High Abundance of Haemoproteus Parasites in Culicoides (Diptera, Ceratopogonidae), with a Confirmation of Culicoides reconditus as a New Vector of These Avian Blood Parasites.

Haemoproteus parasites are the most diverse among Haemosporida. However, their natural vectors (Culicoides) are still poorly investigated and were identified for only a few parasite species and lineages. The application of an integrative approach (insect dissection, microscopic analysis, and molecular-based methods) is necessary in these studies, which have been carried out by a few research groups, mainly in Europe. The aim of this study was (i) to determine the Culicoides species that are naturally infected by Haemoproteus parasites, and which can support its complete sporogonic development, and (ii) to investigate the prevalence of Culicoides species and Haemoproteus parasite lineages in different study sites. In total, 1953 parous Culicoides females, from 11 species, were collected in four different localities in Lithuania and were dissected and analyzed using an integrative approach. The most abundant was C. pictipennis (30.3%). Parasite DNA was found in 7.9% of all investigated Culicoides, of which ~30% had sporozoites in their salivary glands, confirming their vector competence for these parasites. The Botanical Garden presented the highest number of Culicoides parous females, Culicoides species, and parasite lineages, as well as the highest positivity for sporozoites. Culicoides reconditus was confirmed as a natural vector of Haemoproteus parasites, sporozoites of six Haemoproteus lineages were reported for the first time, and 12 new interactions between Haemoproteus parasite lineages and Culicoides species were identified. Haemoproteus parasites seem to be transmitted by a high number of Culicoides species, with C. kibunensis, C. pictipennis, and C. segnis being the most important vectors.

Development of Trypanosoma everetti in Culicoides biting midges.

Trypanosoma species (Trypanosomatida, Kinetoplastea) are almost exclusively heteroxenous flagellated parasites, which have been extensively studied as the causative agents of severe trypanosomiasis in humans and domestic animals. However, the biology of avian trypanosomes remains insufficiently known, particularly in wildlife, despite information that some species might be pathogenic and affect the fitness of intensively infected individuals. Avian trypanosomes are cosmopolitans. Due to regular bird seasonal migrations, this host-parasite system might provide new insight for better understanding mechanisms of transcontinental dispersal of pathogens, their ecological plasticity, specificity and speciation. Trypanosoma everetti parasitizes numerous bird species globally, but data on its biology are scarce and its vectors remain unknown. This study aimed to test experimentally whether widespread Culicoides (Diptera: Ceratopogonidae) biting midges are susceptible to infection with this parasite. Two common house martins Delichon urbicum and two sedge warblers Acrocephalus schoenobaenus naturally infected with T. everetti were caught in the wild after arrival from African wintering grounds. Laboratory reared Culicoides nubeculosus and wild-caught Culicoides impunctatus biting midges were exposed by allowing them to take infected blood meals. The experimentally infected and control insects were maintained in the laboratory and dissected at intervals to follow the development of the parasite. Infections were determined using microscopic examination and PCR-based testing. Four closely related haplotypes of T. everetti were found, and each was present in different individual parasite-donor birds. These parasites readily developed and produced metacyclic trypomastigotes in C. nubeculosus and C. impunctatus biting midges. Molecular characterisation of T. everetti was developed. According to Bayesian phylogenetic analysis using a DNA fragment encoding 18S rRNA, the five species of small avian trypanosomes were closely related. Wild caught Culicoides biting midges were also collected and screened for the presence of natural infections. In all, 6.8% of wild-caught biting midges belonging to five Culicoides species were PCR-positive for kinetoplastids, including Trypanosoma species. Culicoides biting midges are readily susceptible and likely naturally transmit avian trypanosomes and thus, should be targeted in epidemiology research of avian trypanosomiasis.