Culicoides biting midges feeding behaviour as a key for understanding avian Haemoproteus transmission in Lithuania.

Investigations of host feeding behaviour in haematophagous insects are critical to assess transmission routes of vector-borne diseases. Understanding if a certain species has ornithophilic or mammalophilic feeding behaviour can facilitate future studies focused on pathogens transmission to and from certain host species. Culicoides Latreille (Diptera: Ceratopogonidae) are vectors of several pathogens, which include arboviruses, bacteria and parasites to a considerable diversity of vertebrate hosts. However, most of the studies focused on feeding habits target Culicoides species that could transmit the Bluetongue virus, consequently with a mammalophilic feeding behaviour, leaving aside the Culicoides species that are involved in the transmission of vector-borne parasites to birds, such as Haemoproteus Kruse (Haemosporida: Haemoproteidae). This study aimed to investigate the source of blood meals of wild-caught Culicoides using molecular-based methods and to correlate our findings with the reports of Haemoproteus parasites in Culicoides species. Engorged Culicoides females were collected using ultraviolet (UV)-light traps at seven different localities in Lithuania in 2021-2023. Biting midges were dissected, and the abdomens of engorged females were used for molecular investigation of the blood meal source. A polymerase chain reaction (PCR) protocol that amplifies a fragment of the Cytochrome B gene of vertebrates was used. Obtained sequences were compared to available information in GenBank database to confirm the source of the blood meal. In total, 258 engorged Culicoides females, representing nine different species, were analysed. The source of blood meal was identified in 29.1% of them with most of the insects having fed on birds (74.7%) and the remaining on mammals (25.3%). Culicoides segnis Campbell, Pelham-Clinton was the only species to feed exclusively on birds; Culicoides from the Obsoletus group, C. pallidicornis Kieffer and C. punctatus Latreille were found to feed exclusively on mammals; C. festivipennis Kieffer, C. kibunensis Tokunaga and C. pictipennis Staeger had an opportunistic feeding behaviour, with the first two preferably feeding on birds. Due to their feeding behaviour and the presence of Haemoproteus parasites reported in the literature, C. festivipennis, C. kibunensis, C. pictipennis, and C. segnis play an important role in the transmission of those avian vector-borne parasite in the wild. These Culicoides species were already confirmed as being able to support the development of several Haemoproteus species and lineages. Future studies focused on understanding the epidemiology of avian pathogens transmitted by Culicoides should target these species.

Avian haemosporidian parasites from wild-caught mosquitoes with new evidence on vectors of Plasmodium matutinum.

Avian haemosporidian parasites are spread worldwide and pose a threat to their hosts occasionally. A complete life cycle of these parasites requires two hosts: vertebrate and invertebrate (a blood-sucking insect that acts as a vector). In this study, we tested wild-caught mosquitoes for haemosporidian infections. Mosquitoes were collected (2021-2023) in several localities in Lithuania using a sweeping net and a CDC trap baited with CO2, morphologically identified, and preparations of salivary glands were prepared (from females collected in 2022-2023). 2093 DNA samples from either individual after dissection (1675) or pools (418 pools/1145 individuals) of female mosquito's abdomens were screened using PCR for the detection of haemosporidian parasite DNA. Salivary gland preparations were analyzed microscopically from each PCR-positive mosquito caught in 2022 and 2023. The average prevalence of haemosporidian parasites for all analyzed samples was 2.0 % and varied between 0.6 % (2021) and 3.5 % (2022). DNA of Plasmodium ashfordi (cytochrome b genetic lineage pGRW02), P. circumflexum (pTURDUS1), P. homonucleophilum (pSW2), P. matutinum (pLINN1), P. vaughani (pSYAT05), Haemoproteus brachiatus (hLK03), H. majoris (hWW2), and H. minutus (hTUPHI01) were detected in mosquitoes. Coquilletidia richiardii (3.5 %) and Culex pipiens (2.9 %) were mosquito species with the highest prevalence of haemosporidian parasite DNA detected. Mixed infections were detected in 16 mosquitoes. In one of the samples, sporozoites of P. matutinum (pLINN1) were found in the salivary gland preparation of Culex pipiens, confirming this mosquito species as a competent vector of Plasmodium matutinum and adding it to the list of the natural vectors of this avian parasite.

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.