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.     

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.     

枯草芽孢杆菌基因组DNA对中华绒螯蟹非特异性免疫因子的影响

将枯草芽孢杆菌(Bacillus subtzlis)基因组DNA按5、20和80μg/kg剂量体腔注射中华绒螯蟹(Eriocheir sinensis)(约0.1 mL/只),设注射等体积TE缓冲液为对照组,分别在第1、3、5、7天采样,测定该细菌DNA对中华绒螯蟹的免疫刺激作用。结果显示:注射上述3种剂量的枯草芽孢杆菌基因组DNA均能不同程度地提高中华绒螯蟹的THC、血细胞呼吸爆发活性和血清PO、ACP活性,试验组蟹的上述免疫指标的活性显著高于对照组(P<0.05),并以20μg/kg体重剂量的刺激效果最好;但试验剂量的细菌DNA对血清ALP活性无显著影响(P>0.05)。     

Effect of oxidized fish oil and α‐tocopherol on growth,antioxidation status,serum immune enzyme activity and resistance to Aeromonas hydrophila challenge of Chinese mitten crab Eriocheir sinensis

The impact of dietary α‐tocopherol on juvenile Chinese mitten crab Eriocheir sinensis was experimentally evaluated in a 10‐week study. Crab were fed with nine diets including three levels of α‐tocopherol (0, 100 and 300 mg kg^?1 diet) and three levels of fish oil oxidation (fresh, moderate and high) in triplicates. Fresh and moderate oil oxidization enhanced weight gain, but moderate and high oil oxidization lowered survival and feed efficiency. The 100‐mg α‐tocopherol kg^?1 diet resulted in higher hepatopancreas MDA than other α‐tocopherol diets. High oil oxidization led to the lowest serum superoxide dismutase (SOD) and glutathione peroxidase (GPH‐PX). The serum SOD and GPH‐PX activities in crab fed 100 mg α‐tocopherol were higher than in those fed other α‐tocopherol diets. The diet without α‐tocopherol addition lowered lysozyme and phenoloxidase (PO) activities compared to other α‐tocopherol diets. Fresh fish oil diet increased PO activity compared to oxidized oils. High oil oxidization caused significantly more mortality than fresh or moderate oxidization after 7‐d postchallenge with Aeromonas hydrophila. Supplementation with α‐tocopherol significantly enhanced resistance to bacterial infection. This study indicates that α‐tocopherol can protect lipid from peroxidation and enhance disease resistance.     

Effects of dietary folic acid on growth,antioxidant capacity,non‐specific immune response and disease resistance of juvenile Chinese mitten crab Eriocheir sinensis (Milne‐Edwards, 1853)

An 8‐week study was conducted to determine folic acid requirement and its effect on antioxidant capacity and immunity in juvenile Chinese mitten crab Eriocheir sinensis (Milne‐Edwards, 1853), followed by a challenge assay with the pathogen Aeromonas hydrophila for 2 weeks. Folic acid was added to a basal diet at seven levels (0, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 mg folic acid kg^?1 diet), and a diet free of folic acid and vitamin B₁₂ was also included as a control. Crabs were fed twice daily in 32 tanks with 7.76–8.17 mg oxygen L^?1, 25.0–31.0 °C and 7.5–8.3 pH. Growth and feed efficiency were significantly greater in crabs fed ≥2.0 mg folic acid kg^?1, but not significantly different between crabs fed diets >2.0 mg folic acid. The superoxide dismutase activity and glutathione S‐transferase activity were highest in crabs fed ≥2.0 mg folic acid kg^?1, followed by those fed 0.5 and 1.0 mg folic acid kg^?1, and the control diet. The malondialdehyde content was highest in crabs fed the control diet, followed by those fed 0 mg folic acid kg^?1, and the lowest value occurred in those fed ≥0.5 mg folic acid kg^?1. Phenoloxidase activity and total haemocytes were significantly higher in crabs fed ≥2.0 mg folic acid kg^?1 than other diets. Crabs fed 2.0 mg folic acid kg^?1 had the highest lysozyme, acid phosphatase and alkaline phosphatase activities but the lowest cumulative mortality. The optimum dietary folic acid requirement by E. sinensis was estimated at 2.29–2.90 mg kg^?1 diet.     

Growth and immune response of Chinese mitten crab (Eriocheir sinensis) fed diets containing different lipid sources

A 10‐week feeding trial was conducted to evaluate the effects of dietary lipid sources on the growth and immune responses of Chinese mitten crab Eriocheir sinensis. Six isonitrogenous and isoenergetic diets were formulated with fish oil (FO), linseed oil (LO), soybean oil (SO), rapeseed oil (RO), coconut oil (CO) and beef tallow (BT) as the sources of lipid with five replicates each. Thirty crabs (2.35 ± 0.14 g) were stocked into each tank and fed twice daily. Weight gain and specific growth rate of crab fed the FO diet were significantly lower than those fed other diets (P < 0.05), except for crabs fed LO diet (P < 0.05). Crab fed the SO diet weighed more than those fed FO diets (P < 0.05). Serum superoxide dismutase and malondialdehyde of crab fed the FO diet were significantly higher than in other groups (P < 0.05). Crab fed the FO diet had the highest activities of serum phenoloxidase, acid phosphatase, alkaline phosphatase and lysozyme (P < 0.05). The fatty acid composition in the liver of crab reflected the change in test diets. Our results indicate that the use of dietary vegetable or animal oils can achieve similar growth performance to the use of dietary FO in Chinese mitten crab, but non‐FOs may impair crab immunity. Soybean oil is recommended as a suitable replacer for FO in Chinese mitten crab diet.     

Effect of dietary lipids and vitamin E on growth performance,body composition,anti‐oxidative ability and resistance to Aeromonas hydrophila challenge of juvenile Chinese mitten crab Eriocheir sinensis

Vitamin E supplement is important in protecting lipid oxidation and enhancing immunity of aquatic animals. A 10‐week feeding trial was conducted to evaluate the effect of dietary lipid and vitamin E on juvenile Chinese mitten crab Eriocheir sinensis. The experimental diets included three levels of vitamin E (0, 100 and 300 mg kg^?1) and two levels of lipid (6% and 9%). The 9% lipid diet significantly enhanced the body lipid of crabs compared with the 6% lipid diet. The vitamin E concentration of hepatopancreas increased with the increase in vitamin E in the diets regardless of dietary lipid levels. The hepatopancreas fatty acids, especially polyunsaturated fatty acid and highly unsaturated fatty acid, were significantly enhanced by vitamin E supplement or 9% dietary lipid. Vitamin E supplement significantly increased the total antioxidant capacity, superoxide dismutase, lysozyme and phenoloxidase activities of crabs compared with those fed the diets without vitamin E supplement. The hepatopancreas malondialdehyde of crabs fed 100 mg vitamin E kg^?1 was significantly lower than those fed 0 or 300 mg vitamin E kg^?1. The phenoloxidase activity of crabs fed the 9% lipid diet was significantly higher than those fed 6% lipid, irrespective of vitamin E levels. Vitamin E supplementation increased the bacterial resistance of juvenile crab. This study indicates that dietary lipid and vitamin E supplement do not affect crab growth and survival, but vitatmin E supplement at 100 mg kg^?1 can enhance immunity and antioxidant capacity of crab fed 6% or 9% dietary lipids.     

Effect of poly‐β‐hydroxybutyrate on growth,enzyme activity and intestinal microbial community of Chinese mitten crab,Eriocheir sinensis (Milne‐Edwards) juveniles

Poly‐β‐hydroxybutyrate (PHB) is microbial carbon and energy storage polymer, which can be degraded into water‐soluble β‐hydroxybutyric acid in the gastrointestinal tract of aquatic animals. A 60‐day culture experiment was performed using Chinese mitten crab, Eriocheir sinensis (Milne‐Edwards) juveniles with an average initial body weight of 0.74 ± 0.06 g which were fed a diet supplemented with 0%, 0.5%, 1%, 3% or 5% PHB. A PHB dietary supplementation of 1% and 3% significantly improved the body weight gain, moulting frequency and concomitantly reduced 2nd–3rd moulting intervals of the crabs (P < 0.05). The dietary PHB level positively related to hepatopancreatic pepsin, trypsin and lipase activity (P < 0.05). Increasing the dietary PHB also improved total superoxide dismutase activity, but reduced alkaline phosphatase and acid phosphatase activity in the serum of hemolymph (P < 0.05). A 16S rRNA gene analysis by denaturing gradient gel electrophoresis indicated that PHB supplementation led to a significantly higher range‐weighted richness, diversity and evenness of the gut bacterial community when dosed at 3% in the feed. The beneficial effects of PHB are discussed in terms of immune defense, metabolism and gut microbiota of the crabs.     

Effects of dietary protein to energy ratios on growth,body composition and digestive enzyme activities in Chinese mitten‐handed crab,Eriocheir sinensis

This study aimed to determine the optimal protein to energy ratio (P/E ratio) and evaluate the effect of dietary protein and lipid levels on growth performance, body composition and digestive enzymes activities in Chinese mitten‐handed crab, Eriocheir sinensis. Nine practical diets containing three levels both for protein (DP 30%, 35% and 40%) and lipid (DL 2%, 7% and 12%) with P/E ratios ranging from 13.69 to 19.79 mg KJ^?1 were fed to four replicates of crabs (3.39 ± 0.10 g) for 10 weeks. Weight gain increased significantly with the increase in DP level at each DL level. Moreover, weight gain increased in crabs fed with diets containing DL level from 2% to 12% and DP level from 30% to 35%. However, the diet containing 40% DP and 12% DL levels significantly decreased the growth performance and protein efficiency of the crabs. The whole crab and hepatopancreas lipid contents also increased as dietary lipid increased, but not dietary protein. The total protease activity increased significantly with the increase in dietary protein at each lipid level. The lipase activity was statistically comparable among different DL levels at each DP level. Taken together, the crab fed the diet containing 35% protein and 12% lipid levels with P/E 15.77 mg KJ^?1 revealed optimal growth, feed utilization efficiency and digestive enzymes activities. Moreover, our study indicated that the higher dietary lipid level at a relatively lower dietary protein level could provide protein sparing effect in Eriocheir sinensis.     

Enhancement of non‐specific immune responses and disease resistance on oral administration of Nyctanthes arbortristis seed extract in Oreochromis mossambicus (Peters)

The immunomodulatory effect of the seed extract of an Indian medicinal plant Nyctanthes arbortristis L. (NAT) on non‐specific immune responses and disease resistance of tilapia, Oreochromis mossambicus was investigated. Chloroform extract was administered orally as a feed supplement at doses of 0.01%, 0.1% or 1% for 3 weeks. Non‐specific immune parameters such as serum lysozyme and alternate complement haemolytic (ACH₅₀) activities, and cellular reactive oxygen species (ROS), reactive nitrogen intermediate (RNI) and myeloperoxidase production and disease resistance against live virulent Aeromonas hydrophila were investigated after 1, 2 or 3 weeks of feeding with chloroform extract‐supplemented diet. The results of this study indicated that feeding tilapia for 2 weeks with selected doses of chloroform extract of NAT seeds significantly enhanced serum lysozyme, alternate complement activities and cellular ROS, RNI and MPO production. It was evident from the disease resistance test that feed supplemented with NAT seed extract at 0.1% or 1% level significantly reduced the mortality of O. mossambicus and a 3‐week feeding with 0.1% extract‐supplemented diet appears to be the optimal regimen for maximal disease resistance. Thus, the study indicates the scope of using the NAT extract as an immunoprophylactic in finfish aquaculture.     

α-苯基苯并吡喃对中华绒螯蟹免疫相关酶的影响

用α-苯基苯并吡喃作为饲料添加剂饲喂中华绒螯蟹(Eriocheir sinensis)10 d后,测定了其对中华绒螯蟹血淋巴细胞中相关免疫因子的影响。实验设3个α-苯基苯并吡喃添加重复组,分别为A组(2 mg/kg)、B组(4 mg/kg)、C组(6 mg/kg)和1个对照重复组(0 mg/kg)。结果表明:α-苯基苯并吡喃添加剂量为4 mg/kg(B组)可显著地提高中华绒螯蟹血淋巴细胞中酚氧化酶(PO)、溶菌酶(LSZ)和超氧化物歧化酶(SOD)的活力(P<0.05),说明具有生物活性的α-苯基苯并吡喃可刺激中华绒螯蟹机体免疫相关酶活性的提高。     

感染溶藻弧菌对日本蟳肝胰腺免疫功能的影响

摘 要：为探讨日本蟳感染溶藻弧菌的致病机理,采用溶藻弧菌人工感染健康日本蟳,分析了感染前后日本蟳肝胰腺 PO、SOD和LSZ活性变化及注射免疫多糖后的免疫保护率,并研究了细胞超微结构的病理组织学。结果表明：日本蟳感染溶藻弧菌后,肝胰腺中PO、SOD和 LSZ的活性均受到不同程度的影响,感染6~12 h,3种酶的活性均有一个上升的过程,但之后随时间的延长呈现下降趋势;免疫感染组因感染弧菌前注射了免疫多糖,日本蟳肝胰腺中PO、SOD和LSZ酶活性均显著高于感染组的酶活性,感染7 d后的免疫保护率高达72.73％,表明免疫多糖可提高酶的活性,使机体的抗病菌能力增强。超微结构显示：对照组日本蟳肝胰腺细胞结构形态正常,上皮细胞表面的微绒毛排列整齐,各细胞器结构完整;免疫组内质网、糖原颗粒和线粒体丰富,微绒毛排列致密;感染组肝胰腺组织受到明显的损伤,细胞和部分微绒毛脱落、受损;线粒体和粗面内质网变形、水肿或仅剩一空泡;核高度异染色质化,核膜破裂。与感染组相比,免疫后感染组具有形态结构较正常的细胞核、整齐致密的微绒毛和丰富的溶酶体,但依然可见水肿的内质网和狭长畸变的线粒体。     

Dietary astaxanthin supplementation for hatchery‐cultured red king crab,Paralithodes camtschaticus,juveniles

We conducted large‐scale production trials in Seward, Alaska, USA to investigate effects of dietary astaxanthin supplementation on survival, growth and shell colouration of recently settled juvenile (C1–C4) red king crabs (Paralithodes camtschaticus). We supplemented a control diet of commercial crustacean feeds with astaxanthin, and fed these diets to juvenile king crabs at densities of 2000 and 4000 crabs m^?2 for 56 days. We assessed survival and growth by counting crabs and individually measuring carapace width and weighing crabs at the start and end of the experiment, and quantified crab colour (hue, saturation, brightness) in digital photographs. Diets containing astaxanthin had higher survival, suggesting that astaxanthin may provide nutritional or immune system benefits. Crabs had lower hue, higher saturation and lower brightness values when fed diets containing astaxanthin, suggesting that red king crab colouration is plastic and responds to diet. Astaxanthin is likely an important dietary component for hatchery or laboratory reared red king crab juveniles, and should be considered for aquaculture and other rearing of this and possibly other crustacean species.     

Does the wild‐caught Chinese mitten crab megalopae perform better than the hatchery‐produced seed during the juvenile culture?

Although wild Yangtze Eriocheir sinensis juveniles (coin‐sized seed) have better culture performance than the pond‐reared juveniles during the adult crab culture stage, the culture performance of wild‐caught megalopae (WC) remains unknown. Therefore, this study was designed to compare the growth, survival rate, crab yield, feed conversion rate (FCR), precocity, final weight and size distribution between WC and hatchery‐produced megalopae (HP) during the juvenile culture stage. Wild‐caught megalopae had significantly higher body weight, weight gain rate (WGR) and specific growth rate (SGR) during 0–4 weeks but lower WGR and SGR than HP during 4–8 weeks (p < .05). Although WC had a significantly lower survival rate and yield of normal juvenile crabs than HP, total yields of normal juveniles and precocious crabs were similar. Wild‐caught megalopae had a significantly higher precocious rate and yield of precocious crabs than HP (p < .05). Regardless of sex, no significant differences were found in the gonadosomatic index (GSI), hepatosomatic index (HSI) and final weight of normal juveniles and precocious crabs between WC and HP. As for the size distribution of normal crabs, WC had a higher percentage of small female juveniles (<3 g) but a lower proportion of middle size ranges (3–5.99 g and 6–8.99 g) than HP for females (p < .05), and there was no significant difference for males. In conclusion, wild megalopae of the Yangtze E. sinensis had worse culture performance than the hatchery‐produced seeds; therefore, wild megalopae would not be recommended for commercially juvenile culture of E. sinensis.     

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.     

Identification of SNPs in the 5′‐flanking region and 3′‐UTR of the MIH gene and their association with precocity of the Chinese mitten crab Eriocheir sinensis

Moult‐inhibiting hormone (MIH), an important regulator of steroidogenesis, inhibits the synthesis of ecdysteroid in Y‐organ (YO) and plays a significant role in the regulation of moulting and post‐embryonic development of crustacean. Because unsuccessful moulting have been widely observed in precocious crabs, we investigated whether genetic variants in the 5′‐flanking region and 3′‐untranslated region (3′‐UTR) of the MIH gene are associated with precocity of the Chinese mitten crab. Thirty individual DNA samples were sequenced to search for SNPs in the 5′‐flanking region and 3′‐UTR of the MIH gene. Five SNPs (g.196 T>A, g.230 C>T, g.305 T>C, g.323 C>A and g.372 C>T) in the 5′‐flanking region and 6 SNPs (g.2677 C>T, g.2759 T>A, g.2807 T>C, g.3042 A>G, g.3088 T>G and g.3295 T>G) in the 3′‐UTR of the MIH gene were selected for the individual genotyping in a two‐stage association study. We found that a SNP g.3088 T>G in the 3′‐UTR of MIH gene was consistently associated with precocity of the Chinese mitten crab in stage 1 and stage 2, with a per‐allele OR (Odds Ratio) of 1.469 (95% CI: 1.169–1.844) after two stages combined (P = 0.001). However, no significant associations were observed between the other 10 SNPs and precocity of the Chinese mitten crab. To our best knowledge, this is the first association study between various SNP genotypes and phenotype attributes in Chinese mitten crab. Our findings suggest that the SNP g.3088 T>G may be a candidate marker for effective marker‐assisted selection to decrease the precocity of the Chinese mitten crab in future studies.     

注射中华绒螯蟹及锯缘青蟹促雄性腺提取物对中华绒螯蟹雌蟹雄性化的影响

该论文首次报道了经由促雄性腺提取物的注射而在蟹类中引起的性逆转现象。此前关于蟹类促雄性腺活性研究的报道极少，而且蟹类的雄性化均是由促雄性腺的移植所产生。本实验中将锯缘青蟹和中华绒螯蟹的促雄性腺提取物分别注射到刚刚完成性别分化的中华绒螯蟹雌性幼蟹体内，此时幼蟹处于4至5期，壳宽为5～8 mm。注射之后，幼蟹经过大约1～2次蜕皮，此时在注射锯缘青蟹以及中华绒螯蟹的促雄性腺提取物的两组实验幼蟹中均能观察到雄性化现象，而注射生理盐水的对照组实验幼蟹中未能观察到相同现象。由此本实验可以证明促雄性腺确实是蟹类的一种雄性激素，注射促雄性腺提取物能引起雌性幼蟹发生性逆转；同时根据锯缘青蟹和中华绒螯蟹的促雄性腺提取物均能引起中华绒螯蟹雌性幼蟹发生性逆转的现象推测，锯缘青蟹和中华绒螯蟹两种间可能存在促雄性腺的交叉活性；不仅如此，性逆转还能在极低的注射剂量下获得，相当于中华绒螯蟹0.14促雄性腺当量和锯缘青蟹0.06促雄性腺当量。     

Effects of dietary phospholipids and highly unsaturated fatty acids on the precocity,survival, growth and hepatic lipid composition of juvenile Chinese mitten crab,Eriocheir sinensis (H. Milne‐Edwards)

Precocious puberty is one of the major constraints to the further development of Chinese mitten crab (Eriocheir sinensis) farming industry. Although dietary phospholipids (PL) and highly unsaturated fatty acids (HUFA) supplementation have been shown to enhance the growth of larval E. sinensis in other studies, it is still unknown whether this also leads to a higher precocity rate for juvenile E. sinensis. This study was conducted to investigate the effects of dietary PL and HUFA on precocity, survival, growth and hepatic lipid composition of juvenile E. sinensis. Two diets were formulated with PL [3.95% dry weight (DW)] and HUFA (0.98% DW) supplementation (diet A) and without PL and HUFA supplementation (diet B) and fed to juvenile E. sinensis. Although dietary PL and HUFA levels did not significantly affect the survival and growth performance of juvenile E. sinensis, compared with crabs fed diet A, a higher precocity rate was found among juvenile E. sinensis fed diet B (P=0.051). A higher total lipid content, but significantly lower levels of HUFA and PL (P<0.05) were found in the hepatopancreas of crabs fed diet B than in those fed diet A. Meanwhile, the precocious females had significantly lower hepatosomatic index, arachidonic acid (20:4n‐6), eicosapentaenoic acid (20:5n‐3) and docosahexaenoic acid (22:6n‐3) contents in their hepatopancreas when compared with that of the normal immature juveniles (P<0.05). The results suggest that the occurrence of precocious puberty among farmed juvenile E. sinensis could be reduced by the inclusion of appropriate level of dietary PL and HUFA.     

Innate immune response and gene expression of Scylla paramamosain under Vibrio parahaemolyticus infection

An epidemic of ‘milky disease’ in the mud crab (Scylla paramamosain) generally breaks out in the fall when the crab is near maturity, resulting in large economic losses in crab farming. Vibrio parahaemolyticus has been proven to be one of the major pathogens. In this study, the mud crabs were challenged with V. parahaemolyticus, and their innate immune responses were investigated in terms of total haemocyte counts (THCs), haemocytic enzyme activities and gene expression levels during a 114‐h period. The THCs of the mud crabs decreased significantly after 42 h of exposure. The activities of the haemocytic enzymes, including acid phosphatase‐alkaline phosphatase, phenoloxidase, superoxide dismutase (SOD), peroxidase and nitric oxidase synthethase, were significantly enhanced during the challenge course. The gene expression levels also significantly increased for all tested genes (proPO, Cu/Zn‐SOD, Prx, LYS, CRU and ALF) with the exception of CAT down‐regulated expression. The results may imply that the immune responses of the mud crab could be activated by the pathogens, and the data here will provide many clues for further systematic investigation of ‘milky disease’ caused by V. parahaemolyticus and the disease prevention in mud crab S. paramamosain.