| Background:Circadian rhythms are biological processes that follow a chronological oscillation synchronized?entrained?by daily and seasonal cycles,which was regulated by exogenous environmental changes and endogenous clock genes.Melatonin?N-acetyl-5-methoxy-tryptamine?is a conserved and commonly occurring biogenic amine,and widely occurs in vertebrates and invertebrates.Melatonin is also an indicator and regulator of circadian rhythms in organisms.Although the circadian rhythms of crustacean are followed by daily and seasonal cycles,the study of regulation mechanism still lack,and the difference between different types is large.Objective:This study described the circadian rhythm of the melatonin content and oxidative immune enzymes in haemolymph and the eyestalk of Chinese mitten crab?Eriocheir sinensis?,revealed the diurnal differences in the transcription level of the eyestalk of E.sinensis,and laid the foundation for studying the physiological and molecular mechanisms of circadian rhythm,while emphasizing the diurnal difference is also an important factor in studying the oxidative immune defense of crustaceans.Methods:In this study,E.sinensis was used as the experimental animals,and the daily concentration of melatonin in hemolymph and the eyestalk under natural light conditions and artificially set different photoperiod conditions was analyzed by using high performance liquid chromatography?HPLC?.To obtain a more complete description of eyestalk biology in E.sinensis,we conducted comparative transcriptome analysis of eyestalks during the day and at night using high-throughput sequencing on an Illumina Hi Seq 4000 platform.Result:1.The daily variation of melatonin concentration in E.sinensis haemolymph exhibited significant?p<0.05?peaks in melatonin at 16:00(0.180±0.020?g·m L-1)and 24:00(0.244±0.055?g·m L-1),while eyestalks had two significant peaks at 16:00(72.377±18.100?g·eyestalk-1)and 24:00(98.756±30.271?g·eyestalk-1),and melatonin levels are highest at night in both haemolymph and eyestalks.2.The circadian rhythms of melatonin in the eyestalk of E.sinensis were influenced by external light stimulation.When under consistent light?24L:0D?and consistent dark?0L:24D?,the melatonin rhythm in eyestalks and hemolymph was disturbed with respect to that of the control group?12L:12D?.However,disturbance owing to different photoperiod treatments decreased with increasing treatment duration.3.Comparative transcriptome analysis of eyestalks obtained 47,092 unigenes,including 4771 differentially expressed genes?DEGs?from eyestalks during the day and at night.We found that 4269 DEGs were up-regulated during the day and 502 DEGs were up-regulated at night.We identified five DEGs that may contribute to molting,including molt-inhibiting hormone,cuticle,catalase,aquaporin,and ubiquitin-conjugating enzyme.We further identified eight DEGs related to behavior regulation,including three glutamate dehydrogenase genes that were up-regulated during the day.48 unigenes involved in circadian regulation were identified using BLAST search of the NR and KEGG databases and the published literature.Among them,9 unigenes were identified as participating in the‘circadian rhythm–fly'pathway.In the‘circadian rhythm–fly'pathway,all nine unigenes were matched,but none showed significantly different expression between day and night.Conclusion:1.The circadian rhythm of the melatonin content in the hemolymph and and eyestalks of E.sinensis showed a bimodal pattern and was synchronized.2.The circadian rhythms of melatonin content in the hemolymph and and eyestalks of E.sinensis were influenced by external light stimulation.Light inhibited melatonin synthesis.However,disturbance owing to different photoperiod treatments decreased with increasing treatment duration.3.Comparative transcriptome analysis of eyestalks during the day and at night identified five DEGs that may contribute to molting and eight DEGs related to behavior regulation,hence,similar to other crustaceans,E.sinensis may molt at night.Thus,changes in the eyestalks may partially compensate for daily changes in illumination in the Chinese mitten crab's normal environment. |
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