Skip to main content
Download PDF

Expression of sex-specific molecular markers by Babesia bovis gametes

Parasites & Vectors volume 17, Article number: 75 (2024) Cite this article

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 and 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 pka, hap2, α-tubulin II and znfp2. However, α-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 confirmed surface expression of HAP2 by male and TRAP2-4 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.

Graphical Abstract

Background

Bovine babesiosis, caused by the intraerythrocytic protozoan parasite Babesia bovis, is an acute tick-borne hemolytic disease that affects cattle throughout the world [1, 2]. Babesia parasites are transmitted by the one-host tick Rhipicephalus microplus [3]. 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 [4]. Inside the tick midgut, morphologically distinct male and female B. bovis gametes [5] mate and fuse to form zygotes. Zygotes develop into the kinete stage that circulates in tick hemolymph [6]. After kinete invasion of eggs, the parasites are transmitted transovarially [4] resulting in larval progeny containing B. bovis sporozoites that can infect cattle [4]. Bovine babesiosis is poorly controlled, and new vaccines are urgently needed [7]. Vaccines targeting blood stages of Babesia parasites have proven to be largely ineffective [7]. Transmission blocking vaccines may provide alternative strategies to control the spread of Babesia. However, transmission blocking vaccine development is currently limited by our poor understanding of parasite biology in the tick, especially at the molecular level [7]. To date, there are no methods to isolate B. bovis sexual stages from infected tick midgut. We induced B. bovis sexual stage development in in vitro cultures using xanthurenic acid (XA), an intermediate metabolite derived from tryptophan metabolism [8, 9]. In vitro induction of B. bovis sexual stages allowed identifying sexual stage-specific genes and gene families [9,10,11]. In this study, we examined the gene expression of B. bovis sex-specific markers for male and female gametes. Examined genes were selected based on previously identified gamete-specific genes in Plasmodium falciparum [12,13,14]. The corresponding proteins in Babesia parasites are conserved and related to the development of sexual stages among all Apicomplexan parasites [15]. Six target genes, including cyclic adenosine 3',5'-monophosphate (cyclic AMP)-dependent protein kinase cAMPDPK (pka), Hapless2/gcs1 (hap2), α-tubulin I, α-tubulin II, zinc finger C3H1 protein2 (znfp2), and ABC transporter and two gene families, thrombospondin-related anonymous proteins (trap1-4) and LCCL domain-containing proteins (ccp1-3), were selected. In the present study, we tested the expression patterns of B. bovis target genes and gene families in male gametes and in female-enriched gametes to identify sex-specific molecular markers for B. bovis male and female gametes.

Methods

In silico target gene identification using bioinformatic analysis

Bioinformatic analysis was performed based on amino acid identity using NCBI Blastp (https://blast.ncbi.nlm.nih.gov/Blast) to identify B. bovis homologs of P. falciparum male and female gamete-specific proteins, including PKA, HAP2, α-tubulin II, ZNFP2, α-tubulin I, ABC transporter, TRAP1-4 and CCp1-3. SignalP-5.0 was used to predict putative signal peptides [16]. Transmembrane domains were predicted for the target proteins using the Transmembrane Hidden Markov Model Package 2 (TMHMM2) [17] (http://www.cbs.dtu.dk/services/TMHMM-2.0). Clustal Omega analysis (http://www.ebi.ac.uk/Tools/msa/clustalo/) was used to evaluate the percent amino acid identity of proteins. Protein domains conserved among B. bovis homologs were determined using the Simple Modular Architecture Research Tool (http://smart.embl-heidelberg.de/).

Cattle and parasite cultures

A splenectomized 3-month-old male Holstein calf tested negative for B. bovis by PCR [4], and cELISA [18] was used in this study. The calf was inoculated intravenously with B. bovis S74-T3Bo strain stabilate containing approximately 1 × 107 B. bovis-infected erythrocytes [4, 6]. The infected calf was monitored daily for B. bovis in peripheral blood and clinical signs of babesiosis. The animal was maintained according to protocols approved by the University of Idaho Institutional Animal Care and Use Committee (IACUC #2018–16).

Babesia bovis blood stages

Defibrinated blood was collected from the calf at 11 days post-B. bovis inoculation. Blood was collected into flasks containing glass beads and shaken to prevent blood coagulation. Red blood cells from defibrinated blood were washed with Puck’s Saline G to remove white blood cells. Some washed infected RBCs were pelleted by centrifugation at 500 ×g, 10 min at 4 °C, and suspended in TRIzol (ThermoFisher Scientific, Waltham, MA, USA). To establish B. bovis in vitro culture, infected RBCs were placed into flasks with culture medium as previously described [19] and incubated at 3% O2 and 5% CO2. After the in vitro incubation of B. bovis blood stages, B. bovis cultures were used to induce sexual stages.

In vitro induction of B. bovis sexual stages

To induce sexual stages, in vitro cultured B. bovis-infected erythrocytes with 10% PPE were suspended in a medium with 100 μM XA (Sigma, St. Louis, MO, USA) at 26 °C with 5% CO2 as previously described [9]. Induced in vitro sexual stage parasites were isolated at 24 h post-induction by differential centrifugation at 400 × g for 1 min.

Separation of B. bovis male gametes

Male gametes were allowed to swim from the pellet of induced parasites that contained extracellular male and female gametes by incubation at room temperature for 15 min. The supernatant containing male gametes was harvested and spun at 12,000 ×g for 10 min at 4 °C and washed twice in phosphate buffered saline (PBS). The purity of the isolated males was determined by microscopy to be at 94%. Slides were stained using the Hema 3 Stat Pack (Thermo Fisher Scientific, Inc., Waltham, MA, USA). A portion of the male gametes and female-enriched gametes was suspended in TRIzol and stored at − 20 °C. Another portion of the parasites was used for immunofluorescence assays.

RNA extraction and cDNA synthesis

Total RNA was extracted from B. bovis blood stages, male and female-enriched gametes collected from in vitro induced parasites in TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol and RNA pellets suspended in 20 µl DEPC-treated water (Invitrogen, Waltham, MA, USA). RNA samples were treated with DNase I (Invitrogen, Waltham, MA, USA) following the manufacturer’s protocol to remove contaminating genomic DNA and quantified by Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA). The removal of genomic DNA was confirmed by PCR targeting rap1 as previously described [20] using non-reverse transcribed samples. cDNA was synthesized from 150 ng of total RNA of each sample with a Superscript® First-strand cDNA synthesis kit (Invitrogen, Waltham, MA, USA) following the manufacturer’s protocol.

Quantitative PCR assay

Specific primers for each gene were designed using the PrimerQuest® Tool (Integrated DNA Technologies) (Table 1) following recommended guidelines for qPCR primer design. Standard PCR was performed to amplify all target genes from cDNA samples using primers listed in Table 1. PCR cycling conditions consisted of 95 °C for 3 min followed by 35 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s, with a final extension of 72 °C for 5 min. PCR products were visualized by 1% agarose gel electrophoresis. PCR amplicons were cloned into PCR 2.1-TOPO® (Thermo Fisher Scientific) and submitted for sequencing (Eurofins MWG Operon, Louisville, KY). Standard curves were generated for each gene using specific quantities of each plasmid. For the normalization of qPCR data, B. bovis actin (BBOV_IV009790) was used as a reference gene candidate [11]. CFX Manager™ software (Bio-Rad, Hercules, CA, USA) [21] was used to examine the stability of expression of the reference gene. The qPCRs for the genes of interest and reference gene were performed in a CFX96™ Real-Time PCR Detection System (C1000 Touch™ Thermal Cycler) (Bio-Rad, Hercules, CA, USA) using the SsoFast™ EvaGreen® Supermix Kit (Bio-Rad, Hercules, CA, USA). The cycling conditions consisted of an initial denaturation at 95 °C for 2 min followed by 40 cycles of 95 °C denaturation for 15 s and annealing at 55 °C for 30 s. Reactions were performed in triplicate in 20 μl using 300 nM of each primer and 2 μl of 1/20 dilution of cDNA as template. CFX Manager™ software (Bio-Rad, Hercules, CA, USA) was used to analyze the qPCR data. Relative expression was calculated by dividing each gene’s detected expression by the detected actin expression within each life stage. Pairwise differences of stages were tested with Tukey or Tukey-Kramer adjustment.

Table 1 Gene identification, primer sets of Babesia bovis genes of interest used for quantitative RT-PCR
Full size table

Antibody production

Polyclonal antibodies against HAP2 and TRAP1-4 were produced as described previously [9, 22] by immunizing rabbits with synthetic peptides. For each protein, three synthetic peptides predicted to be surface-exposed B-cell epitopes using a proprietary algorithm were synthesized (BioSynthesis, Inc. Lewisville, TX, USA) and used to immunize rabbits (Table 2).

Table 2 Peptides used for rabbit immunization to generate specific antibodies against Babesia bovis HAP2 and TRAP1-4 proteins
Full size table

Detection of B. bovis surface exposed proteins on male and female gametes

A transfected B. bovis line tfBbo5480 expressing eGFP [23] was used to generate sexual stages for immunofluorescence assay (IFA). Live B. bovis male and female-enriched gametes separated from induced sexual stage cultures were washed in PBS plus 3% bovine serum albumin (BSA). Cells were then incubated for 1 h with a 1:100 dilution of primary antibodies (anti-HAP2 or anti-TRAP1-4) in blocking solution. The cells were then washed twice in PBS by 400 ×g centrifugation and incubated for 30 min with 1:1000 goat anti-rabbit IgG Alexa Fluor 647 secondary antibody (Thermo Fisher Scientific) diluted with PBS containing 10% normal goat serum. The cells were again washed twice with PBS and incubated with nucleic acid stain Hoechst 33342 (Thermo Fisher Scientific, CA, USA) for 30 min. Finally, cells were washed twice with PBS and air dried on slides. Identically produced negative controls were performed using pre-immune (PI) rabbit serum as the primary antibodies. All samples were independently visualized by fluorescent microscopy using a Leica microscope with LAS-X software.

Results

In silico analysis of target genes

In silico analysis was performed to select B. bovis homologs to previously identified P. falciparum male and female gamete-specific proteins. In silico predictions suggested the presence of a signal peptide in B. bovis HAP2, CCP1, TRAP1-4 proteins and transmembrane domains in B. bovis HAP2, ABC transporter and TRAP1-4 proteins. Babesia bovis target proteins shared from 22 to 90% amino acid identity with their orthologous proteins from P. falciparum (Table 3). Overall, B. bovis proteins appeared well conserved compared with homologs in P. falciparum. Babesia bovis target protein IDs and functional annotation are shown (Table 3).

Table 3 The protein sequence identities (%) and domains of Babesia bovis homologs to Plasmodium falciparum target proteins
Full size table

Quantitative PCR and gene expression

Quantitative PCR was used to analyze the transcription pattern of target genes and gene families in B. bovis male gametes and female-enriched gametes collected from in vitro induced sexual stage parasites as well as blood stages. The transcription levels of all target genes were normalized to B. bovis actin expression level. The data demonstrated that pka, hap2, α-tubulin II and znfp2 were significantly upregulated in male gametes compared to female-enriched gamete samples or blood stages (P < 0.001) (Fig. 1). However, α-tubulin I and ABC transporter (Fig. 1), trap2-4 (Fig. 2) and ccp1-3 genes (Fig. 3) were significantly downregulated in male gametes compared to female-enriched gamete samples or blood stages (P < 0.05).

Fig. 1
figure 1

Transcriptional analysis of the Babesia bovis pka, α-tubulin II, hap2, znfp2, α-tubulin I and ABC transporter genes in blood stages and male and female-enriched gametes. The data represent the mean of three experiments, each containing three technical replicates. Asterisk (**) indicates statistical pairwise differences (P < 0.001)

Full size image
Fig. 2
figure 2

Transcriptional analysis of the Babesia bovis trap1-4 genes in blood stages and male and female-enriched gametes. The data represent the mean of three experiments, each containing three technical replicates. Asterisk (*) indicates statistical pairwise differences (P < 0.05)

Full size image
Fig. 3
figure 3

Transcriptional analysis of the Babesia bovis ccp1-3 genes in blood stages and male and female-enriched gametes. The data represent the mean of three experiments, each containing three technical replicates. Asterisk (*) indicates statistical pairwise differences (P < 0.05)

Full size image

Surface protein expression by B. bovis parasites

Anti-HAP2 polyclonal antibodies reacted to live male gametes separated from in vitro induced sexual stages (Fig. 4a) while Anti-TRAP1-4 polyclonal antibodies were undetectable on B. bovis male gametes (Fig. 4b-e). Anti-HAP2 polyclonal antibodies were undetectable on B. bovis female-enriched gamete samples (Fig. 5a) as well as anti-TRAP1 polyclonal antibodies were undetectable on female-enriched gametes (Fig. 5b), while anti-TRAP2-4 polyclonal antibodies reacted to live female-enriched gamete samples (Fig. 5c–e).

Fig. 4
figure 4

Live immunofluorescence detection of HAP2 expression on the surface of male gametes. a Babesia bovis male gamete incubated with anti-HAP2 and b-e anti-TRAP1-4 proteins. GFP-TF5480: transfected B. bovis parasites. Alexa Fluor 647: goat anti-rabbit antibody. Hoechst: nucleic acid stain. Negative control preimmune (PI) rabbit serum as primary antibody and stained with Hoechst. Bars, 5 μm

Full size image
Fig. 5
figure 5

Live immunofluorescence detection of TRAP2-4 expression in the surface of female-enriched gametes. a Babesia bovis male gamete incubated with anti-HAP2 and b–e anti TRAP1-4 proteins. GFP-TF5480: transfected B. bovis parasites. Alexa Fluor 647: goat anti-rabbit antibody. Hoechst: nucleic acid stain. Negative control preimmune (PI) rabbit serum as primary antibody and stained with Hoechst. Bars, 5 μm

Full size image

Discussion

Recently, we described the induction of B. bovis sexual stages in vitro using Babesia culture recovered from an acutely infected animal. The induction method yields high numbers of clean sexual stages, free of tick antigens. Induction of B. bovis sexual stages resulted in parasite differentiation into large spherical shapes (female gametes) and a smaller shape that displayed one or more projections (male gametes) [9]. Male gametes have up to 10 flagella randomly distributed around the cell. Swimming movement of B. bovis male gametes allowed us to separate male gametes for further analysis. In addition, we demonstrated that pka, hap2, α-tubulin II and znfp2 genes were upregulated in B. bovis male gametes. We also showed that α-tubulin I and ABC transporter were upregulated in female-enriched B. bovis gametes. Our data are consistent with previous studies in P. falciparum that showed genes were differentially expressed in male and female gametocytes during sexual development including pka, hap2, α-tubulin 2, znfp2, α-tubulin I and ABC transporter [12, 14, 24,25,26]. PKA has several functions in the cell, including regulation of glycogen, sugar and lipid metabolism. PfPKA is a key regulator of P. falciparum development [27]. In P. falciparum, PKA is a male gamete-specific gene [12]. HAP2 of Plasmodium is a protein known to be on the surface of microgametes (male gamete-specific protein) and HAP2-mediated gamete fusion in P. falciparum [28]. HAP2 in B. bovis and B. bigemina were found to be expressed by in vitro induced sexual stages and during the development of Babesia parasites in the tick midgut [9, 29, 30]. In Plasmodium, α-tubulin-2 is expressed abundantly by the male gametocytes but is present at low levels in female gametocytes or ookinetes [25, 30]. Alpha-tubulin 2 was found to be highly expressed in P. falciparum male gametocytes and form microtubules of the axoneme of male gametes [25]. ZNFP2:CCCH-type zinc finger proteins are involved in RNA stability and transcriptional repression [31, 32] functioning in both DNA and RNA regulation [33]. Babesia bovis ZNFP2 is an ortholog to P. falciparum CCCH-ZNF4, which is important for exflagellation of gametocytes [33], while α-tubulin 1 is expressed in female gametes and ookinetes [26]. Antibodies against P. falciparum α-tubulin-1 inhibited P. falciparum oocyst development in mosquito midguts [26]. The genome of the B. bovis parasite encodes multiple members of ATP-binding cassette (ABC) transporters [15], one of which is an ortholog to P. falciparum gABCG2, which is transcribed predominantly by the female gametocyte [24].

Additionally, we demonstrated that trap2-4 and ccp1-3 genes were upregulated in female-enriched gametes in agreement with the previous P. falciparum RNA seq data [12], which showed TRAP and CCp families were upregulated in female gametocytes. The TRAP family [15] contains the vWFA and TSP-1 domains, which assist in erythrocyte invasion [34,35,36]. Babesia bovis TRAP1-4s were recommended to be potential candidates for vaccine development [15, 22]. Members of the widely conserved CCp family, CCp1-3, are multidomain adhesion proteins containing LCCL motifs. CCp1-3s are differentially expressed on gametocytes of apicomplexans, including Plasmodium spp. and Babesia spp. [10, 37, 38], and are expressed on female gametes of P. falciparum [12]. Knocking out ccp genes in P. falciparum led to the blocking of sexual stage development of the parasite in the mosquito vector [37, 39]. Members of the CCp family have been previously identified in Babesia bovis [10], B. bigemina [38] and B. ovata [40] as adhesion proteins and expressed upon gamete induction. Here, we demonstrate the expression of HAP2 and TRAP2-4 proteins on the surface of male and female-enriched gametes, respectively, consistent with our previous studies performed with B. bovis that demonstrated HAP2 [9] and TRAP2-4 [22] were expressed on in vitro induced sexual stages. The qPCR and IFA data confirm the lack of expression of B. bovis TRAP1 in either male or female gametes, which agrees with our previous study [22] that demonstrated expression of TRAP1 in B. bovis kinetes only with no expression by induced sexual stages.

Conclusions

In this study, we document that pka, hap2, α-tubulin II and znfp2 are male gamete-specific molecular markers and α-tubulin I, ABC transporter, trap2-4 and ccp1-3 are molecular markers for female gametes. In addition, HAP2 and TRAP2-4 proteins were found to be expressed on the surface of male and female gametes, respectively. The possible involvement of these proteins in the mechanism of gamete maturation and fusion strongly suggests their future use as potential candidates for developing Babesia transmission blocking vaccines.

Availability of data and materials

All data generated or analyzed in this study are included within the article.

References

  1. McCosker PJ: The global importance of babesiosis. Babesiosis 1981:1–24.

  2. Bock R, Jackson L, de Vos A, Jorgensen W. Babesiosis of cattle. Parasitology. 2004;129:S247-269.

    Article  PubMed  Google Scholar 

  3. Oliveira-Sequeira TC, Oliveira MC, Araujo JP Jr, Amarante AF. PCR-based detection of Babesia bovis and Babesia bigemina in their natural host Boophilus microplus and cattle. Int J Parasitol. 2005;35:105–11.

    Article  CAS  PubMed  Google Scholar 

  4. Howell JM, Ueti MW, Palmer GH, Scoles GA, Knowles DP. Transovarial transmission efficiency of Babesia bovis tick stages acquired by Rhipicephalus (Boophilus) microplus during acute infection. J Clin Microbiol. 2007;45:426–31.

    Article  PubMed  Google Scholar 

  5. Mehlhorn H, Schein E. The piroplasms: life cycle and sexual stages. Adv Parasitol. 1985;23:37–103.

    Article  Google Scholar 

  6. Johnson WC, Taus NS, Reif KE, Bohaliga GA, Kappmeyer LS, Ueti MW. Analysis of stage-specific protein expression during Babesia Bovis development within female Rhipicephalus Microplus. J Proteome Res. 2017;16:1327–38.

    Article  CAS  PubMed  Google Scholar 

  7. Florin-Christensen M, Schnittger L, Bastos RG, Rathinasamy VA, Cooke BM, Alzan HF, et al. Pursuing effective vaccines against cattle diseases caused by apicomplexan protozoa. CABI Reviews. 2021;16:1–23.

    Google Scholar 

  8. Billker O, Lindo V, Panico M, Etienne AE, Paxton T, Dell A, et al. Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito. Nature. 1998;392:289–92.

    Article  ADS  CAS  PubMed  Google Scholar 

  9. Hussein HE, Bastos RG, Schneider DA, Johnson WC, Adham FK, Davis WC, et al. The Babesia bovis hap2 gene is not required for blood stage replication, but expressed upon in vitro sexual stage induction. PLoS Negl Trop Dis. 2017;11:e0005965.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Bastos RG, Suarez CE, Laughery JM, Johnson WC, Ueti MW, Knowles DP. Differential expression of three members of the multidomain adhesion CCp family in Babesia bigemina, Babesia bovis and Theileria equi. PLoS ONE. 2013;8:e67765.

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hussein HE, Johnson WC, Ueti MW. Differential paired stage-specific expression of Babesia bovis cysteine-rich GCC2/GCC3 domain family proteins (BboGDP) during development within Rhipicephalus microplus. Parasit Vectors. 2023;16:16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lasonder E, Rijpma SR, van Schaijk BC, Hoeijmakers WA, Kensche PR, Gresnigt MS, et al. Integrated transcriptomic and proteomic analyses of P. falciparum gametocytes: molecular insight into sex-specific processes and translational repression. Nucleic Acids Res. 2016;44:6087–101.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Bennink S, Kiesow MJ, Pradel G. The development of malaria parasites in the mosquito midgut. Cell Microbiol. 2016;18:905–18.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Walzer KA, Kubicki DM, Tang X, Chi JT. Single-cell analysis reveals distinct gene expression and heterogeneity in male and female Plasmodium falciparum gametocytes. mSphere. 2018. https://doi.org/10.1128/mSphere.00130-18.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Ueti MW, Johnson WC, Kappmeyer LS, Herndon DR, Mousel MR, Reif KE, et al. Comparative analysis of gene expression between Babesia bovis blood stages and kinetes allowed by improved genome annotation. Int J Parasitol. 2021;51:123–36.

    Article  CAS  PubMed  Google Scholar 

  16. Almagro Armenteros JJ, Tsirigos KD, Sønderby CK, Petersen TN, Winther O, Brunak S, et al. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat Biotechnol. 2019;37:420–3.

    Article  CAS  PubMed  Google Scholar 

  17. Krogh A, Larsson B, von Heijne G, Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001;305:567–80.

    Article  CAS  PubMed  Google Scholar 

  18. Goff WL, Molloy JB, Johnson WC, Suarez CE, Pino I, Rhalem A, et al. Validation of a competitive enzyme-linked immunosorbent assay for detection of antibodies against Babesia bovis. Clin Vaccine Immunol. 2006;13:1212–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Levy MG, Ristic M. Babesia bovis: continuous cultivation in a microaerophilous stationary phase culture. Science. 1980;207:1218–20.

    Article  ADS  CAS  PubMed  Google Scholar 

  20. Figueroa JV, Chieves LP, Johnson GS, Buening GM. Multiplex polymerase chain reaction based assay for the detection of Babesia bigemina, Babesia bovis and Anaplasma marginale DNA in bovine blood. Vet Parasitol. 1993;50:69–81.

    Article  CAS  PubMed  Google Scholar 

  21. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:research0034.0031.

    Article  Google Scholar 

  22. Masterson HE, Taus NS, Johnson WC, Kappmeyer L, Capelli-Peixoto J, Hussein HE, et al. Thrombospondin-related anonymous protein (TRAP) family expression by Babesia bovis life stages within the mammalian host and tick vector. Microorganisms. 2022;10:2173.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Johnson WC, Hussein HE, Capelli-Peixoto J, Laughery JM, Taus NS, Suarez CE, et al. A transfected Babesia bovis parasite line expressing eGFP is able to complete the full life cycle of the parasite in mammalian and tick hosts. Pathogens. 2022;11:623.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Tran PN, Brown SHJ, Mitchell TW, Matuschewski K, McMillan PJ, Kirk K, et al. A female gametocyte-specific ABC transporter plays a role in lipid metabolism in the malaria parasite. Nat Commun. 2014;5:4773.

    Article  ADS  CAS  PubMed  Google Scholar 

  25. Kooij TW, Franke-Fayard B, Renz J, Kroeze H, van Dooren MW, Ramesar J, et al. Plasmodium berghei alpha-tubulin II: a role in both male gamete formation and asexual blood stages. Mol Biochem Parasitol. 2005;144:16–26.

    Article  CAS  PubMed  Google Scholar 

  26. Zhang G, Niu G, Hooker-Romera D, Shabani S, Ramelow J, Wang X, et al. Targeting plasmodium α-tubulin-1 to block malaria transmission to mosquitoes. Front Cell Infect Microbiol. 2023. https://doi.org/10.3389/fcimb.2023.1132647.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Wurtz N, Chapus C, Desplans J, Parzy D. cAMP-dependent protein kinase from Plasmodium falciparum: an update. Parasitology. 2011;138:1–25.

    Article  CAS  PubMed  Google Scholar 

  28. Pinello JF, Clark TG. HAP2-mediated gamete fusion: lessons from the world of unicellular eukaryotes. Front Cell Dev Biol. 2022. https://doi.org/10.3389/fcell.2021.807313.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Camacho-Nuez M, Hernández-Silva DJ, Castañeda-Ortiz EJ, Paredes-Martínez ME, Rocha-Martínez MK, Alvarez-Sánchez ME, et al. Hap2, a novel gene in Babesia bigemina is expressed in tick stages, and specific antibodies block zygote formation. Parasit Vectors. 2017;10:568.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Yeoh LM, Goodman CD, Mollard V, McFadden GI, Ralph SA. Comparative transcriptomics of female and male gametocytes in Plasmodium berghei and the evolution of sex in alveolates. BMC Genomics. 2017;18:734.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Hajikhezri Z, Darweesh M, Akusjärvi G, Punga T. Role of CCCH-type zinc finger proteins in human adenovirus infections. Viruses. 2020. https://doi.org/10.3390/v12111322.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Ngwa CJ, Farrukh A, Pradel G. Zinc finger proteins of Plasmodium falciparum. Cell Microbiol. 2021;23:e13387.

    Article  CAS  PubMed  Google Scholar 

  33. Hanhsen B, Farrukh A, Pradel G, Ngwa CJ. The Plasmodium falciparum CCCH Zinc Finger Protein ZNF4 Plays an Important Role in Gametocyte Exflagellation through the Regulation of Male Enriched Transcripts. Cells. 2022;11:1666.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Gaffar FR, Yatsuda AP, Franssen FF, de Vries E. A Babesia bovis merozoite protein with a domain architecture highly similar to the thrombospondin-related anonymous protein (TRAP) present in Plasmodium sporozoites. Mol Biochem Parasitol. 2004;136:25–34.

    Article  CAS  PubMed  Google Scholar 

  35. Song G, Koksal AC, Lu C, Springer TA. Shape change in the receptor for gliding motility in Plasmodium sporozoites. Proc Natl Acad Sci USA. 2012;109:21420–5.

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  36. Terkawi MA, Ratthanophart J, Salama A, AbouLaila M, Asada M, Ueno A, et al. Molecular characterization of a new Babesia bovis thrombospondin-related anonymous protein (BbTRAP2). PLoS ONE. 2013;8:e83305.

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  37. Pradel G, Wagner C, Mejia C, Templeton TJ. Plasmodium falciparum: Co-dependent expression and co-localization of the PfCCp multi-adhesion domain proteins. Exp Parasitol. 2006;112:263–8.

    Article  CAS  PubMed  Google Scholar 

  38. Bohaliga GAR, Johnson WC, Taus NS, Hussein HE, Bastos RG, Suarez CE, et al. Identification of proteins expressed by Babesia bigemina kinetes. Parasit Vectors. 2019;12:271.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Simon N, Kuehn A, Williamson KC, Pradel G. Adhesion protein complexes of malaria gametocytes assemble following parasite transmission to the mosquito. Parasitol Int. 2016;65:27–30.

    Article  CAS  PubMed  Google Scholar 

  40. Nguyen TT, Dang-Trinh MA, Higuchi L, Mosqueda J, Hakimi H, Asada M, et al. Initiated Babesia ovata Sexual Stages under In Vitro conditions were recognized by anti-CCp2 antibodies, showing changes in the DNA content by imaging flow cytometry. Pathogens. 2019;8:104.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Paul Lacy, Gavin Scoles, Sara Davis, Megan Jacks and Nicholas Durfee of the US Department of Agriculture, Animal Disease Research Unit, for excellent technical assistance.

Funding

This work was supported by the United States Department of Agriculture-Agricultural Research Service project no. 2090–32000-040-000D and USDA National Institute of Food and Agriculture-Agriculture and Food Research Initiative grant no. 2020–67030-31476.

Author information

Authors and Affiliations

  1. Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA

    Hala E. Hussein, Wendell C. Johnson, Naomi S. Taus & Massaro W. Ueti

  2. Department of Biology, College of Arts and Sciences, Gonzaga University, Spokane, WA, USA

    Hala E. Hussein

  3. The U.S. Department of Agriculture-ARS-Animal Disease Research Unit, Pullman, WA, USA

    Naomi S. Taus & Massaro W. Ueti

Authors
  1. Hala E. Hussein
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wendell C. Johnson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Naomi S. Taus
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Massaro W. Ueti
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

HEH, WCJ and MWU designed the study. HEH and WCJ performed the experiments, the data analysis and drafted the manuscript. HEH, WCJ, NST and MWU read and critically revised the manuscript. All authors approved the final manuscript.

Corresponding author

Correspondence to Hala E. Hussein.

Ethics declarations

Ethics approval and consent to participate

This study was approved by the Institutional Animal Care and Use Committee of the University of Idaho, Moscow, Idaho, in accordance with institutional guidelines based on the US Department of Agriculture Animal Welfare Act and Animal Welfare Regulations and US National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hussein, H.E., Johnson, W.C., Taus, N.S. et al. Expression of sex-specific molecular markers by Babesia bovis gametes. Parasites Vectors 17, 75 (2024). https://doi.org/10.1186/s13071-024-06185-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13071-024-06185-w

Keywords

Parasites & Vectors

ISSN: 1756-3305

Contact us