| Aedes aegypti is an important vector for disease transmission.It belongs to the Diptera mosquito family of the Arthropodidae,and is widely distributed throughout the world.It is a completely metamorphic insect.Its life history includes four periods:eggs,larvae,pupae and adults.Adult female mosquitoes need to draw blood from spinal animals to reproduce.Aedes aegypti can transmit a variety of diseases,including dengue fever,yellow fever,Zika,Chikungunya,and malaria,which constantly threaten human health.MicroRNA(MiRNA)is a type of short non-coding small molecule with a length of about 21-25 bp,which can play an important role in various life activities such as development,metabolism,and disease.Our previous results show that mosquito-specific and midgut-specific miR-1174 plays an important role in regulating mosquito blood feeding,blood digestion,fluid excretion and ovarian development,and miR-1174 negatively regulates the target gene Serine hydroxymethyltransferase(SHMT).After interference SHMT,blood digestion in the midgut of mosquitoes is blocked and ovarian development is abnormal.Further studies have shown that SHMT has low or no expression in the midgut,and digestive enzymes are highly expressed in the midgut.However,down-regulation of SHMT can affect the expression of digestive enzymes in the midgut of mosquitoes.How SHMT affects the expression of digestive enzyme genes in the midgut,we speculate that miRNA may be used as a bridge between SHMT and digestive enzymes,through direct or indirect ways to maintain the steady state and function of digestive enzymes in the midgut of mosquitoes,and participated in regulation of digestive enzymes by SHMT.1.Functional verification of SHMT geneWe first synthesized two double-stranded RNAs,dsSHMT and ds EGFP,which were injected approximately 20 h after the mosquito emerged as an adult,and fed blood on the third day after injection(PIJ 72 h).When SHMT was interfered,compared with the WT group and the ds EGFP group,the blood digestion of mosquitoes was inhibited,ovarian development was blocked,and the egg production was significantly reduced.2.Small RNA sequencing and differentially expressed miRNAsThe individual female mosquitoes were collected at 72 h after injection(before blood meal)and 15 h after blood feeding.Total RNA was extracted for small RNA sequencing,and differentially expressed miRNA was screened.A total of 196 miRNAs were identified,including 151 known miRNAs of Aedes aegypti and 45 newly identified miRNAs.There were 24 miRNAs differently expressed between the SHMT-depleted treatment group and the control group before the blood meal,15 miRNAs were differently expressed between the treatment group and the control group after blood meal,19 miRNAs were only differently expressed before blood meal,10 miRNAs were only expressed differently after blood meal,and 5 miRNAs were differently expressed between the treatment group and the control group both before and after blood meal.Those differently expressed before the blood meal are aae-let-7,miR-1,miR-100,miR-11894 a,miR-11898,miR-13-3p,miR-13-5p,miR-278-3p,miR-279,miR-281-5p,miR-2941,etc.Those differently expressed post blood meal include aae-miR-11-5p,miR-1174,miR-252-5p,miR-278-5p,miR-2b,miR-8-5p,miR-iab-3p and three newly identified miRNAs.Those differently expressed both before and post blood meal are aae-miR-14,miR-2940-3p,miR-2940-5p,miR-305-5p and novel?22.In the control group,there are 69 miRNAs differently expressed both before and after blood meal.In the treatment group,there are 65 miRNAs differently expressed both before and after blood meal.There are 46 miRNAs differently expressed both in the control group and the treatment group before and after blood meal,which shows that blood meal has a great impact on the expression of mosquito miRNAs.Among the 24 differential miRNAs before blood meal,12 were up-regulated and 12 down-regulated;among the 15 differential miRNAs after blood meal,8 were up-regulated and 7 down-regulated.3.MiRNA pathway genes respond to SHMT RNAiBecause miRNAs have their own special pathways,some important nucleases are needed.We took the material injected with dsSHMT and 24 hours after blood feeding for q PCR detection.The RT-q PCR results showed that AGO 1 was down-regulated,AGO 2 was up-regulated,AGO 3 was unchanged,Dicer-1 was down-regulated,Dicer-2 was up-regulated,and Drosha was down-regulated.The genes contronling the biogenes of miRNAs were regulated by SHMT.However,the specific mechanisms invloved need further exploration.4.Functional study of miRNA in response to SHMTThe sequencing results were confirmed by RT-q PCR.miR-1174,miR-1175,miR-2940-3p,miR-2940-5p,miR-306-5p,miR-34-5p,let-7,miR-8-3p and miR-184 were up-regulated;miR-263a-5p,miR-286 b and miR-2941 were down-regulated;miR-9a,miR-279,miR-309 a,miR-375,miR989,miR-2944b-5p and miR-2946 were not regulated by SHMT.We depleted several miRNAs by injection of their antagomirs to reveal their potential roles.We did not observe any obvious abnormal phenotypes for aae-miR-2940-3p,miR-2940-5p,miR-306-5p and miR-2941.However,when aae-miR-1174 was depleted,the blood feeding rate significantly decreased,blood entered the crop and could not be digested,ovary development was blocked,and finally egg-laying was inhibited.This is consistent with our previous conclusions. |
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