Yssouf Amina, Socolovschi Cristina, Flaudrops Christophe, Ndiath Mamadou Ousmane, Sougoufara Seynabou, Dehecq Jean-Sebastien, Lacour Guillaume, Berenger Jean-Michel, Sokhna Cheikh Sadibou, Raoult Didier, Parola Philippe
Aix Marseille Université, Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes, UM63, CNRS 7278, IRD 198, Dakar, Sénégal, Inserm 1095, Marseille, France.
PLoS One. 2013 Aug 15;8(8):e72380. doi: 10.1371/journal.pone.0072380. eCollection 2013.
The identification of mosquito vectors is typically based on morphological characteristics using morphological keys of determination, which requires entomological expertise and training. The use of protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which is increasingly being used for the routine identification of bacteria, has recently emerged for arthropod identification.
To investigate the usefulness of MALDI-TOF-MS as a mosquito identification tool, we tested protein extracts made from mosquito legs to create a database of reference spectra. The database included a total of 129 laboratory-reared and field-caught mosquito specimens consisting of 20 species, including 4 Aedes spp., 9 Anopheles spp., 4 Culex spp., Lutzia tigripes, Orthopodomyia reunionensis and Mansonia uniformis. For the validation study, blind tests were performed with 76 specimens consisting of 1 to 4 individuals per species. A cluster analysis was carried out using the MALDI-Biotyper and some spectra from all mosquito species tested.
Biomarker mass sets containing 22 and 43 masses have been detected from 100 specimens of the Anopheles, Aedes and Culex species. By carrying out 3 blind tests, we achieved the identification of mosquito vectors at the species level, including the differentiation of An. gambiae complex, which is possible using MALDI-TOF-MS with 1.8 as the cut-off identification score. A cluster analysis performed with all available mosquito species showed that MALDI-Biotyper can distinguish between specimens at the subspecies level, as demonstrated for An gambiae M and S, but this method cannot yet be considered a reliable tool for the phylogenetic study of mosquito species.
We confirmed that even without any specific expertise, MALDI-TOF-MS profiling of mosquito leg protein extracts can be used for the rapid identification of mosquito vectors. Therefore, MALDI-TOF-MS is an alternative, efficient and inexpensive tool that can accurately identify mosquitoes collected in the field during entomological surveys.
蚊媒的鉴定通常基于使用形态学鉴定检索表的形态特征，这需要昆虫学专业知识和培训。基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）蛋白质谱分析越来越多地用于细菌的常规鉴定，最近也开始用于节肢动物的鉴定。
为了研究MALDI-TOF-MS作为蚊类鉴定工具的实用性，我们测试了从蚊腿提取的蛋白质以创建参考光谱数据库。该数据库共包含129个实验室饲养和野外捕获的蚊类标本，涵盖20个物种，包括4种伊蚊、9种按蚊、4种库蚊、带纹卢蚊、留尼汪直脚蚊和同型曼蚊。在验证研究中，对76个标本进行了盲测，每个物种有1至4个个体。使用MALDI-Biotyper和所有测试蚊种的一些光谱进行聚类分析。
从100份按蚊、伊蚊和库蚊物种标本中检测到了分别包含22个和43个质量数的生物标志物质量集。通过进行3次盲测，我们实现了在物种水平上对蚊媒的鉴定，包括冈比亚按蚊复合体的区分，使用MALDI-TOF-MS，以1.8作为鉴定得分阈值是可行的。对所有可用蚊种进行的聚类分析表明，MALDI-Biotyper可以在亚种水平上区分标本，如冈比亚按蚊M和S所示，但该方法尚不能被视为蚊种系统发育研究的可靠工具。
我们证实，即使没有任何专业知识，对蚊腿蛋白质提取物进行MALDI-TOF-MS分析也可用于快速鉴定蚊媒。因此，MALDI-TOF-MS是一种替代的、高效且廉价的工具，可在昆虫学调查中准确鉴定野外采集的蚊子。
