Abstract

Abstract: Tick-borne relapsing fever (TBRF) spirochetes are maintained in nature through vector-associated transmission routes primarily in argasid ticks. However, the extent to which strain-level differences influence the maintenance of TBRF spirochetes remains unclear. We evaluated two Borrelia turicatae strains, Bt-SSK1 and Bt-FCB, in Ornithodoros turicata. Both strains were acquired by female ticks, and after mating we unexpectedly observed that they were hyperparasitized by uninfected and unfed male O. turicata. Bt-SSK1 was maintained in males while Bt-FCB was not. Transovarial transmission (TOT) of Bt-SSK1 and Bt-FCB was also evaluated and striking differences were observed between strains. Bt-SSK1 was vertically maintained in F1 progeny but we failed to detect TOT of Bt-FCB. TOT of Bt-SSK1 occurred inefficiently in early ovipositions but increasing in later or delayed reproductive events, indicating a timing-dependent barrier. Using a gfp-expressing Bt-FCB strain, we show that failure of vertical transmission is associated with a lack of persistent oocyte colonization despite dissemination to ovarian tissues. These findings demonstrate strain-dependent differences in oocyte colonization and represent a critical bottleneck governing vertical transmission and persistence of relapsing fever spirochetes in tick populations. Importance: Vector-borne pathogens rely on diverse transmission strategies to persist in nature, yet the biological factors that govern these processes remain poorly understood. In this study, we demonstrated that strains of Borrelia turicatae differ markedly in their ability to persist within Ornithodoros turicata ticks. We also discovered that male ticks frequently hyperparasitized engorged females, creating an unrecognized transmission route in which previously uninfected ticks can acquire, maintain, and transmitted the B. turicatae. We further showed phenotypic differences in vertical transmission and that it is temporally regulated and linked to successful colonization of developing oocytes, identifying a critical bottleneck in pathogen maintenance. Together, these findings revealed that strain-specific traits influence both horizontal maintenance within tick populations and vertical transmission to progeny. This work provides insight into the ecological and evolutionary processes that enable relapsing fever spirochetes to persist in nature.