| The apple snails(Pomacea spp.)are the world’s invasive species.The high adaptability to environmental stresses is essential for their successful invasion.Estivation is a vital survival strategy for the apple snails to withstand high temperatures,drought,and lack of food.They can arise from estivation quickly once the environment is suitable again and produce a large amount of ROS.The regulation of the antioxidant defense is crucial for the survival of the apple snails from estivation.However,there are rare reports on the underlying molecular mechanism of regulation on the antioxidant defense in apple snails.Nrf2 regulates several antioxidant enzymes,and the Nrf2-Keapl signaling pathway is known to be the most critical antioxidant defense mechanism in cells.In this study,the apple snail Pomacea canaliculata was subjected to drought stress for 45 d to enter deep estivation(EV),rehydration for 10 min(AS10),20 min(AS20),and 1 h(AS1h)to enter short arousal,normally growing snails were used as control(CTRL).The antioxidant adjustment strategy and key pathways were understood more fully by testing the activity of the antioxidant enzyme,observing histopathological and profiling transcriptome expression.In this study,the entire length of Nrf2 CDS from P.canaliculata(Pc Nrf2)was cloned and characterized,and the specific expression of Pc Nrf2 in different tissues and the relative expression levels in the estivation-arousal cycle and different concentrations of hydrogen peroxide treatment was measured by q PCR.We used the combination of RNAi and transcriptome sequencing to explore the potential regulatory downstream target genes of Pc Nrf2.The detailed results of this study are as follows:1.Changes in the phenotypic indicators and the antioxidant enzymes in the estivation-arousal cycle of the apple snailsThe mortality rate was 10% in the deep estivation.The survived snails after estivation by submerging in water to recover.After about 10 minutes,the snails detached the operculum;after 20 minutes,the extrusion of an exploring tentacle;after an hour,the head and foot were starting to deploy,and both tentacles were being turned outward.In the CTRL,EV,AS10,AS20,and AS1 h,the SOD activity in the foot decreased firstly,then rose,and finally dropped.There was no significant change in the SOD activity in hepatopancreas.The CAT activity in the foot raised firstly,then decreased,which is the highest at AS20 and decreased at AS1 h but still significantly higher than the CTRL.In the hepatopancreas,the CAT activity first raised and then reduced,with the highest value found at EV.The activity of GST showed no significant change in the foot;in the hepatopancreas,it firstly raised and then fell,with the highest at AS20.These results indicated that different antioxidant enzymes had different adjustment strategies in the estivation-arousal cycle.Histopathological changes revealed that columnar muscle fibers in the foot dehydrated and atrophied columnar muscle cells and decreased lipid vacuoles in deep estivation.Besides,the pigment cells were dehydrated and resulting the increasing area of space nets among the columnar muscle fibers.At AS20,the columnar muscle cells started rehydrating and elongating,but the grid of the space nets among them enlarged.Also,lipid vacuoles were less and smaller.At AS1 h,the columnar muscle cells and pigment cells rehydrated with the small lipid vacuoles dispersed among them.The histopathological changes in the foot were mainly related to the dehydration and rehydration of the columnar muscle fibers in the estivation-arousal cycle.After entering deep estivation,with atrophy of the hepatic ducts leading to narrowing of the tubular lumen in the hepatopancreas;expansion of the hemolymphatic spaces;reduction in the number of basophilic cells and increase in dark granules;smaller digestive cells with increased levels of arrangement and number and rupture of some cell membranes.The dark granules were the most at EV and decreased dramatically in AS1 h,which has indicated the fast speed in removing the metabolite contaminant of the apple snails.2.Tissue expression profiling of the estivation-arousal cycle in apple snailsWe used the program Short Time-series Expression Miner(STEM)to analyze the gene expression patterns during the estivation-arousal cycle.The increased expression pattern of foot and hepatopancreas tissues in the estivation group was enriched in the longevity regulating pathway,which contains two key transcription factors,Fox O and Nrf2,to regulate antioxidant defense,lipid accumulation,and autophagy.In the decreased expression pattern,the foot and hepatopancreas were mainly enriched to metabolism-related pathways.The results show that the snails resist oxidative damage caused by ROS by activating longevity regulating pathways to regulate the expression of antioxidant detoxification enzymes,and heat shock proteins genes in the estivation;maintain energy supply by inhibiting metabolic pathways.In the increased expression pattern of the short-term arousal group,the foot is mainly involved in various signal transduction pathways;the hepatopancreas is mainly involved in various carbohydrate metabolism,protein digestion,and absorption,ABC transporter proteins,etc.It was shown that the snails rely on signal transduction pathways in the foot to mediate defense and immune mechanisms during arousal,and carbohydrate metabolism and protein digestion and absorption in the hepatopancreas to ensure normal activity.In addition,an antioxidant defense pathway,the Nrf2-Keap1 signaling pathway in the foot short-term arousal group was discovered in the cancer pathway.3.Cloning of Pc Nrf2 and its expression in Pomacea canaliculataWe obtained a sequence of 2253 bp of the full coding region,encoding 750 amino acid residues,of which 602-669 aa is the functional domain of CNC_b ZIP.The q PCR results showed that Pc Nrf2 was highly expressed in the foot and lung,and the relative expression of Pc Nrf2 increased and then decreased in all tissues in the estivationarousal cycle,with the highest expression at AS20.The expression of Pc Nrf2 in the foot and hepatopancreas increased and then decreased under different concentrations of hydrogen peroxide treatment,and the highest expression was found at 10 mg/L.The results indicated that external environmental stress was able to induce activation of Pc Nrf2 expression.4.Pc Nrf2 regulates the molecular pathways of antioxidant defense in apple snailsAll three pairs of designed si RNA sequences functioned 48 h after injection,and the highest interference efficiency was achieved by si Nrf2 III with 61.16%.The differentially expressed genes were analyzed by comparison of si Nrf2 Ⅲ interfered and without interfered through transcriptomes,of which 597 genes were up-regulated and 276 genes were down-regulated.Functional enrichment analysis of the downregulated differentially expressed genes revealed longevity regulating pathways and MAPK signaling pathways.The functions of only 60 genes were annotated,including those related to antioxidant defense,detoxification,and heat shock proteins. |
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