PCR detection of malaria parasites and related haemosporidians: the sensitive methodology in determining bird-biting insects

Rasa Bernotienė, Gediminas Valkiūnas

Knowledge about feeding preference of blood-sucking insects is important for the better understanding epidemiology of vector-borne parasitic diseases. Extraction of DNA from blood present in abdomens of engorged insects provides opportunities to identify species of their vertebrate hosts. However, this approach often is insufficiently sensitive due to rapid degeneration of host DNA in midguts. Recent studies indicate that avian malaria parasites (Plasmodium spp.) and related haemosporidians (Haemosporida) belonging to Haemoproteus can persist both in vectors and resistant blood-sucking insects for several weeks after initial blood meals, and these parasites can be readily detected by polymerase chain reaction (PCR)-based methods. Because avian haemosporidians are cosmopolitan, prevalent and strictly specific to birds, the determination of haemosporidian DNA in blood-sucking dipterans can be used as molecular tags in determining bird-biting insects. This hypothesis was tested by investigation of prevalence of natural haemosporidian infections in wild-caught mosquitoes (Culicidae) and biting midges (Ceratopogonidae: Culicoides). Females of mosquitoes (1072 individuals of three species) and biting midges (300 individuals of three species) were collected in wildlife using simple netting. They were identified and tested individually for the presence of both the haemosporidian parasites and the bird blood using PCR-based methods. Seven different Haemoproteus and two Plasmodium lineages were detected, with overall infection prevalence of 1.12 and 1.67 % in mosquitoes and biting midges, respectively. In all, the detection rate of avian haemosporidian parasites was three fold higher compared with the detection of avian blood. Molecular markers of avian malaria parasites and other haemosporidians are recommended for getting additional knowledge about blood-sucking dipterans feeding on bird blood. Many genetic lineages of avian haemosporidians are specific to avian hosts, therefore, the detection of these parasite lineages in blood-sucking insects can indicate their feeding preferences on the level of species or groups of related bird species.