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

Culicoides biting midges (Diptera: Ceratopogonidae) are important vectors of avian haemosporidian parasites. Understanding their host preferences is crucial for elucidating transmission routes of vector-borne pathogens. In Slovakia, such knowledge is limited, particularly in forested wetlands. This study aimed to identify Culicoides species, their host preferences, and haemosporidian parasites in a wetland ecosystem at the Bird Ringing Station in Drienovec. Midges were collected in 2022 using UV light traps at two sites. In total, 2344 Culicoides individuals of 19 species were collected. Host blood was identified and DNA subsequently extracted from 36 engorged females, revealing feeding on three mammal and five bird species. The most frequently identified host was roe deer (Capreolus capreolus), predominantly fed upon by Culicoides obsoletus (Meigen 1818). Notably, avian haemosporidian DNA was detected for the first time in Slovakia in three Culicoides females. In two Culicoides alazanicus Dzhafarov 1961 individuals, DNA of Haemoproteus asymmetricus (TUPHI01) and Plasmodium matutinum (LINN1) was confirmed, both associated with avian blood from Turdus sp. One Culicoides festivipennis Kieffer 1914 female carried Haemoproteus tartakovskyi (HAWF1) and fed on Coccothraustes coccothraustes. These findings highlight the potential role of local Culicoides species in transmitting avian pathogens and underscore the importance of monitoring their ecology.

• Keywords
• biting midges, host blood, molecular detection, potential vectors,
• MeSH
• Ceratopogonidae * parasitology   physiology   MeSH
• Haemosporida * isolation & purification   genetics   MeSH
• Insect Vectors * parasitology   MeSH
• Host Specificity * MeSH
• Wetlands MeSH
• Birds parasitology   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Slovakia MeSH

Sedentary bird species are suitable model hosts for identifying potential vectors of avian blood parasites. We studied haemosporidian infections in the Tengmalm's Owl (Aegolius funereus) in the Ore Mountains of the Czech Republic using molecular detection methods. Sex of owl nestlings was scored using molecular sexing based on fragment analysis of PCR-amplified CHD1 introns. Observed infection prevalences in nestlings and adult owls were 51 and 86 %, respectively. Five parasite lineages were detected. Most of the infections comprised the Leucocytozoon AEFUN02 and STOCC06 lineages that probably refer to distinct Leucocytozoon species. Other lineages were detected only sporadically. Mixed infections were found in 49 % of samples. The main factor affecting the probability of infection was host age. No effect of individual sex on infection probability was evidenced. The youngest infected nestling was 12 days old. High parasite prevalence in the Tengmalm's Owl nestlings suggests that insect vectors must enter nest boxes to transmit parasites before fledging. Hence, we placed sticky insect traps into modified nest boxes, collected potential insect vectors, and examined them for the presence of haemosporidian parasites using molecular detection. We trapped 201 insects which were determined as biting midges from the Culicoides genus and two black fly species, Simulium (Nevermannia) vernum and Simulium (Eusimulium) angustipes. Six haemosporidian lineages were detected in the potential insect vectors, among which the Leucocytozoon lineage BT2 was common to the Tengmalm's Owl and the trapped insects. However, we have not detected the most frequently encountered Tengmalm's Owl Leucocytozoon lineages AEFUN02 and STOCC06 in insects.

• Keywords
• Avian malaria, Blood parasites, Molecular sexing of owls, Strigiformes, Transmission, Vectors, Wildlife diseases,
• MeSH
• Ceratopogonidae parasitology   MeSH
• Haemosporida genetics   isolation & purification   MeSH
• Insect Vectors parasitology   MeSH
• Bird Diseases epidemiology   parasitology   transmission   MeSH
• Polymerase Chain Reaction MeSH
• Prevalence MeSH
• Protozoan Infections, Animal epidemiology   parasitology   transmission   MeSH
• Sex Factors MeSH
• Simuliidae parasitology   MeSH
• Strigiformes parasitology   MeSH
• Age Factors MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic epidemiology   MeSH

Haemosporidians and trypanosomes of the northern goshawk (Accipiter gentilis) population in the Czech Republic were studied by morphological and molecular methods. Despite the wide distribution of these medium-large birds of prey, virtually nothing is known about their blood parasites. During a 5-year period, altogether 88 nestlings and 15 adults were screened for haemosporidians and trypanosomes by microscopic examination of blood smears and by nested PCR. Both methods revealed consistently higher prevalence of blood protists in adults, Leucocytozoon (80.0 % in adults vs. 13.6 % in nestlings), Haemoproteus (60.0 vs. 2.3 %), Plasmodium (6.7 vs. 0 %), and Trypanosoma (60.0 vs. 2.3 %). Altogether, five haemosporidian lineages were detected by cytochrome b sequencing. Two broadly distributed and host nonspecific lineages, Plasmodium (TURDUS1) and Leucocytozoon (BT2), were detected only sporadically, while three newly described northern goshawk host-specific Leucocytozoon lineages (ACGE01-03) represent the absolute majority of the haemosporidians identified by molecular methods. Our findings support evidences that in falconiform birds the Leucocytozoon toddi group is formed by several host-specific clusters, with Leucocytozoon buteonis in buzzards and Leucocytozoon mathisi in hawks. Between-year comparisons revealed that the infection status of adults remained predominantly unchanged and individuals stayed uninfected or possessed the same parasite lineages; however, two gains and one loss of blood parasite taxa were also recorded.

• Keywords
• Avian blood parasites, Birds of prey, Haemosporida, Mixed infection, PCR detection, Raptors, Trypanosoma,
• MeSH
• Cytochromes b genetics   MeSH
• Falconiformes parasitology   MeSH
• Phylogeny MeSH
• Haemosporida classification   genetics   isolation & purification   MeSH
• Host Specificity MeSH
• Malaria, Avian epidemiology   parasitology   MeSH
• Bird Diseases epidemiology   parasitology   MeSH
• Parasitemia epidemiology   parasitology   veterinary   MeSH
• Plasmodium classification   genetics   isolation & purification   MeSH
• Polymerase Chain Reaction veterinary   MeSH
• Prevalence MeSH
• DNA, Protozoan chemistry   isolation & purification   MeSH
• Protozoan Infections, Animal epidemiology   parasitology   MeSH
• Sequence Alignment veterinary   MeSH
• Trypanosoma classification   genetics   isolation & purification   MeSH
• Trypanosomiasis epidemiology   parasitology   veterinary   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic epidemiology   MeSH
• Names of Substances
• Cytochromes b MeSH
• DNA, Protozoan MeSH

The prevalences of heteroxenous parasites are influenced by the interplay of three main actors: hosts, vectors, and the parasites themselves. We studied blood protists in the nesting populations of raptors in two different areas of the Czech Republic. Altogether, 788 nestlings and 258 adult Eurasian sparrowhawks (Accipiter nisus) and 321 nestlings and 86 adult common buzzards (Buteo buteo) were screened for parasites by the microscopic examination of blood smears and by cultivation. We examined the role of shared vectors and parasite phylogenetic relationships on the occurrence of parasites. In different years and hosts, trypanosome prevalence ranged between 1.9 and 87.2 %, that of Leucocytozoon between 1.9 and 100 %, and Haemoproteus between 0 and 72.7 %. Coinfections with Leucocytozoon and Trypanosoma, phylogenetically distant parasites but both transmitted by blackflies (Simuliidae), were more frequent than coinfections with Leucocytozoon and Haemoproteus, phylogenetically closely related parasites transmitted by different vectors (blackflies and biting midges (Ceratopogonidae), respectively). For example, 16.6 % buzzard nestlings were coinfected with Trypanosoma and Leucocytozoon, while only 4.8 % with Leucocytozoon and Haemoproteus and 0.3 % with Trypanosoma and Haemoproteus. Nestlings in the same nest tended to have the same infection status. Furthermore, prevalence increased with the age of nestlings and with Julian date, while brood size had only a weak negative/positive effect on prevalence at the individual/brood level. Prevalences in a particular avian host species also varied between study sites and years. All these factors should thus be considered while comparing prevalences from different studies, the impact of vectors being the most important. We conclude that phylogenetically unrelated parasites that share the same vectors tend to have similar distributions within the host populations of two different raptor species.

• MeSH
• Raptors parasitology   MeSH
• Falconiformes parasitology   MeSH
• Phylogeny MeSH
• Haemosporida classification   genetics   isolation & purification   physiology   MeSH
• Insect Vectors parasitology   physiology   MeSH
• Host Specificity MeSH
• Bird Diseases epidemiology   parasitology   transmission   MeSH
• Prevalence MeSH
• Protozoan Infections, Animal epidemiology   parasitology   transmission   MeSH
• Birds parasitology   MeSH
• Simuliidae parasitology   physiology   MeSH
• Trypanosoma classification   genetics   isolation & purification   physiology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic epidemiology   MeSH

We studied haemosporidian parasites in the scarlet rosefinch Carpodacus erythrinus in a small isolated semicolony during an eight-year period using molecular methods of parasite detection. The scarlet rosefinch is an interesting model of parasite host species. It winters in South Asia which represents a rare exception among European passerines. Males express yellow to red carotenoid-based plumage ornament which is a good predictor of male reproductive success. In 240 blood samples originating from 199 adult individuals, the total parasite prevalence reached 60%. Prevalence varied among years from 36 to 81% in Haemoproteus, 8 to 22% in Plasmodium, and 0 to 14% in Leucocytozoon. Twenty parasite lineages were detected (Haemoproteus: 5 lineages, Plasmodium: 10 lineages, and Leucocytozoon: 5 lineages). Among them, the Haemoproteus ROFI2 lineage, which is a host-specific parasite lineage of the scarlet rosefinch, was the most frequently found. Parasite lineages showed varying degree of lineage specificity. While Haemoproteus lineages detected in the scarlet rosefinch have relatively narrow host breadth restricted mainly to Fringillidae family, Leucocytozoon and Plasmodium lineages generally showed wider host range. The presence of some parasite lineages hitherto detected in sedentary European passerines (SISKIN1, CCF3, BT2) or in Culicoides biting midges at the same locality (ROFI1) suggest local transmission. On the contrary, lineages LK05 and FANTAIL1 that were previously reported exclusively from Asian hosts imply parasite transmission at the scarlet rosefinch wintering sites in South Asia. Mixed infections were found in 17% of infected samples and comprised mainly the most frequent lineages. The pattern of concomitant infections seemed to be rather random and matched expected levels based on lineage frequencies. Between-year comparisons revealed that in a majority of the repeatedly captured individual hosts the infection status remained unchanged (individuals stayed uninfected or possessed the same parasite lineages). However, 16 gains and 8 losses of lineages were also reported. We have not found any effect of haemosporidians on male carotenoid ornament expression or host body mass.

• MeSH
• Haemosporida isolation & purification   MeSH
• Coinfection epidemiology   parasitology   veterinary   MeSH
• Blood parasitology   MeSH
• Bird Diseases epidemiology   parasitology   MeSH
• Passeriformes MeSH
• Prevalence MeSH
• Protozoan Infections, Animal epidemiology   parasitology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Asia MeSH
• Europe MeSH