■Wolbachia,一种内共生细菌,全球用于控制虫媒病毒,因为它能够阻止虫媒病毒复制并操纵Wolbachia宿主的繁殖,埃及伊蚊.最近已经从天然Ae报道了基于聚合酶链反应(PCR)的Wolbachia检测。埃及伊蚊种群。然而,由于PCR的技术限制,如底漆不兼容,基于PCR的测定不够可靠或准确。在这项研究中,我们检查了双消化限制性位点相关DNA测序(ddRAD-Seq)的效率以及在现场收集的Ae中Wolbachia检测和定量的局限性。马尼拉大都会的埃及伊蚊自然种群,菲律宾,与基于PCR的测定相比。
■总共217个人Ae。埃及伊蚊是从马尼拉大都会收集的,菲律宾。我们将其分为14个种群,由7个女性和男性种群组成。我们构建了每个种群的ddRAD-Seq库的文库,并通过使用wsp和16SrRNA的PCR分析筛选了Wolbachia。使用RPS17作为管家基因测量每个种群的Wolbachia密度。
■从获得的146,239,637个序列读段,在整个Wolbachia基因组中绘制了26,299和43,778个读段(使用wAlbA和wAlbB菌株,分别),这表明ddRAD-Seq补充了PCR检测,并从全基因组的角度支持更可靠的Wolbachia检测。根据PCR测定和Ae中Wolbachia的相对密度,每个种群中映射到Wolbachia基因组的读数数量与每个种群中Wolbachia感染的个体数量呈正相关。基于qPCR的埃及伊蚊种群,建议通过ddRAD-Seq对Wolbachia进行基于ddRAD-Seq的半定量。男Ae.埃及伊蚊比雌性展示了更多映射到Wolbachia基因组的读数,表明Wolbachia的患病率更高。我们在Wolbachia基因组中检测到150个单核苷酸多态性位点,允许更准确地检测四种菌株:wPip,wRi,马来西亚Brugia的TRS,还有wmel.
■放在一起,我们的结果证明了从现场收集的Ae中检测基于ddRAD-Seq的Wolbachia的可行性。埃及伊蚊.
Wolbachia, an endosymbiotic bacterium, is globally used to control arboviruses because of its ability to block arboviral replication and manipulate the reproduction of Wolbachia host, Aedes aegypti. Polymerase chain reaction (PCR)-based Wolbachia detection has been recently reported from natural Ae. aegypti populations. However, due to the technical limitations of PCR, such as primer incompatibility, PCR-based assays are not sufficiently reliable or accurate. In this study, we examined double digestion restriction site-associated DNA sequencing (ddRAD-Seq) efficiency and limitations in Wolbachia detection and quantification in field-collected Ae. aegypti natural populations in Metro Manila, the Philippines, compared with PCR-based assays.
A total of 217 individuals Ae. aegypti were collected from Metropolitan Manila, Philippines. We separated it into 14 populations consisting of 7 female and male populations. We constructed a library for pool ddRAD-Seq per population and also screened for Wolbachia by PCR assays using wsp and 16S rRNA. Wolbachia density per population were measured using RPS17 as the housekeeping gene.
From 146,239,637 sequence reads obtained, 26,299 and 43,778 reads were mapped across the entire Wolbachia genome (with the wAlbA and wAlbB strains, respectively), suggesting that ddRAD-Seq complements PCR assays and supports more reliable Wolbachia detection from a genome-wide perspective. The number of reads mapped to the Wolbachia genome per population positively correlated with the number of Wolbachia-infected individuals per population based on PCR assays and the relative density of Wolbachia in the Ae. aegypti populations based on qPCR, suggesting ddRAD-Seq-based semi-quantification of Wolbachia by ddRAD-Seq. Male Ae. aegypti exhibited more reads mapped to the Wolbachia genome than females, suggesting higher Wolbachia prevalence rates in their case. We detected 150 single nucleotide polymorphism loci across the Wolbachia genome, allowing for more accurate the detection of four strains: wPip, wRi, TRS of Brugia malayi, and wMel.
Taken together, our results demonstrate the feasibility of ddRAD-Seq-based Wolbachia detection from field-collected Ae. aegypti mosquitoes.