DNA identification of species of the Anopheles maculipennis complex and first record of An. daciae in Belgium.

The present study aimed at identifying the members of the Anopheles maculipennis complex (Diptera: Culicidae) occurring in Belgium. Therefore, the second internal transcribed spacer of nuclear ribosomal DNA (ITS2) and the mitochondrial cytochrome oxidase subunit I (COI) loci were sequenced in 175 and 111 specimens, respectively, collected between 2007 and 2019. In parallel, the suitability of two species-diagnostic PCR-RFLP assays was tested. The identified specimens included: An. maculipennis s.s. (N = 105), An. daciae (N = 62), An. atroparvus (N = 6) and An. messeae (N = 2). Each species was characterized by unique ITS2 haplotypes, whereas COI only supported the monophyly of An. atroparvus, a historical malaria vector in Belgium. Species identification results were further supported by unique PCR-RFLP banding patterns. We report for the first time An. daciae in Belgium, where it was found to co-occur with An. maculipennis s.s. The latter was the most prevalent in the collection studied (60%) and appears to have the widest distribution in Belgium. As in other studies, An. daciae and An. messeae appeared the most closely related species, up to the point that their species status remains debatable, while their ecological differences, including vector competences, need further study.

An integrated diagnostic setup for the morphological and molecular identification of the Ceratitis FAR complex (C. anonae, C. fasciventris, C. rosa, C. quilicii, Diptera, Tephritidae).

The Ceratitis FAR complex (Diptera, Tephritidae) includes four economically important frugivorous flies (Ceratitis anonae, Ceratitis fasciventris, Ceratitis quilicii, Ceratitis rosa) whose immature stages and adult females cannot be properly resolved through morphological identification. In order to develop a simplified molecular tool for the identification of two of these species (C. rosa, C. quilicii), we selected a subset of six microsatellite markers out of a panel of 16 loci that were previously developed for the molecular differentiation of the taxa within the complex. These six markers were first tested in silico and then used for the actual genotyping of C. quilicii and C. rosa, resulting in the correct identification of all male reference specimens. Here, we propose an integrated morphological and molecular setup for the identification of the four species of the FAR complex. The decision map relies on preliminary DNA barcoding or morphological identification (when possible) to exclude species not belonging to the complex followed by (a) morphological identification of all adult male specimens and female C. anonae, (b) molecular identification via a panel of 16 microsatellite markers for immature stages, damaged vouchers and samples potentially including adult female C. fasciventris/C. quilicii/C. rosa and (c) molecular identification via a reduced panel of six microsatellite markers for samples including only C. quilicii and C. rosa. This simplified diagnostic setup was profitably implemented in the framework of the ERAfrica fruit fly project and will help correctly identify species within the FAR complex for their early detection and monitoring.