| Malpighian tubules?MT?is a crucial organ for insect‘s fluid homeostasis,which dynamically regulates the excretion and water-salt balance via the related fluid homeostasis proteins.If fluid homeostasis is disrupted,the survival ability of insects will be affected.Therefore,MT and the critical fluid homeostasis genes are potential targets for pest management.The brown planthopper?Nilaparvata lugens St?l?is one of the most important piercing-sucking pests on rice.In this study,we used N.lugens as the research object to study the functions of the critical fluid homeostasis genes,in order to find crucial regulatory genes for insect living and excretion.Firstly,RNA-seq technology was used to analyze the gene expression profiles of MT,Midgut?MG?and Hindgut?HG?in N.lugens,to screen highly expressed genes of MT.Then RNAi technology was used to study the fuctions of these genes.The main results in this study are as follows:?1?RNA-seq results showed that these genes were highly expressed in MG,MT and HG:salivap-6,cytochrome c oxidase,esterase E4,ribosomal proteins and so on.Compared to HG and MG,genes with higher expressions in MT mainly included:five aquaporin genes?Eglp1,Eglp2,DRIP,BIB,AQP12L?,one Na+/K+-ATPase?ATP1A1?,one potassium channel gene?Kir3?,solute carrier organic anion transporter?SLCO?,sodium-coupled monocarboxylate transporter?SMCT?and sodium/calcium exchanger?NCX?.Additionally,some neuropeptide genes such as Neuroparsin,ILP2,ILP4 and PDF,and some neuropeptide receptors such as Nl?B5 DH receptor and Nl?A25CAP2b receptor were also relatively highly expressed in MT.The q RT-PCR technology was used to validate the expression levels of eight interested genes.The correlation analysis proved the validity of the transcriptome data.?2?Based on the above transcriptomic data,the eight genes enriched in N.lugens MT?Nl Eglp1,Nl Eglp2,Nl PRIP,Nl DRIP,Nl AQP12L,Nl?A25,Nl?B5 and Nl ATP1A1?were selected for the RNA interference experiment.Survival rates and the amount of excreted honeydew were significantly reduced when silencing Nl Eglp2,Nl AQP12L and Nl ATP1A1.On the 7th day after the injection of ds Nl ATP1A1,ds Nl DRIP and ds Nl Eglp2,the corrected mortalities of N.lugens were 95.7%,63.6%and 42.9%,respectively.On the 5th day after the injection of ds Nl ATP1A1,ds Nl DRIP,and ds Nl Eglp2,the weight of honeydew significantly decreased by 85.9%,53.9%and84.7%,respectively.These results showed that Nl ATP1A1,Nl DRIP and Nl Eglp2genes have the potential for pest management.?3?Temporal expression patterns of Nl ATP1A1 were analyzed by q RT-PCR.The results showed that the expression level of Nl ATP1A1 gene was the highest in 1stinstar nymphs,and the lowest in adults,indicating that this gene may be important in the early development stages of N.lugens.After RNAi,RNA-seq technology was used to analyze the gene expression profiles of N.lugens injected with the ds Nl ATP1A1 and the ds GFP.It was found that 9441 genes were relatively up-regulated and 285 genes were relatively down-regulated.There were some down-regulated genes after RNAi which were also enriched in MT,such as two amino acid transporters and a carbonic anhydrase,a sucrose transporter and a neuropeptide gene?NPLP3?.We speculated that these genes and ATP1A1 may have functional interactions in MT of N.lugens.?4?Temporal expression patterns of Nl DRIP and Nl Eglp2 were analyzed by q RT-PCR.The data showed that the expression level of Nl DRIP was highest in 1stinstar nymphs.The expression level of Nl Eglp2 gene was highest in females.These results indicated that there was a significant functional differentiation of the aquaporin genes in N.lugens.In the present study,gene expression profiles in the MT of N.lugens were analyzed.Then the functions of aquaporins and Na+/K+-ATPase genes were studied.These results can help us know more about the functions of MT in N.lugens and also provide a theoretical basis for the development of novel targets for pest control based on the fluid homeostasis system in N.lugens. |
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