| The Chinese mitten crab,Eriocheir sinensis,has become a very important aquaculture species in China because its rich nutritional value and broad market demand.However,in the pond culture process,various factors lead to higher limb autotomy rate,especially in the juvenile crab(larval crab)cultivation stage.Although autotmy has brought direct survival benefits,which seriously affect and restrict the quality and economic benefits of E.sinensis.In China,pond culture and rice-crab coculture are the main culture models for E.sinensis.Pyrethroid pesticides such as Del)is widely used in agricultural activities,and it enters aquaculture waters through farmland drainage and surface runoff.Therefore,the contradiction between the use of Del in agricultural areas and the culture of E.sinensis is particularly serious,which seriously threatens the healthy culture of E.sinensis.In order to artificially promote the limb regeneration,enhance the immunity and anti-stress ability,and anti-pesticide toxicity of E.sinensis,we gradually explored the effects of MT and its precursor substance—L-trp on the limb regeneration,immune performance,anti-stress and anti-pesticide toxicity of E.sinensis,in order to explore a new way for artificial regulation of limb regeneration,immune and healthy culture of E.sinensis.(1)Effects in immune levels after autotomyThis study compared the effects of cheliped loss with autotomy and ablation on the short-term(3 h)hematological immunity of juvenile E.sinensis.Compared with the crabs before treatment,the THC,GC,hemocyte agglutination and phosphatase activity were significantly increased,while HC and T-AOC were significantly decreased within3h.However,the THC levels,hemocyte agglutination and hemocyte proliferation were significantly higher in the ablation group within 3h compared to the autotomy group.The bacterial challenge showed that the mortality rate of crabs with cheliped autotomy significantly higher than the control group,whereas the mortality rate of the ablation group was significantly higher than that of the autotomy group.Therefore,the observations in this study indicate that cheliped autotomy significant reduced hematological immunity and antibacterial ability,whereas compared with the passive trauma response of ablation,autotomy as a congenital,efficient,and active trauma response mechanism,the damage is lower,the stress response is more sensitive and rapid.(2)Hemolymph transcriptome analysis after autotomyTo detect the important functional genes after autotomy related to the hematological molecular immunity of E.sinensis,we compared and analyzed the hemolymph transcriptome data of the intact crab,left cheliped autotomized crabs and bilateral eyestalk ablation crabs with high-throughput sequencing techniques.A total of9 567 differentially expressed genes were obtained by multiple comparison of the three groups’libraries.The differentially expressed genes in the three libraries were enriched to 344 specific KEGG metabolic pathways by KEGG enrichment analysis.To verify the expression levels of DEGs identified by RNA-Seq,six hematological immune-related genes were selected for qRT-PCR validation,the qRT-PCR results were consistent with the DEGs results.(3)The changes in bud morphology,growth-related gene expression and nutritional status during cheliped regenerationThe study found that the new cheliped was pre-formed in the bud and then regenerated with the completion of molting of E.sinensis.The new cheliped was similar in morphology to the normal cheliped after the first molting but smaller in size.The expression of growth-related genes showed that the expression levels of EcR-mRNA and Chi-mRNA were significantly up-regulated,whereas the expression of MIH-mRNA was significantly down-regulated(P<0.05).The nutritional status during the regeneration process showed that the hepatopancreas total lipid content decreased significantly within 28 days and was significantly lower in the autotomy group than in the control group at 14 d and 21 d(P<0.05).The hepatopancreas fatty acid composition results showed that SFA,HUFA and n-3/n-6 were significantly higher in the autotomy group than in the control group at 21 d(P<0.05),whereas the∑n-6PUFA and∑n-3 PUFA at 1 d and 7 d,and the MUFA at 28 d in the autotomy group were significantly lower than in the control group(P<0.05).Moreover,the levels of ARA,EPA and DHA showed that DHA was significantly lower at 7 d and significantly higher at 21 d in the autotomy group than in the control group(P<0.05),whereas ARA and EPA were not significantly different between the two groups.Muscle L-tryptophan content was significantly lower at 1 d and significantly higher at 7 d in the autotomy group than in the control group(P<0.05).These results indicate that during the cheliped regeneration process,crabs could accelerate molting and regeneration by regulating growth-related gene expression and nutrient metabolism.(4)The effects of MT on cheliped regeneration,immunity and anti-pesticide toxicityThe result was found that the injection of melatonin significantly increased the limb regeneration rate(P<0.05).The levels of EcR and Chi expression was significantly increased following the melatonin injection,while the expression of MIH was significantly decreased(P<0.05).Melatonin significantly increased lipase activity(P<0.05).We observed that the survival rates of limb-impaired and unilateral eyestalk-ablated crabs were substantially improved following melatonin treatment,whereas the survival of the unilateral eyestalk-ablated crabs was significantly decreased compared with the control group(P<0.05).MT significantly increases blood immunity and antioxidant capacity(P<0.05).Del exposure test results showed that Del-exposure significantly induced oxidative stress and functional damage of the mitochondria,for example,Del-exposure resulted in a significant increase in MDA content and a significant decrease in GSH content in mitochondria,and the ATP content,Na~+-K~+-ATPase activity and Cyt-C content in mitochondria decreased significantly.Moreover,hemocyte apoptosis was significantly induced by Del-exposure.However,MT pretreatment can effectively prevent oxidative damage and functional damage caused by Del-exposure to mitochondria and hemocyte.Therefore,the observations in this study indicate that MT can not only promote the cheliped regeneration of E.sinensis,but also enhance immunity and prevent toxic damage caused by Del.(5)The effects of dietary supplementation of L-trp on the cheliped regeneration and hematologic immunityDietary supplementation of L-trp significantly increases limb regeneration rates(P<0.05).The expression of EcR and Chi is significantly up-regulated with the dietary supplementation of L-trp while the expression of MIH is significantly down-regulated(P<0.05).Moreover,digestive enzyme activities are significantly enhanced with dietary supplementation of L-trp(P<0.05).Further,dietary supplementation of L-trp significantly increases MT levels in eyestalks and L-trp levels in muscles of E.sinensis(P<0.05).Interestingly,our study preliminarily explores the role of 5-HT and DA receptors in the cheliped regeneration of E.sinensis.We find that dietary L-trp supplementation regulates the expression of 5-HT and DA-related receptors in digestive organs(hepatopancreas and intestines)and nervous tissues(cranial and thoracic ganglia),and we speculate that it may be involved in the improvement of digestion and absorption function and the regulation of nerve tissue repair during the cheliped regeneration of E.sinensis.In addition,we counted the mortality after 14 days of feeding and found that dietary supplementation of L-trp significantly lowered the mortality of E.sinensis.Moreover,L-trp significantly improves blood immunity,antioxidant and antibacterial capabilities.Taken together,dietary supplementation of L-trp not only can promote cheliped regeneration by regulating MT levels and the binding of 5-HT and DA to corresponding receptors,but also can enhance the antioxidant capacity and improve the hematological immune status and antibacterial ability of E.sinensis under the cheliped autotomy stress,thereby increasing the survival rate of E.sinensis under cheliped autotomy stress. |
PDF Full Text Request