Background

Piroplasmosis is a group of human and veterinary tick-borne diseases caused mainly by parasites belonging to two Apicomplexa genera, Babesia and Theileria [1]. Theileria parasites, which can infect a number of domesticated and wild ruminants around the world, kill millions of cattle each year and cause enormous economic losses to the livestock industry; of these, Theileria annulata and Theileria parva are the most common pathogens, while Babesia can infect a range of warm-blooded vertebrates, including humans and wild and domestic animals [2, 3]. Human babesiosis is a malaria-like disease caused by a wide spectrum of Babesia species, such as Babesia microti [4], Babesia duncani [5], Babesia divergens [6], Babesia motasi, [7] Babesia crassa [8], Babesia bigemina [9], Babesia venatorum [10] and Babesia bovis [11]. Among these species, B. microti is the most common causative agent [12], while B. duncani is a highly virulent zoonotic pathogen that can cause severe to fatal infections in laboratory mice and hamsters. In humans, symptoms of B. duncani infection range from asymptomatic to mild flu-like illness, and even to fatal, particularly in the elderly, post-splenectomy or immunocompromised individuals [13]. B. duncani and B. microti pose health threats to humans, domestic and wild animals [14, 15]. B. duncani has been found in bighorn sheep and mule deer [16]. Also, B. microti has been detected in domestic cats [17] and dogs [18], and small mammals [19, 20] and baboons [21]. Currently, there is a lack of specific drugs or vaccines to treat piroplasm infections, making it urgent to develop effective therapeutic strategies to improve the management of these parasites.

In Apicomplexa parasites, histone lysine methylation levels are associated with parasite virulence [22], motility [23], stage transition [24] and host-parasite interactions [25]. In addition, many key enzymes downstream of histone lysine methylation pathways have been proven to be ideal targets for cancer drug development [26,27,28,29]. Intriguingly, several studies have demonstrated that methyltransferase inhibitors show promise as therapeutic drugs for treating malaria parasites [30, 31], Trypanosoma brucei [32, 33] and Leishmania [34]. Currently, SET (Su(var)3–9, E(z), Trithorax) domain-containing histone lysine methyltransferases (HKMTs) and JmjC (Jumonji C) domain-containing histone demethylases (JHDMs) have been identified and characterized at the genome-wide level in several Apicomplexa parasites, such as Plasmodium falciparum [35], Cryptosporidium parvum [25] and Toxoplasma gondii [36]. The SET domain, a sequence of about 130 amino acid residues first discovered in the Drosophila genome, is the enzymatic activity domain of HKMTs and is highly evolutionarily conserved [37, 38], while, the evolutionarily highly conserved JmjC domain is the functionally active region of the JHDM family [39]. Based on sequence similarity and substrate specificity, SET domain-containing HKMTs can be classified into several subfamilies: SETD1, SETD2, SUV3D9, SMYD, SET7/9 and SETD8 [40], and JHDM can be divided into JHDM1, PHF2/PHF8, JARID, JHDM3/JMJD2, UTX/UTY, JHDM2 and JmjC domain only groups [41]. However, little is known about the classification, structural and physical characteristics, and function of HKMTs and JHDMs in human pathogenic babesia, including B. duncani, B. microti, and in veterinary important pathogens, such as B. bigemina, B. bovis, T. annulata and T. parva.

Here, we conducted systematic and comprehensive research on putative KMT and JHDM genes in several representative piroplasm genomes to disclose the information focusing on their physicochemical properties, chromosome distribution, phylogenetic classification and interspecies collinearity. Furthermore, the expression profiles of the retrieved genes at different developmental stages of B. duncani were determined by real-time quantitative PCR. Our study will provide the scientific community with new viewpoints and clues to better understand HKMTs and JHDMs and pave the way for research on the role of these methylation regulators in the intraerythrocytic development of human pathogenic and veterinary important piroplasm parasites.

Results

Identification and characterization of the putative KMTs and JHDMs in B. duncani, B. bigemina, B. microti, B. bovis, T. annulata and T. parva

By thoroughly searching the B. duncani, B. bigemina, B. microti, B. bovis, T. annulata and T. parva reference genomes with the SET domain sequences of several well-studied HKMTs, 49 putative SET domain-containing candidates were obtained. However, further structure verification analysis with SMART revealed that 11 hits (GenBank ID: KAK2185947, KAK2197250, XP_001610401, BAN65725, XP_001611356, XP_012768058, XP_952658, XP_954450, XP_955402, XP_061162069 and XP_765804) did not possess any SET domain (data not shown). Consequently, 38 putative KMTs were retrieved from the six genomes. The physicochemical properties including the length of the primary sequence, molecular weight, isoelectric point and subcellular localization are listed in Table 1. The molecular weights of the identified putative KMTs varied from 11.03 kDa (BdSET3) to 549.87 kDa (BbSET1); while, the isoelectric points (PIs) of the 38 putative KMTs fluctuated between 4.43 and 9.04. This finding suggested that the KMT family members of these six species are quite different from each other. Moreover, although the subcellular localization of these 38 putative KMTs varied greatly, the same subfamilies had similar subcellular localization, and most of them had nuclear localization (Table 1).

Table 1 Putative SET domain-containing lysine methyltransferases identified in Babesia duncani, Babesia microti, Babesia bigemina, Babesia bovis, Theileria annulata and Theileria parva
Full size table

Additionally, we identified eight putative JHDMs through systematic BLASTp in the six piroplasm genomes with a series of JmjC domain sequences from representative human JHDMs. On the basis of prediction analysis via the ExPASy website, the molecular weights of these JHDMs vary from 33.86 kDa to 84.5 kDa; while, the PIs of these six putative JHDMs are also different from each other (the PI ranges between 5.48 and 8.29). All the putative JHDMs have nuclear localization (Table 2).

Table 2 Putative JmjC domain-containing histone demethylases identified in Babesia duncani, Babesia microti, Babesia bigemina, Babesia bovis, Theileria annulata and Theileria parva
Full size table

Phylogenetic analysis

To analyze the phylogenetic relationships between the putative KMTs of the six species and several well-known HKMTs from Homo sapiens, Drosophila melanogaster and other Apicomplexa species, a phylogenetic tree was constructed from the SET domain amino acid sequences of these proteins by the maximum likelihood ratio method using MEGA 7.0. As shown in Fig. 1A, the 38 putative KMTs were divided into eight KMT subfamilies. However, BmSET6, BdSET6 and BBOVSET6 clustered together to form a new clade distinct from other known HKMTs, suggesting that these three putative KMTs may be parasite-specific enzymes involved in regulating some of the parasite's unique biological processes. Moreover, a phylogenetic cladogram of the JmjC domain proteins was also generated by MEGA 7.0 based on the maximum likelihood ratio algorithm with the JmjC domain protein sequences of the JHDMs of H. sapiens and T. gondii to examine the evolutionary history of these proteins. Of note, the identified JHDMs were divided into two main subfamilies on the basis of their branching features and bootstrap values. As depicted in Fig. 1B, BdJmjC1 and TpJmjC1 were classified into JmjC domain only subgroup, while the remaining JHDMs were assigned to the JARID subfamily.

Fig. 1
figure 1

Phylogenetic trees of the SET domain sequences of the putative KMTs (A) and the JmjC domain sequences of the JHDMs (B). The SET domain sequences of the KMTs and the JmjC domain sequences of the JHDMs were aligned by Clustal W algorithm, and phylogenetic tree files were generated by MEGA 7.0 by the maximum-likelihood method and visualized by R software. The numbers at each branch point on the evolutionary trees represent the bootstrap values. Hs, Homo sapiens; Dm, Drosophila melanogaster; Tg, Toxoplasma gondii; Pf, Plasmodium falciparum; Sc, Saccharomyces cerevisiae; BESB, Besnoitia besnoiti; Bd, Babesia duncani; Bb, Babesia bigemina; BBOV, Babesia bovis; Bm, Babesia microti; Ta, Theileria annulata; Tp, Theileria parva

Full size image

Chromosome distribution and interspecies collinearity analysis of the identified putative KMTs and JHDMs

Chromosome distribution analysis of the screened putative KMTs and JHDMs in the six Piroplasm species based on the GFF genome annotation files revealed that the identified genes were mainly concentrated on three to five chromosomes in each parasite's genome (Fig. 2A-F). The genes identified in B. duncani, B. bovis and T. annulata were mainly distributed on 3 chromosomes, in B. bigemina on 5 chromosomes and in B. microti and T. parva on 4 chromosomes.

Fig. 2
figure 2

Chromosomal distributions (A to F) and interspecies collinearity analysis (G) of putative KMT and JHDM genes. Panels A to F are the chromosomal maps of the putative KMTs and JHDMs in the six piroplasm genomes: Babesia bovis (A), Babesia microti (B), Theileria annulata (C), Babesia bigemina (D), Babesia duncani (E) and Theileria parva (F). The upper part of (G) indicates the interspecies collinearity relationship of putative KMT and JHDM genes between Babesia bigemina, Babesia duncani and Theileria annulata, while the middle part indicates the interspecies relationship of putative KMT and JHDM genes between Babesia bovis, Babesia duncani and Babesia microti; the lower part indicates the interspecies relationship of putative KMT and JHDM genes between Babesia duncani, Theileria parva and Theileria annulata. The yellow lines indicate the collinearity relationship of putative KMT genes between species, and the green lines indicate the collinearity relationship of putative JHDM genes between species

Full size image

To further explore the synteny relationships of the putative KMTs and the JHDMs among B. duncani, B. bigemina, B. bovis, B. microti, T. annulata and T. parva, we conducted an interspecies collinearity analysis. As shown in Fig. 2G, B. duncani and B. bovis share six pairs of collinear putative KMT genes and one pair of collinear JHDM genes; two pairs of collinear putative KMT genes were found between B. duncani and B. microti; seven putative KMT and one JHDM homologous genes were found between B. duncani and B. bigemina, whereas, B. duncani and T. annulata share five putative KMT and one JHDM homologous genes; besides, five putative KMT and two JHDM homologous genes were identified between B. duncani and T. parva and T.parva and T. annulata, respectively.

Conserved motif, gene architecture and domain analysis of the identified putative KMTs and JHDMs

Based on the MEME search, the conserved motifs and the corresponding distributions in the putative KMTs and JHDMs were analyzed. A total of ten conserved motifs were retrieved for the putative KMTs and JHDMs, separately. Notably, the distribution and constitution of the conserved motifs in the putative KMTs vary significantly. Generally, proteins belonging to the same KMT subfamily have similar motif constitutions and distributions. Noteworthily, all of the identified putative KMTs have a motif 1 sequence that contains a core “ELxFDY”, an AdoMet binding and catalytic site (Fig. 3A). Additionally, motif 2, with a “RFINHSCxPN” core, and motif 3, with “GxG” and “YxG” cores, were distributed on most of the identified putative KMTs. “RFINHSCxPN”, “GxG” and “YxG” are also canonical AdoMet catalytic motifs present in the majority of HKMTs from different species [25, 35, 40]. Another thing worth noting is that BdJmjC2, BbJmjC1, BBOVJmjC1, BmJmjC1, TaJmjC2 and TpJmjC2 share a similar composition and distribution of conserved motifs, however, BdJmjC1 has no common motifs (Fig. 3B). What’s more, JHDM motifs 1 and 2, which contain “HxE/ExnH” Fe (II) and “N and K” α-KG binding sites, were highly conserved. The amino acid sequences of the conserved motifs essential for enzyme activity in the identified putative KMTs and JHDMs are shown as sequence logos (Fig. 4).

Fig. 3
figure 3

Conserved motif (left), domain (middle) and gene structure (right) analysis of putative KMTs (A) and JHDMs (B) in Babesia duncani, Babesia bigemina, Babesia bovis, Babesia microti, Theileria annulata and Theileria parva

Full size image
Fig. 4
figure 4

The sequence logo of the conserved motifs found in the putative KMTs (A) and JHDMs (B) in Babesia duncani, Babesia bigemina, Babesia bovis, Babesia microti, Theileria annulata and Theileria parva

Full size image

Gene structure exploration of the CDS, UTR and intron in the SET domain and JmjC domain gene families was carried out based on the genome annotation file (GFF). According to the gene structure analysis results, the number of exons in the SET gene family members fluctuated considerably (Fig. 3A). For example, BBOVSET7 has 15 exons, while nine of the putative KMTs have only one exon. Interestingly, UTRs were found in the six putative KMT genes of B. bovis. For the JmjC gene family, the exon number of each member also varied drastically, ranging between 4 (BdJmjC1) and 12 (TaJmjC2 and TpJmjC2) (Fig. 3B).

The functional conserved domains of the identified putative KMTs and JHDMs were obtained by a conserved domain search in the NCBI-CDD database. As shown in Fig. 3A, the domain constitutions in the same subfamily are obviously conserved. Among these 38 putative KMTs, with the exception of TaSETup3, additional conserved domains were found only in SETD1 family members, and all other subfamily members contained only one conserved SET domain. On the other hand, except for BdJmjC1 and TpJmjC1, all the identified JHDMs also contain a JmjN domain in addition to a JmjC domain. Moreover, a zf-C5HC2 domain was found to exist in BmJmjC1 and TaJmjC2 (Fig. 3B).

Gene expression profile of the putative KMTs and JHDMs in B. duncani

To figure out the gene expression patterns of the putative KMTs and JHDMs in B. duncani, we conducted real-time qPCR to determine the relative expression levels of these genes at different developmental stages. All the amplification reactions were performed in parallel in three duplicates. The primers, reaction systems and amplification conditions for RT‒qPCR used in our study are listed in Tables S3 and S4, respectively. Meanwhile, the detailed information on the S1/S2/S3 stage samples and the results of relative expression levels as well as the statistical estimates are shown separately in supplementary Table S5 and S6. According to the heatmap of the relative gene expression levels (Fig. 5A), the expression patterns of the putative KMTs and the JHDMs varied significantly at different developmental stages. The majority of the putative KMT and JHDM genes exhibited increased expression with parasite development, with the exception of BdSET1, whose expression decreased as B. duncani parasites developed from S1 to S2 and increased from S2 to S3. These results imply that the identified putative KMT and JHDM genes may be involved in the intraerythrocytic development of B. duncani, especially BdSET1 which may be a crucial regulator of stage transition.

Fig. 5
figure 5

Expression profiles of the putative KMT and JHDM genes at different developmental stages in Babesia duncani. A Heatmap of the relative expression levels of the putative KMT and JHDM genes in S1-, S2- and S3-stage Babesia duncani. B Images of S1, S2 and S3 intraerythrocytic stages of Babesia duncani from Giemsa-stained thin blood smears

Full size image

Discussion

Histone methylation plays an essential role in various biological processes in Apicomplexa parasites, including transcriptional regulation, chromatin remodeling, parasite-host interactions, parasite differentiation and pathogenicity [22, 25, 42, 43]. HKMTs and JHDMs are the “writers” and “erasers” of histone methylation, respectively, and are critical for maintaining dynamic changes in histone methylation levels. The widely studied functions and evolutionary history of the members of the HKMT and JHDM families in several Apicomplexa parasites, including P. falciparum [22, 35, 44, 45], T. gondii [36] and C. parvum [25], have stimulated our interest in the identification and characterization of the SET domain-containing or JmjC domain-containing gene families in the human pathogenic genus Babesia, such as B. microti and B. duncani, and in piroplasms of veterinary importance such as B. bovis, B. bigemina, T. annulata and T. parva.

In the current study, we conducted genome-wide identification and characterization of SET domain-containing KMTs and JHDMs in B. duncani, B. bovis, B. bigemina, B. microti, T. annulata and T. parva. A total of 38 putative KMT and eight JHDM genes were obtained, and SMART confirmed the presence of the SET domain and JmjC domain in each putative KMT and JHDM, respectively. However, the number of putative KMTs and JHDMs in the B. microti, B. duncani, B. bovis and B. bigemina genomes is close to that in the P. falciparum [35] and C. parvum [25] genomes, much less than that in H. sapiens (HKMTs > 50 and HKDMs > 30) [46], which may be due to the relatively compact size of the genome of Apicomplexa parasites (genome size of B. duncani 10.4 Mb, B. bigemina 13.8 Mb, B. bovis 8.2 Mb, B. microti 6.4 Mb, T. annulata 8.4 Mb, T. parva 8.3 Mb, P. falciparum 23.3 Mb and C. parvum 9.1 Mb, whereas H. sapiens 3.1 Gb) and demonstrates the importance of the HKMT and JHDM gene families in Apicomplexa parasite evolution. The phylogenetic tree revealed that the 38 putative KMTs can be grouped into eight distinct subfamilies: SETD1, SETD2, SETD8, SVU39, SET7/9, SMYD, LSMT and others (a clade distinct from other known HKMT subfamilies). In T. gondii, TgAKMT can affect parasite motility, invasion and parasite egress by methylating non-histone proteins, and when TgAKMT is knocked out, parasite motility is inhibited and invasion and parasite egress are reduced [23, 36, 47]. Putative KMTs (BdSET5, BBOVSET5, BbSET5, BmSET5, TaSETup1 and TpSET5) that cluster with TgAKMT have been identified in each of these six Apicomplexan piroplasm genomes, and these genes are likely involved biological processes similar to those of TgAKMT. Interestingly, in the study by Cheeseman et al., TaSETup1 showed the ability to methylate H3K18 and may be involved in the regulation of stage differentiation in T. annulata [42]. These findings indicate that putative KMTs clustering with TgAKMT may be able to catalyze both non-histone and histone substrates to exert effects on parasite behavior. Since AKMT (Apicomplexa lysine methyltransferase) is a methyltransferase that does not exist in mammals but only in Apicomplexa parasites, investigating its mechanism in parasite development and pathogenesis could help develop new therapeutic strategies to control parasites. In addition, in P. falciparum, PfSET2 regulates var gene expression by modulating H3K36me3 levels on var genes, and in the absence of PfSET2, all var genes are expressed [22, 48, 49]. In this study, a SETD2 HKMT homologue was found in the genomes of each of the six piroplasm species. Future research to confirm whether these genes are involved in modulating the expression of var antigens in these six piroplasm strains, as they are in P. falciparum, will provide some theoretical support for the development of vaccines against these parasites. Moreover, we did not observe any intraspecies collinearity between these screened genes (data not shown), indicating that no duplicate events of these methylation-related genes occurred during evolution. Importantly, interspecies collinearity analysis of piroplasm species also unraveled the significance of the HKMT and JHDM genes in species evolution.

Due to the limited number of transcription factors in Apicomplexa protozoan genomes, other epigenetic regulatory mechanisms, such as histone lysine/arginine methylation or acetylation may also contribute greatly to protozoan development [43, 50, 51]. Previous studies have demonstrated that histone methylation levels are involved in orchestrating the cell differentiation of Apicomplexa parasites and their complex life cycle [25, 42, 52]. Accordingly, dynamic changes in histone lysine methylation levels usually occur together with alterations in gene expression throughout the parasite development stages [44, 53,54,55]. Therefore, we analyzed the expression patterns of the putative KMTs and JHDMs in different developmental stages of B. duncani. According to the relative expression levels of the genes identified by RT‒qPCR (Fig. 5), the putative KMT and JHDM genes exhibit distinct expression profiles in different intraerythrocytic developmental stages. The expression level of BdSET1 gradually decreased with parasite development from S1 to S2, and increased as B. duncani parasites developed from S2 to S3. BdSET1 is a SETD1 superfamily member supported by a high bootstrap value according to the phylogenetic tree constructed from well-studied HKMTs (Fig. 1A). Furthermore, BdSET1 possesses four additional PHDs and one bromodomain, similar in domain number and constitution to other SETD1 family members, such as BmSET1, BbSET1 TaSETup5 and TpSET1 (Fig. 3A). The PHD and bromodomain can bind to the H3K4 site and are essential for the HKMT activity of SETD1 family members [56,57,58]. Therefore, we speculate that BdSET1 may be able to catalyze H3K4 to form mono-, di- or trimethylated H3K4. Besides, H3K4 methylation is an active marker of transcription and several studies have demonstrated that the methylation level of H3K4 is associated with parasite life cycle progression [58]. These findings raise the possibility that BdSET1 might be a critical regulator of B. duncani parasite development. However, more well-designed studies should be performed to support this speculation.

Conclusions

In general, this study could help the scientific community acquire more basic information on the HKMTs and JHDMs of human pathogenic and veterinary important Piroplasm parasites and lay a theoretical foundation for future works on the mechanisms of histone lysine methylation regulation in Apicomplexa babesia parasite differentiation.

Materials and methods

Identification of putative KMTs and JHDMs

The SET motifs of several well-studied histone lysine methyltransferases (shown in supplementary Table S1) from different species, including human, Drosophila, and Apicomplexa protozoans like Toxoplasma gondii and Plasmodium falciparum [25], were used as queries with BLASTp algorithm (https://blast.ncbi.nlm.nih.gov/) to search for potential SET domain-containing KMTs in B. duncani (accession number: GCA_028658345.1), B. microti (GCF_000691945.2), B. bigemina (GCF_000981445.1), B. bovis (GCF_000165395.2), T. annulata (GCF_000003225.4) and T. parva (GCF_000165365.1) genomes. Parallelly, the amino acid sequences of the JumonjiC (JmjC) domain of representative human histone lysine demethylases [41] (Table S2) were also subjected to a search for putative JHDMs in the same genome sets. Moreover, the presence of SET domain in putative KMTs and the presence of JmjC domain in putative JHDMs of these six species were confirmed using the Simple Modular Architecture Research Tool (SMART, http://smart.embl.de/). The basic characteristics of the identified proteins, including molecular weight (kDa) and isoelectric point (PI), were obtained by the ExPASy website tool (https://prosite.expasy.org/). Besides, Euk-mPLoc 2.0 online software [59] (http://www.csbio.sjtu.edu.cn/bioinf/euk-multi-2/) was used to predict the subcellular localization of the putative KMTs and JHDMs.

Phylogenetic analysis

Multiple sequence alignment was performed using the Megalign tool by the Clustal W algorithm imbedded in MEGA 7 software (Molecular Evolutionary Genetics Analysis Version 7.0) to compare the conserved SET domain or JmjC domain sequences of the identified proteins with the representative HKMTs or JHDMs listed in Tables S1 and S2, respectively. Subsequently, phylogenetic tree files for the SET domain or the JmjC domain sequences of these proteins were generated using MEGA 7 with the maximum likelihood (ML) algorithm due to its relatively higher accuracy and faster computing speed. The reliability was evaluated by 1000 bootstrap replications. Thereafter, visualization and beautification of the tree files were performed on the R-project platform using the “ggplot2”, “treeio” and “ggtree” packages [60, 61].

Chromosomal distribution and interspecies collinearity analysis

The genome sequence (FASTA) and annotation (GFF) files of the six piroplasm strains were downloaded from GenBank. The chromosome length and gene distribution information obtained from the genome annotation file (GFF) and the gene IDs of the identified putative KMTs and JHDMs were visualized with the “Gene Location Visualize from GTF/GFF” program imbedded in TBtools software. The interspecies collinearity relationships of the putative KMT and JHDM genes among B. duncani, B. bovis, B. microti, B. bigemina, T. annulata and T. parva were evaluated via the Multiple Collinearity Scan toolkit (MCScanX) (e-value threshold = 1e-10) and visualized via the Multiple Synteny Plot program in TBtools [62].

Conserved motif, architecture and gene structure analysis

The conserved motifs of putative KMTs or JHDMs of the six species were searched using the Multiple Em for Motif Elicitation (MEME) online website tool (https://meme-suite.org/meme/tools/meme) [63] with the parameters set to a maximum number of motifs of 10 and an optimal motif width of 6—50. The gene structure of putative KMTs and JHDMs, including CDSs, introns and UTRs, were predicted and visualized by TBtools using the corresponding genome annotation files of these species. The conserved domains of all the identified proteins were analyzed and searched with the NCBI Batch CD-search Tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi). Moreover, sequence logos for the conserved motif of the identified proteins were generated through a MEME search and visualized on the R-project platform using the “ggplot2” and “ggseqlogo” packages [64].

In vivo parasite culture and collection of merozoites at different developmental stages

A vial of B. duncani WA1 purchased from ATCC and cryopreserved in liquid nitrogen at Vector and Vector-Borne Diseases (VVBD) Laboratory, Lanzhou Veterinary Research Institute (LVRI) (CAAS Lanzhou, China) was thawed quickly in a water bath at 37℃. Immediately, a 10-week-old female Golden Syrian hamster (Chales River Laboratories, Beijing, China) was inoculated with the whole vial of parasite via intraperitoneal injection. Parasitemia was monitored daily from day 5 post infection (p.i.) by thin blood smears stained with Giemsa prepared via lateral saphenous venipuncture through microscopic examination with a × 1000 oil-immersion lens. When the parasitemia increased to approximately 10%, the hamster was anesthetized with isoflurane (Orbiepharm, Qingdao, China) in an ABS Rodent Gas Anesthesia System (Yuyan Instruments Corporation, Shanghai) with consumption of isoflurane set at 5 ml/hour. The hamsters were then euthanized by collecting blood via cardiac puncture. Fresh infected blood was diluted in RPMI-1640 medium. Immediately following dilution, six 10-week-old female Golden Syrian hamsters (Chales River Laboratories, Beijing, China) were inoculated with diluted blood (2500 infected erythrocytes/500 μL/hamster). Thin blood smears were prepared and stained with Giemsa to monitor the parasite morphology daily after 5 dpi. Based on the parasite morphology within erythrocytes under microscopy and the intraerythrocytic cell division cycle of Babesia spp. proposed by Rezvani, et al. [65], the developmental stages of B. duncani could be artificially divided into several phases. Distinct morphologies (e.g. single-ring, double-ring and tetradic forms) could represent different developmental stages (named as S1/S2/S3 stages, respectively). In the early stages of infection, single-ring forms predominate (> 90%), and as the infection progresses, double-ring and tetradic forms progressively increase, and when double-ring forms predominate over tetradic forms, the developmental stage is assumed to be S2, whereas when tetradic forms predominate over single-ring forms, it can be considered to be S3. Hamsters were anesthetized and euthanized as aforementioned, and blood samples were collected in EDTA-K2 anticoagulant vacuum tubes. Immediately thereafter, B. duncani merozoites were extracted from infected erythrocytes and purified as previously described [66].

Total RNA extraction, cDNA library establishment and real-time quantitative PCR (RT‒qPCR)

For all merozoites at different developmental stages, 100 μl of purified merozoites was used to extract total RNA using TRIzol Reagent (Life Technologies) according to the manufacturer’s instructions. After removing the potentially contaminating genomic DNA by a gDNA eraser kit (Takara), 1 μg of total RNA was reverse transcribed to establish a cDNA library using PrimScript™ RT reagent kit (Takara) in a 20 μl reaction. RT‒qPCR was carried out on an Agilent Mx3005P instrument (Agilent Technologies) to determine the relative gene expression levels using a TB Green® Kit (Takara Bio USA, Inc.) in accordance with the manufacturer’s instructions. A housekeeping gene of B. duncani HSP70 (BdWA1_003044) was used as the internal reference gene [42] and the relative expression levels of the identified putative KMTs and JHDMs were calculated using the 2−∆ΔCt method [67]. The average relative expression of the putative KMT and JHDM genes at different developmental stages was visualized using the Heatmap Illustrator program embedded in TBtools software.