Background Trypanosoma theileri species complex includes parasites of Bovidae (cattle, sheep, goat, etc.) and Cervidae (deer) transmitted mainly by Tabanidae (horse flies and deerflies) and keds (Hippoboscidae). While morphological discrimination of species is challenging, two big clades, TthI and TthII, each containing parasites isolated from bovids and cervids, have been identified phylogenetically. To date, the development in the vector has been studied in detail only for the ked-transmitted sheep parasite T. melophagium (TthII), while the fate of trypanosomes in tabanids was described only briefly by light microscopy. Methods We collected infected tabanids of various species and identified trypanosomes by molecular phylogenetic analysis. The morphology and development of trypanosomes was studied using the combination of statistical analyses as well as light and electron microscopy. Results Two trypanosome species belonging to both TthI and TthII clades of the T. theileri complex were identified. The phylogenetic position of these two trypanosomes suggests that they parasitize deer. Both species were indiscernible by morphology in the vector and showed the same development in its intestine. In contrast to the previously described development of T. melophagium, both trypanosomes of tabanids only transiently infected midgut and settled mainly in the ileum, while pylorus and rectum were neglected. Meanwhile, the flagellates developing in the tabanid ileum (pyriform epimastigotes and metacyclic trypomastigotes) showed similarities to the corresponding stages in T. melophagium by morphology, mode of attachment to the host cuticle and formation of the fibrillar matrix surrounding the mass of developing parasites. In addition, for the first time to our knowledge we documented extraintestinal stages in these trypanosomes, located in the space between the epithelium and circular muscles. Conclusions The development of different species of flagellates of the T. theileri complex in their insect vectors shows many similarities, which can be explained not only by their common origin, but also the same transmission mode, i.e. contamination of the oral mucosa with the gut content released after squashing the insect either by tongue or teeth. The observed differences (concerning primarily the distribution of developmental stages in the intestine) are associated rather with the identity of vectors than the phylogenetic position of parasites. Graphical Abstract

Monoxenous (insect-restricted) trypanosomatids are highly diverse and abundant in nature. While many papers focus on the taxonomy and distribution of these parasites, studies on their biology are still scarce. In particular, this concerns trypanosomatids inhabiting the ubiquitous mosquitoes. To shed light on the circulation of monoxenous trypanosomatids with the participation of mosquitoes, we performed a multifaceted study combining the examination of naturally- and experimentally-infected insects using light and electron microscopy and molecular identification of parasites. Our examination of overwintering mosquitoes (genera Culex and Culiseta) revealed that their guts contained living trypanosomatids, which can be spread during the next season. Experimental infections with Crithidia spp. demonstrated that imagines represent permissive hosts, while larvae are resistant to these parasites. We argue that for the parasites with wide specificity, mosquitoes act as facultative hosts. Other trypanosomatids may have specific adaptations for vertical transmission in these insects at the expense of their potential to infect a wider range of hosts and, consequently, abundance in nature.

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