Haemoproteus (Parahaemoproteus) parasites are transmitted by Culicoides biting midges. However, the natural vectors of only six of the almost 180 recognized Haemoproteus species have been identified. The aim of this study was to investigate wild biting midges naturally infected with Haemoproteus and to understand the interaction network between Culicoides and Haemoproteus in Europe. Culicoides were collected with UV light traps from different sites in Lithuania. Parous females were morphologically identified based on their wings and heads. PCR-based methods were used to detect the Haemoproteus DNA, and salivary gland preparations were analyzed for the presence of sporozoites. Of the 580 Culicoides analyzed, 5.9% were positive for Haemoproteus DNA, and sporozoites were found in two of 11 sampled biting midge species: Culicoides kibunensis and Culicoides segnis. The interaction network revealed that C. kibunensis and C. segnis are frequently associated with several Haemoproteus lineages. On the other hand, some Haemoproteus lineages were found to interact with only one Culicoides species. This was the first report of C. segnis being a competent vector for H. minutus TURDUS2, H. asymmetricus TUPHI01, H. majoris PHSIB1, and H. fringillae CCF3; and of C. kibunensis being a competent vector for H. belopolskyi HIICT1. Culicoides segnis and C. kibunensis are both important vectors of Haemoproteus 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.

Culex pipiens mosquitoes (Diptera: Culicidae) are widespread during warm periods and actively feed on blood while serving as vectors for various human and animal pathogens. Culex mosquitoes overwinter as adults in temperate zones, raising the question of whether hibernating Cx. pipiens can act as pathogen reservoirs. In this study, hibernating mosquitoes and mosquitoes collected during the warm season were tested for the presence of trypanosomatids and avian haemosporidian parasites using PCR. Midgut preparations were made from Cx. pipiens females in order to search for trypanosomatids morphologically. In total, 1037 Cx. pipiens mosquitoes (556 collected during the warm season and 481 overwintering mosquitos) were investigated. The parasite prevalence differed for mosquitoes collected during the warm season and hibernating ones for both Haemosporida (2.9% in warm-season and no infections in overwintering mosquitoes) and Trypanosomatida (1.6% and 0.4%, respectively) parasites. A phylogenetic analysis confirmed that the trypanosomatids found in hibernating mosquitoes were monoxenous and were not parasites of vertebrates. The peak prevalence of Haemosporida parasites was detected in July (4.9%) and August (2.8%), and for Trypanosomatida, it was detected in May (3.5%). The results of the present study show that overwintering Cx. pipiens mosquitoes are questionable reservoirs for avian haemosporidian parasites, but some monoxenous trypanosomatids can be found in overwintering females.

Four new crane fly species of the subgenus Tipula (Vestiplex) Bezzi, 1924, T. (V.) apteroides sp. nov. (Yunnan), T. (V.) bidentata sp. nov. (Sichuan), T. (V.) spinaventralis sp. nov. (Sichuan) and T. (V.) ventrobasilata sp. nov. (Sichuan) are described and illustrated, providing discussion on separation of the newly described species from other closely related species. The male genitalia of T. (V.) aestiva Savchenko, 1960 is illustrated and redescribed.