PCR identification of five species from the Anopheles maculipennis complex (Diptera: Culicidae) in North-Eastern Romania.

The members of the Anopheles maculipennis complex have been incriminated for the transmission of the malaria in Europe, which was endemic until the middle of the century. The global warming and the intensification of the intercontinental travel constitute a risk of the re-emergence of the malaria in Europe, given the presence of the Anopheles vectors. The study has attempted the identification by using the PCR (Polymerase Chain Reaction) of the members of the Anopheles maculipennis complex from the North-eastern area of Romania from the city of Iasi. In total there have been identified by using the PCR amplifying the ITS2 sequence of the ribosomal DNA, 217 specimens belonging to the complex of A. maculipennis among which: 58 A. atroparvus, 18 A. melanoon, 2 A. labranchiae, 52 A. maculipennis and 87 A. messeae. The ITS2 sequences of the ribosomal DNA have been compared to those of the species belonging to the A. maculipennis available in GenBank. The Species A. labranchiae is reported for the first time in Romania, being identified in the larval stage IV. The adaptation of a new species to the climatic conditions present in the North-eastern Romania, confirms the phenomenon of global warming and also the intensification of the travelling. As a result of the analysis of the A. labranchiae sequence, this one corresponds to the extent of 96% to the species from Italy, registered in GenBank, given the fact that a high number of the inhabitants of the municipality of Iasi are working in this country.

A rapid and robust selection procedure for generating drug-selectable marker-free recombinant malaria parasites.

Experimental genetics have been widely used to explore the biology of the malaria parasites. The rodent parasites Plasmodium berghei and less frequently P. yoelii are commonly utilised, as their complete life cycle can be reproduced in the laboratory and because they are genetically tractable via homologous recombination. However, due to the limited number of drug-selectable markers, multiple modifications of the parasite genome are difficult to achieve and require large numbers of mice. Here we describe a novel strategy that combines positive-negative drug selection and flow cytometry-assisted sorting of fluorescent parasites for the rapid generation of drug-selectable marker-free P. berghei and P. yoelii mutant parasites expressing a GFP or a GFP-luciferase cassette, using minimal numbers of mice. We further illustrate how this new strategy facilitates phenotypic analysis of genetically modified parasites by fluorescence and bioluminescence imaging of P. berghei mutants arrested during liver stage development.