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Little is known about the infection ecology and host specialization within this tritrophic interaction.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Methods<\/jats:title>\n            <jats:p>In this study, we explore the prevalence, diversity and specificity of <jats:italic>Trypanosoma<\/jats:italic> infections in <jats:italic>Corethrella<\/jats:italic> across various localities in tropical America and Borneo by sequencing both midge and trypanosome markers in midge samples.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>Bayesian phylogenetic analyses and ASAP species delimitation of <jats:italic>Corethrella<\/jats:italic> (cytochrome c oxidase I [COI]) and <jats:italic>Trypanosoma<\/jats:italic> (18S, glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) revealed a previously unknown high diversity of frog-biting midge-associated trypanosomes. Across regions and localities, the infection prevalence in midges caught by acoustic midge traps ranged from 2.9% to 23.5%, suggesting that a notable proportion of midges carried trypanosomes, likely acquired from a previous blood meal. At one locality, La Gamba (Costa Rica), the infection prevalence in trap-caught midges was 10.9%, while it was even higher in midges collected directly from frog hosts (20.7%), in agreement with the hypothesis that midges ingest trypanosomes from infected frogs. Bipartite network analyses revealed high degrees of specialization of <jats:italic>Trypanosoma<\/jats:italic> in trap-caught <jats:italic>Corethrella,<\/jats:italic> both across all localities (H2\u2032\u2009=\u20090.87) and when analyzed for our most sampled locality (Cahuita, Costa Rica) alone (H2\u2032\u2009=\u20090.94).<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusions<\/jats:title>\n            <jats:p>Our data suggest that most trypanosomes detected in trap-caught midges are established, host-competent (i.e., specialist) parasites in an infective stadium.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Graphical Abstract<\/jats:title>\n          <\/jats:sec>","DOI":"10.1186\/s13071-025-06993-8","type":"journal-article","created":{"date-parts":[[2025,8,15]],"date-time":"2025-08-15T07:06:25Z","timestamp":1755241585000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Uninvited guests: diversity and specificity of Trypanosoma infections in frog-biting midges (Corethrella spp.)"],"prefix":"10.1186","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0009-0004-3484-0021","authenticated-orcid":false,"given":"Maximilian","family":"Sigl","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4395-7737","authenticated-orcid":false,"given":"Jonas","family":"Virgo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ulmar","family":"Grafe","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0281-2291","authenticated-orcid":false,"given":"Jonas","family":"Henske","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maximilian","family":"Schweinsberg","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8465-7944","authenticated-orcid":false,"given":"Thomas","family":"Eltz","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2025,8,15]]},"reference":[{"key":"6993_CR1","first-page":"522","volume":"37","author":"S McKeever","year":"1977","unstructured":"McKeever S. 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