| Circadian rhythms are fundamental biological mechanisms that regulate physiological activities in organisms and are influenced by external environmental factors.Crustaceans are one group of organisms that are affected by circadian rhythms.With recent advances in highthroughput sequencing technology and histological studies,multi-omics approaches can be used to understand the circadian regulatory mechanisms in these organisms.In this study,we used a multi-omics approach to explore the circadian regulatory mechanisms in Chinese sawtooth rice shrimp,with four sampling time points(0:00,6:00,12:00,and 18:00)as the experimental subjects.The main findings are as follows:(1)Determination of intestinal flora.In this study,the intestinal flora macrogenes were determined by measuring the mitochondrial 16 s ribosomal DNA of the intestinal flora.The number of OTUs identified in the gut microbiome between 12:00 and 24:00 was significantly different,and the results of alpha and beta diversity analysis showed significant differences in the composition of the gut bacterial community.indices indicated that the abundance and diversity of shrimp gut communities sampled at midnight(24:00)were higher than those of samples sampled at noon(12:00).Conditionally pathogenic bacteria including Flavobacterium spp.,Immunobacterium spp.and Pseudomonas spp.were significantly present in the 18:00 and24:00 groups(P<0.05).(2)Transcriptome determination.Transcriptome enrichment analysis by comparing different time sampling groups showed that the GO pathway with the highest degree of enrichment in DEGs was associated with sugar and chitin metabolism.In the KEGG enrichment analysis,DEGs were mainly enriched in the phototransduction-fly pathway and the thermogenesis pathway.The enrichment results showed that many unigenes related to circadian rhythms were annotated,including many core biological clock genes such as Per,Tim,Clk,Cyc,Cry and 5-HT.It can be inferred that the circadian rhythms of physiological activities in Chinese sawtooth rice shrimp are endogenously regulated but influenced by external conditions such as light and heat.(3)Fluorescence PCR validation.To verify the accuracy of the transcriptome data results and to explore the real-time expression of circadian-rhythm-regulated genes under circadian rhythm,the expression levels of four important circadian-rhythm-regulated genes(Per,Tim,Clk and Cry)were evaluated by q RT-PCR.The validation results showed that the expression of Clk genes fluctuated significantly with circadian rhythm.In addition the expression trends of each gene were consistent with the results of transcriptome data.(4)Enzyme activity assay.The results showed that all the enzymes of the participating species tested had circadian rhythms.The activity of alkaline phosphatase in the hepatopancreas of the Chinese sawtooth rice shrimp showed significant variation by circadian rhythm.Significant differences(P<0.05)were observed in each tested group during the daytime.24:00 o’clock samples were significantly different(P<0.05)from 6:00 o’clock samples.The results of the acid phosphatase experiment showed that the difference was not significant only in the "afternoon".The rest of the groups were affected by circadian rhythms.The trends of the two related antioxidant enzymes(CAT and T-AOC)were basically the same,with significant changes in the samples at 24:00 at night.Amylase,which is related to digestive function,had a significant decrease in activity throughout the day only in the 18:00 sample(P<0.05).This macroscopically confirmed the prevalence of circadian rhythm regulation in Chinese sawtooth rice shrimp. |
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