Culicoides (Diptera: Ceratopogonidae) biting midges are a diverse group of insect vectors that transmit pathogens affecting humans, livestock, and wild animals. Among them, Oropouche virus, African Horse sickness virus, and bluetongue virus are the most notable pathogens. However, comparatively little is known about which Culicoides species serve as vectors of wildlife parasites affecting wild birds globally, including the malaria-like parasite of the genus Haemoproteus (Haemosporida: Haemoproteidae) and kinetoplastid Trypanosoma (Trypanosomatida: Trypanosomatidae). Beyond the direct impact of their bites, infections by these parasites negatively affect wild birds from early developmental stages, significantly influencing their ecology and evolution. Here, we present a comprehensive review of the role of Culicoides species in the transmission of these two genera of avian parasites in Europe: Haemoproteus and Trypanosoma. We identify key information and methods used to study Culicoides-bird-parasite interactions, from insect sampling to vector competence assessment. Additionally, we highlight key knowledge gaps and propose future research directions in this area.

• Keywords
• Culicoides, Haemoproteus, Trypanosoma, Avian malaria, Birds, Blood parasites, Vectors,
• MeSH
• Ceratopogonidae * parasitology   physiology   MeSH
• Animals, Wild parasitology   MeSH
• Haemosporida physiology   isolation & purification   MeSH
• Insect Vectors * parasitology   MeSH
• Bird Diseases * transmission   parasitology   epidemiology   MeSH
• Protozoan Infections, Animal * transmission   parasitology   epidemiology   MeSH
• Birds * parasitology   MeSH
• Trypanosoma physiology   isolation & purification   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Geographicals
• Europe epidemiology   MeSH

BACKGROUND: Traditionally, blood meal analysis has been the primary method used to assess feeding patterns of insects. In contrast, parasite detection is commonly applied to monitor parasite circulation and prevalence in vectors, but rarely to study host feeding patterns. Our study aimed to test whether broad-target screening for haemosporidian and trypanosome parasites could complement blood barcoding by revealing additional host associations. We hypothesised that combining both methods would provide a more comprehensive understanding of vector feeding behaviour than either method alone. In addition to evaluating the two methods, we also analysed the vector species composition and their abundance, providing important faunistic and prevalence data that contribute to the broader understanding of local vector-parasite dynamics. METHODS: Mosquitoes and biting midges were trapped over a 5-year period at three localities in Czechia. Blood-fed individuals underwent blood meal barcoding analysis. In parallel, parasite detection was conducted using nested polymerase chain reaction (PCR) and gut dissection techniques. RESULTS: A total of 10,152 mosquitoes were collected, with Culex pipiens (66%) and Aedes vexans (18%) being the predominant species. In addition, 1701 biting midges, primarily Culicoides pictipennis (61%) and C. festivipennis (12%), were captured. Among the collected samples, 281 mosquitoes (3%) and 52 biting midges (3%) were blood-fed. Parasites were detected in 468 mosquito pools (5%, 341 trypanosomes, 127 haemosporidians) and 21 midge pools (1%, 8 trypanosomes, 13 haemosporidians). Blood meal barcoding of engorged Aedes, Anopheles, Culiseta, and Mansonia samples revealed only mammalian hosts; however, parasite detection indicated previous feeding on birds. Culex displayed stronger ornithophily according to parasite detection, although blood meal analysis showed a more opportunistic behaviour, with the detection of avian, mammalian and even amphibian blood. Avian parasites were detected in five Culicoides species (Culicoides alazanicus, C. festivipennis, C. kibunensis, C. nubeculosus and C. pictipennis) while human blood was detected only in C. pictipennis. Overall, four Haemoproteus lineages and 15 Plasmodium lineages were identified, 11 of which were new records for Czechia and 4 were newly described. CONCLUSIONS: Integrating blood meal analysis with parasite detection provides a more comprehensive understanding of insect feeding patterns and vector-host dynamics. Blood meal analysis remains the gold standard for identifying recent host interactions, offering direct and often species-level evidence of feeding events. In addition, parasite detection extends the window of detectability beyond the digestion of host blood and can reveal additional or otherwise-overlooked host associations. Together, these complementary approaches increase the likelihood of detecting interactions with a broader range of hosts, including humans, who might be missed by parasite screening alone.

• Keywords
• T. theileri, Avian trypanosomes, Biting midge, Blood meal, Haemosporidians, Host feeding patterns, Method comparison, Mosquito,
• MeSH
• Ceratopogonidae * parasitology   physiology   MeSH
• Culicidae * parasitology   physiology   MeSH
• Haemosporida isolation & purification   genetics   MeSH
• Insect Vectors * parasitology   physiology   MeSH
• Host-Parasite Interactions MeSH
• Mosquito Vectors * parasitology   physiology   MeSH
• Blood * parasitology   MeSH
• Polymerase Chain Reaction MeSH
• Feeding Behavior * MeSH
• Trypanosoma isolation & purification   genetics   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic MeSH

Avian trypanosomes (Trypanosoma, Kinetoplastea) are successful blood parasites occurring worldwide. These parasites are usually non-pathogenic to their avian hosts, thus neglected in studies regarding their life cycles and vectors. Several families of blood-sucking dipteran insects, including mosquitoes, have been identified as vectors of avian trypanosomes. Mosquitoes have been experimentally confirmed as vectors of Trypanosoma culicavium and Trypanosoma thomasbancrofti. In this study, we describe a third species of avian trypanosomes occurring in mosquitoes, designated as Trypanosoma tertium n. sp. This species can be distinguished from related trypanosome species based on morphology and small subunit rRNA gene sequence. Two isolates of T. tertium n. sp. obtained from a mosquito and a bird host were able to infect two subspecies of laboratory Culex pipiens mosquitoes, with infection rates reaching 60% and heavy infections in 90% of positive females. In infected mosquitoes, trypanosomes occurred as long epimastigotes in the midgut and short epimastigotes and rosettes in the hindgut. Putative infectious stages were detected in the diuretic liquid of infected mosquitoes, suggesting, besides transmission through ingestion of the infected vector, a possible transconjunctival infection. Among wild mosquitoes, avian trypanosomes were detected exclusively in Cx. pipiens with 3.3% total prevalence, while T. tertium n. sp. prevalence was only 0.08% among 1128 dissected Cx. pipiens individuals. In birds, T. tertium n. sp. was detected in 8 species within which the prevalence was 1.3% (686 birds), while it was 0.3% in total (3084 birds). We discuss the relationship of the newly described T. tertium n. sp. with other mosquito-transmitted trypanosomes.

• Keywords
• Culex, avian blood parasite, monoxenous Kinetoplastea, morphology, phylogeny, transmission, vector,
• MeSH
• Culex * parasitology   MeSH
• Phylogeny MeSH
• Mosquito Vectors * parasitology   MeSH
• Bird Diseases * parasitology   epidemiology   transmission   MeSH
• Prevalence MeSH
• Trypanosoma * genetics   isolation & purification   classification   growth & development   MeSH
• Trypanosomiasis * veterinary   parasitology   epidemiology   transmission   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Parasites of the genus Porcisia, together with the genus Endotrypanum, form a sister clade to the species-rich and medically important genus Leishmania. Both Porcisia species, P. hertigi and P. deanei, are dixenous parasites of Neotropical porcupines. Almost 50 years after their first discovery, knowledge of their life cycle remains poor and their insect vectors are unknown. Because competent vectors of their closest phylogenetic relatives, genera Endotrypanum and Leishmania, are phlebotomine sand flies (Diptera: Psychodidae) and/or biting midges (Diptera: Ceratopogonidae), we examined here the potential of both sand flies and biting midges to transmit Porcisia parasites. The insects (Lutzomyia longipalpis, L. migonei and Culicoides sonorensis) were exposed to parasites through the chicken skin membrane and dissected at various time intervals post bloodmeal. Potentially infected females were also allowed to feed on the ears of anaesthetized BALB/c mice and the presence of parasite DNA was subsequently confirmed in the mice by PCR. Porcisia hertigi did not survive defecation in L. longipalpis or L. migonei, suggesting that these sand fly species are unlikely to serve as natural vectors of this parasite. Similarly, P. hertigi infections were lost in Culicoides midges. In contrast, mature P. deanei infections developed in 51-61% of L. longipalpis females, 7.3% of L. migonei females and 7.7% of Culicoides sonorensis females. In all three vector species, P. deanei colonized predominantly Malpighian tubules and produced metacyclic infective forms. Transmission of P. daenei to BALB/c mice was demonstrated via the prediuresis of L. longipalpis females. This mode of transmission, as well the colonization of Malpighian tubules as the dominant tissue of the vector, is unique among trypanosomatids. In conclusion, we demonstrated the vector competence of L. longipalpis for P. deanei but not for P. hertigi, and further studies are needed to evaluate competence of other Neotropical vectors for these neglected parasites.

• Keywords
• Culicoides, Lutzomyia, Malpighian tubules, Porcisia deanei, Porcisia hertigi, contaminative transmission, prediuresis,
• MeSH
• Phylogeny MeSH
• Insect Vectors MeSH
• Leishmania * MeSH
• Mice MeSH
• Psychodidae * MeSH
• Life Cycle Stages MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Louse flies (Hippoboscidae) are permanent ectoparasites of birds and mammals. They have a cosmopolitan distribution with more than 200 described species. The aim of this study was to reveal host-vector-parasite associations between louse flies, birds, and trypanosomes. A total of 567 louse fly specimens belonging to 7 species were collected from birds at several localities in Czechia, including the rare species Ornithophila metallica and Ornithoica turdi. There was a significant difference in the occurrence of Ornithomya avicularia and Ornithomya fringillina on bird hosts according to their migratory status, O. fringillina being found more frequently on long-distance migrants. Trypanosomes were found in four species, namely, Ornithomya avicularia, O. fringillina, O. biloba, and Ornithoica turdi; the later three species are identified in this paper as natural trypanosome vectors for the first time. The prevalence of trypanosomes ranged between 5 and 19%, the highest being in O. biloba and the lowest being in O. fringillina. Phylogenetic analysis of the SSU rRNA gene revealed that a vast majority of trypanosomes from hippoboscids belong to the avian T. corvi/culicavium group B. Four new lineages were revealed in group B, with louse flies being probable vectors for some of these trypanosome lineages. We also confirmed the transcontinental distribution of several trypanosome lineages. Our results show that hippoboscids of several genera are probable vectors of avian trypanosomes.

• Keywords
• Hippoboscidae, Ornithoica, Ornithomya, Ornithophila, Trypanosoma, avian parasite, host specificity, transmission,
• Publication type
• Journal Article MeSH