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.

Avian trypanosomes are cosmopolitan and common protozoan parasites of birds; nevertheless, knowledge of their life cycles and vectors remains incomplete. Mosquitoes have been confirmed as vectors of Trypanosoma culicavium and suggested as vectors of T. thomasbancrofti; however, transmission has been experimentally confirmed only for the former species. This study aims to confirm the experimental transmission of T. thomasbancrofti to birds and its localization in vectors. Culex pipiens were fed on blood using four strains of T. thomasbancrofti, isolated from vectors and avian hosts; all strains established infections, and three of them were able to develop high infection rates in mosquitoes. The infection rate of the culicine isolates was 5–28% for CUL15 and 48–81% for CUL98, 67–92% for isolate OF19 from hippoboscid fly, while the avian isolate PAS343 ranged between 48% and 92%, and heavy infections were detected in 90% of positive females. Contrary to T. culicavium, trypanosomes were localized in the hindgut, where they formed rosettes with the occurrence of free epimastigotes in the hindgut and midgut during late infections. Parasites occurred in urine droplets produced during mosquito prediuresis. Transmission to birds was achieved by the ingestion of mosquito guts containing trypanosomes and via the conjunctiva. Bird infection was proven by blood cultivation and xenodiagnosis; mature infections were present in the dissected guts of 24–26% of mosquitoes fed on infected birds. The prevalence of T. thomasbancrofti in vectors in nature and in avian populations is discussed in this paper. This study confirms the vectorial capacity of culicine mosquitoes for T. thomasbancrofti, a trypanosome related to T. avium, and suggests that prediuresis might be an effective mode of trypanosome transmission.

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. Graphical