Identification of Anopheles daciae in Germany through ITS2 sequencing

Abstract

Until the middle of the twentieth century, malaria was frequently endemic in parts of Germany; Anopheles maculipennis complex species were considered the primary vectors. Three species of this complex have been identified in Germany: A. maculipennis s.s., Anopheles messeae and Anopheles atroparvus; the last predominantly from the coastal regions of Northern Germany. Anopheles daciae is a recently described member of the A . maculipennis complex and resembles the well-characterised species A. messeae, although the two species can be distinguished through their egg morphology and sequencing of the internal transcribed spacer 2 (ITS2) region of their nuclear rDNA. In this study, we harvested larval and adult mosquito samples from five breeding sites and ten CO₂ trap collection sites in the Upper Rhine Valley of Southwestern Germany to analyse the complement of anopheline species present. Mosquito ITS2 DNA was extracted and polymerase chain reaction (PCR)-amplified using established protocols. Genomic analysis was performed by a species-diagnostic restriction fragment length polymorphism assay as well as by sequencing of PCR products; the data obtained were aligned against nucleic acid sequences from English mosquitoes retrieved from GenBank. Additionally, the larval breeding sites of A. messeae were characterised through water quality measurement. Forty-seven samples were successfully processed, of which 6 were identified as A. daciae and 41 as A. messeae. All samples of A. daciae, which has not previously been found in Central Europe, originated from one CO₂ trap collection site in Dettenheim, close to Karlsruhe, Southwestern Germany. The identification of this malarial vector in a novel area may have implications for the re-emergence of disease subsequent to climatic changes.