BACKGROUND: Lyme disease, caused by Borrelia burgdorferi sensu lato (s.l.), is the most common vector-borne disease in the Northern Hemisphere, with Ixodes ticks as its primary vectors. However, many patients do not recall tick bites, fueling speculation about alternative transmission routes, particularly via mosquito bites. This belief is reinforced by studies reporting Borrelia presence in mosquitoes. This study evaluates whether three mosquito species can acquire, maintain, and transmit Borrelia spirochetes. METHODS: Mosquitoes (Aedes aegypti, Culex quinquefasciatus, and Culex pipiens biotype molestus) were fed on Borrelia-infected mice to assess pathogen acquisition. Additional experiments involved ex vivo feeding on Borrelia-enriched blood to examine spirochete persistence in the mosquito gut. The potential for mechanical transmission was tested by simulating interrupted feeding between infected and naive hosts. The role of trypsin in Borrelia survival and infectivity was also investigated. RESULTS: Mosquitoes exhibited low efficiency in acquiring Borrelia from infected hosts. Spirochetes artificially introduced through ex vivo blood meals were rapidly eliminated during digestion, primarily due to trypsin activity. Inhibition of trypsin prolonged spirochete persistence and infectivity in the mosquito gut. Mechanical transmission experiments revealed no evidence of Borrelia transmission from infected to naive hosts. CONCLUSIONS: Our findings demonstrate that mosquitoes lack the biological capacity to efficiently acquire and maintain B. burgdorferi s.l. spirochetes and are unable to transmit them through natural or mechanical means. This study provides compelling evidence against mosquito-borne transmission of Lyme disease and reinforces Ixodes ticks as the sole competent vectors, which is crucial for targeted public health interventions and accurate risk communication.

Bioptic Laboratory Mikulášské náměstí 4 326 00 Plzeň Czech Republic

Department of Biological Sciences and Biomolecular Science Institute Florida International University Miami FL 33199 USA

Department of Parasitology Faculty of Science Charles University Viničná 7 128 44 Prague Czech Republic

Department of Pathology Faculty of Medicine in Plzen Charles University and University Hospital E Beneše 13 305 99 Plzeň Czech Republic

Faculty of Science University of South Bohemia Branišovská 1645 31a 370 05 České Budějovice Czech Republic

Institute of Parasitology Biology Centre of the Czech Academy of Sciences Branišovská 31 370 05 České Budějovice Czech Republic

Institute of Vertebrate Biology Czech Academy of Sciences Květná 8 603 65 Brno Czech Republic

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