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

Trypanosomes belonging to Trypanosoma theileri group are mammalian blood parasites with keds and horse fly vectors. Our aim is to study to vector specificity of T. theileri trypanosomes. During our bloodsucking Diptera survey, we found a surprisingly high prevalence of T. theileri trypanosomes in mosquitoes (154/4051). Using PCR and gut dissections, we detected trypanosomes of T. theileri group mainly in Aedes mosquitoes, with the highest prevalence in Ae. excrucians (22%), Ae. punctor (21%), and Ae. cantans/annulipes (10%). Moreover, T. theileri group were found in keds and blackflies, which were reported as potential vectors for the first time. The vectorial capacity was confirmed by experimental infections of Ae. aegypti using our isolates from mosquitoes; sand fly Phlebotomus perniciosus supported the development of trypanosomes as well. Infection rates were high in both vectors (47-91% in mosquitoes, 65% in sandflies). Furthermore, metacyclic stages of T. theileri trypanosomes were observed in the gut of infected vectors; these putative infectious forms were found in the urine of Ae. aegypti after a second bloodmeal. On the contrary, Culex pipiens quinquefasciatus was refractory to experimental infections. According to a phylogenetic analysis of the 18S rRNA gene, our trypanosomes belong into three lineages, TthI, ThII, and a lineage referred to as here a putative lineage TthIII. The TthI lineage is transmitted by Brachycera, while TthII and ThIII include trypanosomes from Nematocera. In conclusion, we show that T. theileri trypanosomes have a wide range of potential dipteran vectors, and mosquitoes and, possibly, sandflies serve as important vectors.

• Keywords
• Phlebotomus, Trypanosoma melophagium, Trypanosoma theileri, ked, mosquito, phylogeny, prediuresis, tabanid, transmission, vector,
• Publication type
• Journal Article MeSH

RNA of Kyzylagach virus (KYZV), a Sindbis-like mosquito-borne alphavirus from Western equine encephalitis virus complex, was detected in four pools (out of 221 pools examined), encompassing 10,784 female Culex modestus mosquitoes collected at a fishpond in south Moravia, Czech Republic, with a minimum infection rate of 0.04%. This alphavirus was never detected in Central Europe before.

• Keywords
• Culex modestus, Sindbis, alphaviruses, arboviruses, mosquito, reedbeds,
• MeSH
• Culicidae virology   MeSH
• Alphavirus Infections transmission   virology   MeSH
• Mosquito Vectors virology   MeSH
• Sindbis Virus * MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Europe epidemiology   MeSH