Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, 27100, Pavia, Italy.
Department of Biosciences and Pediatric Clinical Research Center "Romeo Ed Enrica Invernizzi", University of Milan, 20113, Milan, Italy.
Parasit Vectors. 2024 Sep 28;17(1):407. doi: 10.1186/s13071-024-06478-0.
Mosquitoes (Culicidae), as disease vectors, represent a risk for human health worldwide. Repeated introductions of alien mosquito species and the spread of invasive species have been recorded in different countries. Traditionally, identification of mosquitoes relies on morphological observation. However, morphology-based identification is associated with a number of potential disadvantages, such as the high level of specialisation of the operator and its limited applicability to damaged samples. In these cases, species identification is achieved through molecular methods based on DNA amplification. Molecular-based taxonomy has also enabled the development of techniques for the study of environmental DNA (eDNA). Previous studies indicated the 16S mitochondrial ribosomal RNA (rRNA) gene as a promising target for this application; however, 16S rRNA sequences are available for only a limited number of mosquito species. In addition, although primers for the 16S rRNA gene were designed years ago, they are based on limited numbers of mosquito sequences. Thus, the aims of this study were to: (i) design pan-mosquito 16S rRNA gene primers; (ii) using these primers, generate a 16S rRNA gene mosquito reference library (with a focus on mosquitoes present in Italy); and (iii) compare the discriminatory power of the 16S rRNA gene with two widely used molecular markers, cytochrome c oxidase subunit 1 mitochondrial gene (COI) and internal transcribed spacer 2 (ITS2).
A total of six mosquito genera (28 mosquito species) were included in this study: Aedes (n = 16 species), Anopheles (5 species), Coquillettidia (1 species), Culex (3 species), Culiseta (2 species) and Uranotaenia (1 species). DNA was extracted from the whole mosquito body, and more than one specimen for each species was included in the analysis. Sanger sequencing was used to generate DNA sequences that were then analysed through the Barcode of Life Data Systems (BOLD). Phylogenetic analyses were also performed.
Novel 16S rDNA gene, COI and ITS2 sequences were generated. The 16S rRNA gene was shown to possess sufficient informativeness for the identification of mosquito species, with a discriminatory power equivalent to that of COI.
This study contributes to the generation of DNA barcode libraries, focussed on Italian mosquitoes, with a significant increase in the number of 16S rRNA gene sequences. We hope that these novel sequences will provide a resource for studies on the biodiversity, monitoring and metabarcoding of mosquitoes, including eDNA-based approaches.
蚊子（蚊科）作为疾病传播媒介，对全球人类健康构成威胁。在不同国家，已记录到外来蚊子物种的反复引入和入侵物种的传播。传统上，蚊子的鉴定依赖于形态观察。然而，基于形态的鉴定存在许多潜在的缺点，例如操作人员的专业化程度高，以及对受损样本的适用性有限。在这些情况下，物种鉴定是通过基于 DNA 扩增的分子方法来实现的。基于分子的分类学还为环境 DNA（eDNA）的研究开发了技术。先前的研究表明，16S 线粒体核糖体 RNA（rRNA）基因是一种很有前途的应用目标；然而，只有有限数量的蚊子物种具有 16S rRNA 序列。此外，尽管多年前就设计了用于 16S rRNA 基因的引物，但它们是基于有限数量的蚊子序列设计的。因此，本研究的目的是：（i）设计泛蚊子 16S rRNA 基因引物；（ii）使用这些引物，生成 16S rRNA 基因蚊子参考文库（重点关注意大利存在的蚊子）；（iii）比较 16S rRNA 基因与两种广泛使用的分子标记，细胞色素 c 氧化酶亚基 1 线粒体基因（COI）和内部转录间隔 2（ITS2）的区分能力。
本研究共纳入 6 个蚊子属（28 个蚊子种）：Aedes（n = 16 种）、Anopheles（5 种）、Coquillettidia（1 种）、Culex（3 种）、Culiseta（2 种）和 Uranotaenia（1 种）。从整个蚊子体中提取 DNA，每种物种都包含多个标本进行分析。使用 Sanger 测序生成 DNA 序列，然后通过生命数据系统条码（BOLD）进行分析。还进行了系统发育分析。
生成了新的 16S rDNA 基因、COI 和 ITS2 序列。16S rRNA 基因在蚊子种鉴定方面具有足够的信息量，其区分能力与 COI 相当。
本研究有助于生成意大利蚊子的 DNA 条码文库，16S rRNA 基因序列数量显著增加。我们希望这些新序列将为蚊子多样性、监测和 metabarcoding 研究提供资源，包括基于 eDNA 的方法。
