Expression of sex-specific molecular markers by Babesia bovis gametes

Authors: HUSSEIN, HALA - Gonzaga University; JOHNSON, WENDELL - Retired ARS Employee; Taus, Naomi; Ueti, Massaro

Submitted to: Parasites & Vectors
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/7/2024
Publication Date: 2/19/2024
Citation: Hussein, H.E., Johnson, W.C., Taus, N.S., Ueti, M.W. 2024. Expression of sex-specific molecular markers by Babesia bovis gametes. Parasites & Vectors. 17. Article 75. https://doi.org/10.1186/s13071-024-06185-w.
DOI: https://doi.org/10.1186/s13071-024-06185-w

Interpretive Summary: Bovine babesiosis, caused by the intraerythrocytic protozoan parasite Babesia bovis is an acute tick-borne hemolytic disease that affects cattle throughout the world. Babesia parasites are transmitted by the one-host tick Rhipicephalus microplus. Transmission of Babesia parasites from the bovine host to the tick vector requires the formation and development of parasite sexual stages inside the tick midgut. Inside the tick midgut, morphologically distinct male and female B. bovis gametes mate and fuse to form zygotes. In this study, we developed a method to separate male gametes from in vitro-induced B. bovis culture. We describe the identification of sex-specific molecular markers essential for B. bovis sexual reproduction. These findings will enhance our understanding of the biology of Babesia sexual stages and, consequently, the development of additional strategies to control bovine babesiosis.

Technical Abstract: Background: Bovine babesiosis caused by Babesia bovis is one of the most important tick-borne diseases of cattle in tropical and subtropical regions. Babesia bovis parasites have a complex lifecycle, including development within the mammalian host and tick vector. In the tick midgut, extracellular Babesia parasites transform into gametes that fuse to form zygotes. To date, little is known about genes and proteins expressed by male gametes. Methods & Results: We developed a method to separate male gametes from in vitro induced B. bovis culture. Separation enabled the validation of sex-specific markers. Collected male gametocytes were observed by Giemsa-stained smear and live-cell fluorescence microscopy. Babesia male gametes were used to confirm sex-specific markers by quantitative real-time PCR. Some genes were found to be male gamete specific genes including cyclic adenosine 3',5'-monophosphate (cyclic AMP)-dependent protein kinase cAMPDPK (PKA), hap2, a-tubulin II, zinc finger C3H1 protein2 (ZNFP2). However, a-tubulin I and ABC transporter, TRAP2-4 and CCp1-3 genes were found to be upregulated in culture depleted of male gametes (female-enriched). Live immunofluorescence analysis using polyclonal antibodies against HAP2 and TRAP4 confirmed surface expression of HAP2 by male and TRAP 2-4 by female gametes. These results revealed strong markers to distinguish between B. bovis male and female gametes. Conclusions: Herein we describe the identification of sex-specific molecular markers essential for B. bovis sexual reproduction. These tools will enhance our understanding of the biology of sexual stages and, consequently, the development of additional strategies to control bovine babesiosis.