Analysis on DNA methylation levels in mosquitoes of the Culex pipiens complex
Objective To analyze and compare the DNA methylation levels of three subspecies of Culex pipi-ens complex,including Cx.p.pallens,Cx.p.molestus and Cx.p.quinquefasciatus,and the DNA methylation levels of Cx.p.quinquefasciatus before and after blood-feeding.Methods Mosquitoes of the 3 subspecies were collected at 5 days post-feathering without blood-feeding and Cx.p.quinquefasciatus were collected at 3 days after blood-feeding.DNA was extracted and sonicated into fragments of approximately 250 bp.The fragmented DNA was sequenced,and the data were aligned with the reference genome sequence of Cx.p.quinquefasciatus(Taxonomy ID:7176).Methyla-tion levels of the 3 subspecies were analyzed at the genomic,chromosomal and elemental levels(the methylation level above 3％was considered hypermethylated).The differences in methylation levels among the 3 subspecies mosquitoes without blood-feeding,and in Cx.p.quinquefasciatus before and after blood-feeding were compared.The sites with P＜0.001 and absolute value of methylation difference＞5 were identified as differentially methylated sites.The regions with Q＜0.05 and the absolute value of methylation difference＞3 were identified as differentially methylated regions(DMR).The genes with the nearest transcription start site(TSS)to DMRs were identified as DMR-associated genes,which were subjected to gene ontology(GO)enrichment analysis.Results The genome-wide methylation levels of Cx.p.pallens,Cx.p.molestus and Cx.p.quinquefasciatus were 0.454％-0.672％,0.491％-0.649％and 0.499％-0.655％,respectively,all were below 3％.The methylation levels of CHH of Cx.p.pallens,Cx.p.molestus and Cx.p.quinquefas-ciatus were 0.631％,0.618％and 0.624％,respectively,which were higher than CHG(0.567％,0.559％,0.559％)(t=7.14,83.43,6.87,all P＜0.05)and CG/CpG(0.508％,0.505％,0.505％)(t=10.59,12.52,13.33,all P＜0.05).There were 56 hypermethylated sites and 11 hypermethylated regions present among all 3 subspecies.No significant differ-ences were found among the 3 subspecies(F=0.07,P＞0.05)at genome-wide methylation levels.No significant differ-ences were found in methylation levels of chromosome 1(.568％,0.562％,0.565％),2(0.573％,0.564％,0.566％)and 3(0.575％,0.566％,0.569％)among the 3 subspecies at the chromosome level(F=0.05,0.11,0.13,all P＜0.05)nor in the methylation levels of promoters(0.567％,0.552％,0.556％),exons(0.562％,0.556％,0.558％),introns(0.561％,0.550％,0.555％)and TSS(0.579％,0.506％,0.621％)among the 3 subspecies at the chromosomal elements level(F=0.37,0.06,0.06,0.16,all P＞0.05).There were 178 differentially methylated sites and 4 DMRs between Cx.p.pallens and Cx.p.molestus;209 differentially methylated sites and 8 DMRs between Cx.p.pallens and Cx.p.quinquefasciatus;and 215 differentially methylated sites and 11 DMRs between Cx.p.molestus and Cx.p.quinquefascia-tus.GO enrichment analysis revealed that the DMR-associated genes were mainly enriched in the biological processes with response to radiation,light stimuli and abiotic stimuli.The genome-wide methylation levels of Cx.p.quinquefas-ciatus slightly increased from 0.602％before blood-feeding to 0.617％after blood-feeding,without statistically signifi-cant differences(t=1.21,P＞0.05).The methylation levels of chromosome 1,2 and 3 in Cx.p.quinquefasciatus were 0.569％,0.569％and 0.572％before blood-feeding,and were 0.596％,0.597％and 0.600％after blood-feeding.There were no statistically significant differences before and after blood-feeding(t=1.31,1.33,1.30,all P＞0.05).The methyla-tion levels of the promoters,exons,introns and TSS in Cx.p.quinquefasciatus before blood-feeding were 0.557％,0.561％,0.560％,0.552％,and were 0.585％,0.584％,0.584％,0.594％after blood-feeding,respectively.There were no statistically significant differences before and after blood-feeding(t=1.48,1.35,1.20,1.69,all P＞0.05).There were 6 DMRs in Cx.p.quinquefasciatus between before and after blood-feeding.GO enrichment analysis showed that the DMR-associated genes were mainly enriched in endosomes and vesicles in cell components,and protein binding or small GTPases binding in molecular functions.Conclusion The genome-wide methylation levels of Cx.p.pallens,Cx.p.molestus and Cx.p.quinquefasciatus are relatively low.The DMR-associated genes are mainly related to biologi-cal processes that respond to abiotic stimuli.The methylation level of Cx.p.quinquefasciatus slightly increases after blood-feeding,and the DMR-associated genes before and after blood-feeding are mainly involved in protein binding.
NETL
NSTL
万方数据
