs might play potential key roles in the regulation of gene expression in the brachyurization development of the Chinese mitten crab. Particularly, miR‐2a‐3p, miR‐305‐5p, miR‐263a‐5p and miR‐7‐5p may play major regulatory roles in the metamorphosis of E. sinensis.     

Effects of hypoxia on immune responses and carbohydrate metabolism in the Chinese mitten crab,Eriocheir sinensis

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.     

Comparative study on the nutrition composition of adult male Chinese mitten crab (Eriocheir sinensis) with different coloured hepatopancreases

High lethality of white hepatopancreas syndrome has caused large economic loss of Chinese mitten crab (Eriocheir sinensis). Hepatopancreas colour of diseased crabs is gradually lighter, turning from golden yellow to yellow‐white to white. This study investigated composition of conventional nutrients in three edible parts of different coloured hepatopancreases of adult males, including normal yellow hepatopancreas group (Control), yellow‐white hepatopancreas group (YWH) and white hepatopancreas group (WH). No significant differences were observed in tissue indices and total edible yield among three crab types; however, condition factor level in crabs of YWH and WH was significantly lower. Dry matter content was significantly reduced in crabs of WH, especially crude fat level of hepatopancreas in WH males. Levels of all the amino acids of WH crabs in muscles and gonads were significantly lower than that of control. Proportion of saturated fatty acids and monounsaturated fatty acids were much lower in hepatopancreas of WH crabs than those of control, while proportion of polyunsaturated fatty acids and highly unsaturated fatty acids were significantly higher. Overall, YWH and WH do not affect growth. WH crabs showed poor nutritional quality, and nutritional value of YWH was in the middle as transition state between control and WH.     

Changes in the gut microbiome of the Chinese mitten crab (Eriocheir sinensis) in response to White spot syndrome virus (WSSV) infection

Intestinal microorganisms play important roles in maintaining host health, but their functions in aquatic animal hosts have yet to be fully elucidated. The Chinese mitten crab, Eriocheir sinensis, is one such example. We attempted to identify the shift of gut microbiota that occurred in response to infection of white spot syndrome virus (WSSV), an emerging viral pathogen in the crab aquaculture industry. The microbiota may exert some control over aspects of the viral pathogenesis. We investigated the changes in composition and structure of the crab gut microbiome during various WSSV infection stages of 6 h post‐infection (hpi) and 48 hpi, using a 16S rRNA approach on the MiSeq Illumina sequencing platform. Four phyla (Firmicutes, Proteobacteria, Tenericutes and Bacteroidetes) were most dominant in the gut of E. sinensis regardless of the WSSV infection stages. However, further analysis revealed that over 12 bacterial phyla, 44 orders and 68 families were significantly different in abundance at various states of WSSV infection. Several intriguing aspects of E. sinensis gut bacteria that had not been previously reported were also uncovered, such as class Mollicutes was dominant here, but absent in crabs from Yangtze River estuary and Chongming Islands. Overall, this study provided the first evidence that changes in gut microbiome were closely associated with the severity of WSSV infection and that indicator taxa could be used to evaluate the crab health status.     

Temporal and spatial dynamics of white spot syndrome virus in the Chinese mitten crab,Eriocheir sinensis

White spot syndrome virus (WSSV) is the most severe viral pathogen to the crustacean aquaculture industry worldwide. Recently, serious WSSV outbreaks caused catastrophic losses in the Chinese mitten crab, Eriocheir sinensis in Jiangsu Province, Eastern China. However, to date, little is known about its infection mechanism in the new natural host. This study aimed to reveal the temporal and spatial dynamics of WSSV in E. sinensis. The slow viral growth in the early stage of infection was the light infection stage (from 0 to 24 hpi), and the exponential growth stage that followed was the logarithmic phase (from 24 to 72 hpi). The viral growth curve ended with the plateau phase (from 72 to 144 hpi) which demonstrated a consistent high level of viral load and accompanied heavy crab mortality. The viral load increased as time progressed with similar growth curves, however, at different degrees. The viral copy numbers of tissues at different time intervals, analysed using one‐way analysis of variance (anova ), showed significant differences between tissues at all time points (P < 0.05). Infection was detectable as early as 6 hpi in all the tissues screened. The severity of infection was found to be maximum in gill and pleopods, which could be recommended for diagnostic testing. This study might provide important data to analyse theoretically the interaction between WSSV and the host.     

Cottonseed meal protein hydrolysate stimulates feed intake and appetite in Chinese mitten crab,Eriocheir sinensis

One hundred and twenty crabs (initial weight: 51.32 ± 0.06 g) were fed three experimental diets with cottonseed meal protein hydrolysate (CPH) at 0, 6 and 12 g/kg for 60 days. The results showed that CPH0.6 diet significantly improved the 1‐hr feed intake, protease activity and weight gain of crabs. In thoracic ganglia, 4E‐binding protein (4E‐BP) expression level of crab fed the diets containing CPH was significantly higher than CPH0 group. In cerebral ganglia, protein kinase B and 4E‐BP expression levels of crab fed CPH0.6 diets were significantly lower than CPH0 group. In mid‐intestine, target of rapamycin (TOR) relative expression level of crabs fed the diets containing CPH was significantly lower than CPH0 group. 4E‐BP expression level showed an opposite pattern. In thoracic ganglia, cerebral ganglia and mid‐intestine, leptin receptor expression level of crabs fed CPH0.6 diet was significantly lower than CPH0 group. Neuropeptide Y and neuropeptide Y receptor expression level of crab fed CPH0.6 diet was significantly higher than CPH0 group. In conclusion, our results indicated that dietary supplementation of 6 g/kg CPH could stimulate appetite and quicken feeding rate via the TOR signalling pathway.     

Dietary protease modulates nutrient retention efficiency and hepatopancreatic protease activity in juvenile Chinese mitten crab Eriocheir sinensis

An 8‐week growth trial was conducted with juvenile Chinese mitten crab fed a high fishmeal (FM) diet (PC), a low FM diet (NC) and three other diets (NC125, NC150 and NC175) supplemented with 125, 150 and 175 mg/kg of a dietary protease. In the NC diets, a portion of FM (from PC diet) was replaced by a combination of plant proteins. All diets were isoproteic (404 ± 3 g/kg) and isoenergetic (18.6 ± 0.2 kJ/g). No differences were observed among treatments in most parameters except protein (PRE, %) and lipid (LRE, %) retention efficiencies, where LRE was the highest (37.9%) in NC175 followed by the PC. A linear regression demonstrated significant effects of protease on hepatopancreatic trypsin activity (HPRO, p = .001), and PRE (p = .05) and LRE (p = .014) but not on energy retention efficiency (ERE). Dietary protease level was positively correlated with HPRO and nutrient retention efficiency (p < .05). On the other hand, the specific growth rate (SGR) was positively correlated with portein deposition (PD), lipid deposition (LP) and recovered energy (RE) and negatively correlated with survival (p < .05). This study is first to demonstrate that mainly nutrient retention efficiencies are modulated by specific dietary enzymes in animals.     

Effects of dietary supplementation of Haematococcus pluvialis powder on gonadal development,coloration and antioxidant capacity of adult male Chinese mitten crab (Eriocheir sinensis)

The green algae Haematococcus pluvialis is an important source of natural astaxanthin as feed additive. This study was conducted to investigate the effects of dietary supplementation of H. pluvialis powder on gonadal development, coloration and antioxidant capacity of adult male Chinese mitten crab Eriocheir sinensis, and four experimental diets were formulated to contain 0, 0.2%, 0.4% and 0.6% of H. pluvialis powder. There were four treatments (defined as D1~D4) in this study and each treatment had three replicates. Dietary H. pluvialis contents had no significant effects on survival, body weight gain rate and gonadal development of male E. sinensis. For colour parameters, the total carotenoids content in carapace and hepatopancreas as well as hepatopancreatic lightness (L*) and carapace redness (a*) increased significantly with increasing dietary H. pluvialis (p < 0.05). For the antioxidant indices in the serum, D4 had the lowest activities of superoxide dismutase (SOD) and peroxidase (POD), but the highest glutathione peroxidase (GSH‐Px) and lactic dehydrogenase (LDH), while the malondialdehyde (MDA) in serum and hepatopancreas decreased significantly with the rising content of dietary H. pluvialis (p < 0.05); D1 had the highest levels of SOD, POD and GSH‐Px in hepatopancreas. For the non‐specific immune indices, the highest activities of acid phosphatase (ACP) and γ‐glutamyl transpeptidase (γ‐GT) were found on the serum of D3 and D4 (p < 0.05). D1 had the highest levels of ACP and alkaline phosphatase (ALP) in hepatopancreas, while D2 and D3 had the lowest levels of ALP and ACP respectively. These results suggested the optimal dietary natural astaxanthin level was around 40 mg/kg diets.     

Effects of xylan and cottonseed meal on growth performance,digestion, intestinal morphology and the expression of two peritrophin genes of Chinese mitten crab,Eriocheir sinensis

Xylan is one of the main non‐starch polysaccharides (NSPs) in cottonseed meal. This study aimed to evaluate the anti‐nutrition effect of xylan and to better understand cottonseed meal as a feed ingredient for the Chinese mitten crab, Eriocheir sinensis. A diet containing 280 g/kg fishmeal without cottonseed meal or xylan was formulated as a control (FM diet). A second diet (XYL diet) was supplemented with 53.3 g/kg xylan according to its amount in the third diet, the cottonseed meal diet (CSM diet), which was formulated to include 400 g/kg cottonseed meal. The last diet (CSM + XYLase diet) included 400 g/kg xylanase hydrolysed cottonseed meal, in which most xylan was hydrolysed. Diets were isonitrogenous (370 g/kg crude protein) and isoenergetic (17.58 KJ/g) and were randomly fed to 12 tanks of crabs for 8 weeks. Results showed that no difference was detected among groups in growth performance, serum biochemical index, or digestive and metabolism enzyme activity (p > 0.05). The apparent digestibility coefficient (ADC) of dry matter was FM > XYL > CSM + XYLase > CSM (p < 0.05). The ADC of protein in crab fed diet FM and XYL was significantly higher than CSM and CSM + XYLase (p < 0.05). Cottonseed meal decreased the fold height of the hindgut, upregulated the expression of peritrophin genes of Es‐PL44 and Es‐PP1 (p < 0.05), while xylan decreased the fold height of the hindgut and upregulated the expression of Es‐PP1 (p < 0.05). These results indicated that the negative effect of cottonseed meal and xylan on digestibility partly compensated by enhanced absorption of the hindgut innerly and by supplementation of xylanase externally, and cottonseed meal is an attractive alternative protein source in general for Chinese mitten crab.     

Effect of dietary lipid source and vitamin E on growth,non‐specific immune response and resistance to Aeromonas hydrophila challenge of Chinese mitten crab Eriocheir sinensis

Six purified diets were formulated to contain three lipid sources, fish oil (FO), linseed oil (LO) and soybean oil (SO), at 6% diet lipid crossing two levels of vitamin E (100 and 300 mg α‐tocopheryl acetate/kg diet) for each lipid source (FO100, FO300, LO100, LO300, SO100, SO300). The juvenile Chinese mitten crab, Eriocheir sinensis, respectively, fed on these diets with four replicates for 6 weeks. The crab weight gain (WG) and specific growth rate (SGR) were significantly affected by dietary lipid sources. No difference was found between the crabs fed two levels of vitamin E, but the WG and SGR were numerically higher in crab fed 300 mg/kg vitamin E than those fed the other level of vitamin E. The lipid source and vitamin E level could affect fatty acid composition in the hepatopancreas. The contents of saturated fatty acids (SAFA) and n‐3HUFA were significantly higher in the crab‐fed fish oil. The highest contents of n‐6PUFA and n‐3PUFA were found in the crab‐fed soybean oil and linseed oil respectively. The contents of SAFA, n‐3HUFA and n‐3PUFA were higher in the 300 mg/kg vitamin E treatment. A lower malondialdehyde (MDA) content and higher phenoloxidase (PO) activity were observed in the crab fed 300 mg/kg vitamin E. The results of this study indicate that the Chinese mitten crab fed the diet with 6% fish oil and 300 mg/kg vitamin E showed better growth, antioxidant capacity and resistance to Aeromonas hydrophila.     

Effects of dietary cholesterol levels on moulting performance,lipid accumulation,ecdysteroid concentration and immune enzymes activities of juvenile Chinese mitten crab Eriocheir sinensis

Effects of dietary cholesterol levels on moulting performance, lipid accumulation, ecdysteroid concentration and immune enzymes activities of juvenile Eriocheir sinensis were investigated. Crabs were fed with feeds contained various cholesterols of 1100, 2100 and 3200 mg kg^?1 in both paddy fields and laboratory experiments. In paddy fields trial, the crabs fed with diets contained 3200 mg kg^?1 cholesterol achieved higher growth rate than those fed with diets contained no supplemental cholesterol (1100 mg kg^?1). In laboratory trial, moulting frequencies of crabs fed with diets contained 3200 mg kg^?1 cholesterol were higher than those of crabs fed with diets contained no supplemental cholesterol from the 6th to 10th moult. Further laboratory experiment indicated that intermoults of crabs fed with diets contained 3200 mg kg^?1 cholesterol significantly shortened compared with crabs fed with the basal feeds (1100 mg kg^?1 cholesterol). In the intermoult, total lipid content, ecdysterone concentration and three immune enzymes in crabs were increased with the increment of dietary cholesterol levels between the 7th and the 8th moult in laboratory experiments. Taken together, dietary cholesterol not only enhanced moulting performances of growth, survival and moult frequency, but also enhanced total lipid storage, ecdysterone concentration and three immune enzymes activities in the intermoult period.     

Development of In situ hybridization and real‐time PCR assays for the detection of Hepatospora eriocheir,a microsporidian pathogen in the Chinese mitten crab Eriocheir sinensis

A microsporidian parasite, Hepatospora eriocheir, is an emerging pathogen for the Chinese mitten crab Eriocheir sinensis. Currently, there is scant information about the way it transmits infection in the crustacean of commercial importance, including its pathogenesis, propagation and infection route in vivo. In this study, chromogenic in situ hybridization (ISH) and quantitative real‐time PCR (qPCR) assays were developed to address this pressing need, and we provided an advance in the detection methods available. Pathogens can be seen in situ with associated lesions using ISH. Positive hybridization signals were noted inside the epithelial cells of the hepatopancreas, and putative free parasite spores were observed within the tubule lumen, which were associated with lesions detected by electron microscopy and haematoxylin and eosin (H&E) analysis. qPCR allows the determination of parasite loads in infected tissues, which is important for understanding disease progression and transmission. The hepatopancreas displayed the biggest statistical copy numbers among different tissues of infected crabs, confirming a tissue‐specific pathogen infection characteristic. The qPCR assay also proved to be suitable for the diagnosis of asymptomatic carrier crabs. Combination of the two methods could facilitate the study of H. eriocheir infection mechanism in E. sinensis, enhance the early diagnosis of the pathogen and improve the management of microsporidian diseases in commercial crustaceans.     

Label‐free quantification proteomics reveals the effects of dietary fish oil and soybean oil on the immune response of Chinese mitten crab,Eriocheir sinensis

The replacement of fish oil (FO) in Eriocheir sinensis can significantly reduce the cost of E. sinensis cultivation, while several studies have indicated that replacing FO with soybean oil (SO) could significantly reduce the resistance of E. sinensis to disease. However, the molecular mechanisms underlying these effects remain poorly understood. In this study, crabs were fed two diets containing FO or SO, following which a label‐free quantification proteomic analysis was employed. And the activity of enzymes involved in the nonspecific immune response was also measured. Growth performance was undifferentiated between the crabs fed with FO and SO. A total of 519 proteins were identified, and 70 proteins were significantly altered between the crabs fed the two different diets. Five proteins related to the immune response were identified to be differently expressed. C‐type lectin, haemocyanin subunit 6 and cryptocyanin were significantly downregulated, while fatty acid‐binding protein and catalase were highly expressed in the crabs fed SO. The activities of acid phosphatase, alkaline phosphatase, superoxide dismutase and phenoloxidase were all significantly changed in crab fed with different diets. These findings will provide novel insight into the molecular mechanism regarding the replacement of FO on the immune response of E. sinensis and provide evidences for the relationship between nutrition and immunity in E. sinensis.     

中华绒螯蟹的摄食节律

为了探究中华绒螯蟹(Eriocheir sinensis)最适宜的投喂时间,本实验将120只初重约68 g的幼蟹随机分养在24个塑料缸(70 cm×40 cm×50 cm)中。采用一次饱食投喂(将昼夜分为8个时间点:10:00、13:00、16:00、19:00、22:00、01:00、04:00、07:00,每个时间段作为1个处理组,每天每个处理组饱食投喂1次)的方法研究中华绒螯蟹的昼夜摄食节律。结果显示,幼蟹在不同投喂时间点表现出24 h周期性的摄食节律,且总摄食高峰期在22:00。从其中6d的平均摄食量可以看出,河蟹主要在19:00~22:00时间段摄食;在肝胰腺酶活性方面,淀粉酶活性在19:00~22:00内高于其他各组,且在22:00获得最高值;脂肪酶、碱性磷酸酶和钠钾ATP酶酶活性有相似的变化趋势,在13:00~22:00时间段这些酶的酶活呈现上升的趋势,且最高值均在22:00获得。在相关摄食基因表达方面,肝胰腺中Leptin基因的相对表达量在22:00点显著低于13:00和16:00,与其他各组相比差异不显著(P0.05);肠道中胆囊收缩素(CCK)基因的相对表达量在22:00点显示出最低值,与10:00、13:00、04:00和07:00点相比差异显著(P0.05)。综上所述,中华绒螯蟹存在明显的摄食节律,这可能与光照紧密相关,最适宜的投喂时间为22:00。     

中华绒螯蟹血淋巴细胞的超微结构

改进常规血液细胞电镜制样技术 ,直接取中华绒螯蟹 (Eriocheirsinensis)血淋巴液固定、脱水、包埋、电镜观察。结果显示 ,中华绒螯蟹血淋巴具有 3类细胞 ,即无颗粒细胞、小颗粒细胞和大颗粒细胞。其中 ,无颗粒细胞的核质比最大 ,异染色质高度浓缩、边缘化 ,几乎没有常染色质 ,胞质内线粒体数目相对较多 ,与高等动物的中幼红细胞结构很相似 ,该细胞数目在中华绒螯蟹血淋巴中最多 ,可能担负着血液的能量转换功能 ;小颗粒细胞核内常染色质较多 ,内质网、高尔基体发达 ,胞质中存在 0 .2～ 0 .4 μm的电子致密颗粒 ,而且胞质中有许多大小不一的空泡 ,细胞表面有许多突起似伪足 ,类似高等动物的单核细胞 ,可能具有吞噬和消化异物的功能 ;大颗粒细胞胞质中的颗粒较大 ,直径 1.4～ 2 .0 μm ,胞质中具空泡 ,细胞表面也常见伪足突起 ,类似高等动物的嗜酸性粒细胞     

Common specific indigenous bacteria reside in the intestinal tract of Chinese mitten crab (Eriocheir sinensis)

In our previous study, five bacterial phylotypes of Mollicutes group 1, Mollicutes group 2, Bacteroides, Meniscus and Marinifilum were found to be dominant (abundance > 0.5%) in the intestine of Chinese mitten crab (CMC, Eriocheir sinensis) farmed in Lake Tai, China. To shed light on whether these five bacterial lineages are common specific indigenous intestinal bacteria, samples of adult CMCs collected from eight geographically separated farms in China, juvenile crabs, farming water and crab feed are subjected to analysing by using 16S rRNA gene sequencing and phylotype‐specific, real‐time quantitative PCR (qPCR). Four phylotypes of the Mollicutes group 1, the Mollicutes group 2, Bacteroides and Meniscus are detected, with relatively higher number, in all crab samples, including adults and juveniles. The Mollicutes group 1 and 2 are more dominant, with the group 1 more abundant in midgut while the group 2 in hindgut. The Marinifilum phylotype is almost undetectable in crab samples but abundant in the farming water and feed. By contrast, phylotypes of the Mollicutes group 1, the Mollicutes group 2, Bacteroides and Meniscus are neither detectable in the water nor in feed. These four lineages appear to be the common specific indigenous intestinal bacteria in the entire CMC species, with the Mollicutes group 1 and 2 likely serving as the major symbiotic players in CMCs. Their beneficial contributions to CMC host await future deep investigation.