Four hundred and seventy-seven collected Anopheles specimens were assigned to the Maculipennis group according to morphological characters. Of these, ITS2 rDNA species-specific PCR according to Proft et al. generated 394 An. messeae, 82 An. maculipennis and 1 An. atroparvus. While An. messeae and An. maculipennis have previously been shown to have a widespread distribution in Germany, the salt-tolerant species An. atroparvus mainly occurs in coastal marsh regions but has also been found in inland areas, although at much lower frequencies. In total, “An. messeae” accounted for 80% of our Anopheles PCR identifications.
DNA sequence analysis of the ITS2 region of the “An. messeae” mosquitoes revealed five single nucleotide polymorphisms in ten specimens, nine females and a male (GenBank accession nos.: JX173885, JX416347-52, JX444557-59), identical to those defining An. daciae according to Nicolescu et al.. Three of the females were hand-collected in August 2011 and June 2012 in a domesticated rabbit stall in Maust, Brandenburg, north-eastern Germany, close to the border with Poland. Four An. daciae females were sampled in June 2012 in a stable harbouring sheep in Ralbitz-Rosenthal, Saxony, and one male was caught in August 2011 in a rabbit stall in Schoeneiche, Brandenburg. The two remaining females were trapped by a BG-Sentinel mosquito trap (Biogents, Germany) in August and September 2011 in Trebur, Hesse. In all locations, either An. messeae or An. maculipennis or both were also shown to occur.
This is the first description of An. daciae for Germany. Considering known differences in vector competence and/or vectorial capacity for malaria parasites of different Maculipennis group species in the same geographic region and of the same species in different geographical areas, the status of An. daciae as a vector in Germany and elsewhere should be investigated. Such studies, however, should not remain restricted to malaria parasites but should include further pathogens since Maculipennis group sibling species have been shown to be infected in the field with Ťahyňa virus in Austria, West Nile virus in Portugal, Sindbis and Batai viruses in Germany[17, 18], and Dirofilaria immitis and Setaria labiatopapillosa filaria in Italy[19, 20].
Despite having followed the recent literature and having denominated An. daciae a species, the authors do not consider the evidence given for the species status of An. daciae, separate from An. messeae, as convincing and sufficient. There are three criteria on which the suggested species status of An. daciae is based, most importantly ITS2 rDNA sequence polymorphisms, with An. daciae being described as an ITS2 variant of An. messeae different at five positions out of 435 nucleotides. However, while investigating the intragenomic heterogeneity of the ITS2 region of geographically distinct An. messeae populations, Bezzhonova & Goryacheva found that the An. daciae variant was just one out of various variants in peripheral populations of An. messeae, the other variants not being elevated to species status. Admittedly, the An. daciae variant was the only one found at more than one, geographically distinct location, which indicates that the genetic divergence is stable. In our ITS2 sequence analyses, the An. daciae ITS2 variant was the only one encountered in addition to the An. messeae variant.
A second criterion given by Nicolescu et al. is the egg structure, which is considered different from that of An. messeae. The differences given, however, are minor and have not been shown to be statistically significant, i.e. to be outside the range of natural phenotypic variation within a species. In fact, such variation can be commonly observed in insect specimens of the same species including the Maculipennis group members.
Most ambiguous is the delimitation of An. daciae and An. messeae by means of unique polymorphisms in the COI gene, which, although used for species identification by barcoding, displays a certain degree of sequence variability. While some COI sequence haplotypes are said to represent An. daciae, no data on intraspecific sequence divergence, either for An. messeae or for An. daciae, in contrast to interspecific divergence, have yet been published. Phylogenetic tree construction from GenBank COI sequences to check for clustering is not possible since it is not known without the corresponding ITS2 sequences whether sequence entries running under the name of An. messeae must actually be assigned to the An. messeae or to the An. daciae variant. Studies on correlated COI and ITS2 sequence analyses have therefore been initiated. Preliminary analyses of COI sequences of An. messeae specimens identified in our lab by ITS2 sequences, as compared to An. daciae COI sequences presented by Nicolescu et al., have shown an identical haplotype. In support of such studies, the ecological and/or physiological features of An. daciae should be studied.