1. Introduction
Members of the genus
Spiroplasma are Gram-positive bacteria without cell walls. They are known as symbionts of arthropods and plants and are classified into three major clades based on the 16S ribosomal RNA gene (rDNA) sequence: Ixodetis, Citri-Chrysopicola-Mirum (CCM), and Apis [
1,
2].
Spiroplasma is one of the most common endosymbionts with a wide range of hosts, including insects, arachnids, crustaceans, and plants [
3]. It is estimated that 5–10% of insect species harbor this symbiont group [
2,
4].
Spiroplasma has a wide range of fitness effects and transmission strategies [
2,
5,
6,
7,
8,
9,
10,
11,
12,
13,
14,
15,
16,
17]. Some
Spiroplasma species affect the sex ratio by inducing male killing in hosts such as flies, butterflies, and ladybird beetles [
7,
8,
9,
10]. Several
Spiroplasma species are known to cause disease in arthropods such as bees and plants [
6,
17,
18]. On the other hand, some flies infected with
Spiroplasma can develop resistance to other pathogens [
5,
10,
11,
12]. A wide range of symbiotic relationships involving
Spiroplasma have been observed [
5,
7,
8,
14,
15,
16]. The rapid spread of
Spiroplasma infection in fruit fly natural populations has been reported in some areas of North America, and this phenomenon has been confirmed in laboratory settings [
19]. This characteristic of
Spiroplasma is not only biologically interesting, but also useful for symbiotic control applications among host individuals [
20].
Ticks have long been studied, since they transmit a variety of pathogens to humans and animals.
Spiroplasma mirum is the first reported tick-associated
Spiroplasma, which was obtained from
Haemaphysalis leporispalustris in the United States in 1982 during the search for rickettsiae in ticks [
21]. Another species,
S. ixodetis, was isolated from
Ixodes pacificus in the United States in 1981 [
22]. Thus far, these two species are the only validated
Spiroplasma species detected in ticks. Nevertheless, several alleles or putative new species of
Spiroplasma have been found in various tick species such as
I. arboricola, I. frontalis, I. ovatus, I. persulcatus, I. ricinus, I. uriae, Dermacentor marginatus, Rhipicephalus annulatus, R. decoloratus, R. geigyi, and
R. pusillus [
23,
24,
25,
26,
27,
28,
29,
30].
In Japan, 46 tick species belonging to seven genera (
Amblyomma,
Argas,
Dermacentor,
Rhipicephalus, Haemaphysalis,
Ixodes, and
Ornithodoros) have been recorded [
11,
12]. Several tick-borne diseases such as Lyme disease, relapsing fever, Japanese spotted fever, severe fever with thrombocytopenia syndrome, and tick-borne encephalitis are endemic [
31]. Taroura et al. first detected
Spiroplasma DNA in questing
I. ovatus ticks captured in several prefectures [
24]. Subsequently, a microbiome study revealed the presence of
Spiroplasma in the salivary glands of
I. ovatus and
I. persulcatus [
23]. More recently, several
Spiroplasma isolates were obtained by incubating the homogenates of
I. monospinosus,
I. persulcatus, and
H. kitaokai with tick and mosquito cells [
32]. These studies collectively indicate that there is a close relationship between
Spiroplasma and ticks in Japan; however, no comprehensive studies have been conducted to determine the genetic diversity and prevalence of tick-associated
Spiroplasma.
The aim of this study was to identify and genetically characterize Spiroplasma in different tick species in Japan. A linear mixed model (LMM) was developed to resolve the correlation among several extrinsic and intrinsic factors associated with Spiroplasma infection in ticks.
4. Discussion
Prior to this study, there was only limited information available on the prevalence and genetic diversity of tick-associated
Spiroplasma in Japan. In addition to three tick species (
H. kitaokai, I. ovatus, and
I. persulcatus) that were previously revealed to harbour
Spiroplasma [
24,
32], five additional species, i.e.,
A. testudinarium,
D. taiwanensis,
H. flava,
I. pavlovsky, and
I. turdus, were found to be infected with
Spiroplasma, thus expanding our knowledge of the host range of tick-associated
Spiroplasma in Japan.
The infection rate of
Spiroplasma ranged from 0% to 84% depending on the tick species. To investigate whether this difference in infection rate is determined by the tick species or other factors, LMM analysis was performed. The results indicated that
Spiroplasma infection was mainly influenced by the species of ticks but less likely to be influenced by temporal and seasonal factors (
Table 5). Although the prevalence of
Spiroplasma in tick populations has not been well understood, several previous studies reported that the
Spiroplasma infection rates are variable between populations such as in
I. arboricola,
I. ricinus, and
R. decoloratus [
28,
43]. A study investigating
Spiroplasma infection rates in natural
Drosophila populations in the southwestern United States and northwestern Mexico observed varying infection rates depending on the fly species [
44]. In the same study, there was a difference in
Spiroplasma infection rates in two fly species between the two collection sites. Similarly, in our LMM analysis, the introduction of district as the random effect variable improved the models significantly (
Table 4), indicating that the
Spiroplasma infection status in ticks may be partially influenced by the sampling location.
The highest infection rate was observed in
I. ovatus; 82% (32/39) of males and 85% (35/41) of females were positive based on PCR amplification of
Spiroplasma 16S rDNA (
Figure 1). Sequencing analysis of PCR amplicons identified 11
Spiroplasma alleles in this tick species (
Table 3). Furthermore,
H. kitaokai, the second most infected species (28% (11/40) of males and 42% (16/38) of females), had four different
Spiroplasma alleles. The association between specific 16S rDNA alleles (G1, G9, and G11) and their host tick species was statistically confirmed (
Table 6). The presence of these alleles resulted in the high overall infection rates in
I. ovatus and
H. kitaokai. These
Spiroplasma alleles may have adapted to the tick environment, which is important for symbionts [
45]. The transmission of symbionts occurs mainly through the vertical or horizontal route. Vertical transmission involves the dispersal of symbionts and occurs primarily from the mother to offspring. Horizontal transmission occurs via host-to-host contact and acquisition from the environment [
45]. The high infection rates observed in
I. ovatus and
H. kitaokai suggest the vertical transmission of
Spiroplasma in these tick species. Symbionts can positively affect the nutrition, reproduction, and defence of their hosts. These positive effects may promote the coexistence or coevolution of symbionts and their hosts [
45]. Therefore, it is of particular interest to investigate whether
Spiroplasma affects tick fitness, as it may help understand the close association between
Spiroplasma and ticks.
Among the three
Spiroplasma clades, tick-associated
Spiroplasma has only been identified in the Ixodetis and CCM groups. In the present study, most of the samples were classified as belonging to the Ixodetis group (
n = 98), and only three samples were classified as belonging to the CCM group (
Figure 3). Considering that most of the
Spiroplasma species from ticks identified in previous studies belong to the Ixodetis group [
21,
22,
24,
25,
29,
30,
43,
46], this group of
Spiroplasma may be widely distributed in the world. On the other hand, there is a lack of information on the geographic distribution and host range of tick-associated
Spiroplasma in the CCM group. The alleles G10 and G17 obtained in the present study showed high sequence identities (99.7% and 99.4%, respectively) to
S. mirum, which has been found to cause persistent infection in the mouse brain [
47] and neurological deterioration and spongiform encephalopathy in suckling rats [
48,
49]. Furthermore, several ruminants such as deer, sheep, and goats developed spongiform encephalopathy in a dose-dependent manner when experimentally inoculated with
S. mirum in their brains [
50]. The alleles G10 and G17 were obtained from
A. testudinarium,
I. pavlovsky, and
I. persulcatus, whose primary hosts include domestic and wild ruminants such as cattle and sika deer in Japan [
51,
52]. Furthermore,
A. testudinarium and
I. persulcatus are human-biting species that serve as main vectors for human tick-borne diseases [
53,
54]. Hence, it is important to investigate the potential of these
Spiroplasma alleles as agents of human and animal diseases.
The 16S rDNA-based genotyping of 101
Spiroplasma-positive samples identified 17 alleles, some of which were observed in more than two different tick species (
Table 2). However, further characterization by sequencing additional genes (ITS,
dnaA, and
rpoB) divided them into 31 haplotypes, and only one of them (SP24) was observed in two tick species (
A. testudinarium and
I. persulcatus) (
Table 3). A previous study suggested the possible horizontal transmission of
Spiroplasma between different ticks and other arthropods, considering that tick-derived
S. ixodetis did not form a tick species-specific clade [
30]. Our results indicated that horizontal transmission among tick species is not common, at least among the tested tick species. However, the fact that certain alleles (G2, G9, and G15) in the Ixodetis group were more related to
Spiroplasma found in other arthropods than other alleles found in ticks highlights the important role of horizontal transmission between arthropods in the spread of
Spiroplasma in ticks, as suggested previously [
30].
The genes
dnaA and
rpoB are frequently used in the detection and characterization of
Spiroplasma alleles in various arthropods [
1,
29,
36,
40,
46,
55]. In this study,
dnaA and
rpoB were not amplified in nearly half of the haplotypes tested (
Table 3). This may be attributed to nucleotide mismatches in the primer annealing sites. To understand the genetic diversity of
Spiroplasma and clarify the mode of horizontal transmission in ticks, further assays using different gene targets and primer sets are necessary. A previous study developed a multi-locus sequence typing method based on five genes (16S rDNA,
rpoB,
dnaK,
gyrA, and
EpsG) by referring the daft genome of
S. ixodetis Y32 type [
30]. Considering high PCR success rates reported for ticks and other arthropods, the method might be useful to genotype
Spiroplasma in ticks.
Some species of
Spiroplasma are known to affect host reproductive systems through mechanisms such as male killing [
7,
8,
9,
10]. For instance,
Spiroplasma kills
Drosophila males by inducing male X chromosome-specific DNA damage and activating p53-dependent abnormal apoptosis in male embryos [
56]. In this study, 49 male ticks and 60 female ticks were infected with
Spiroplasma, and there was no statistically significant difference for any of the tested tick species (
Figure 1). This result is consistent with that of LLM analysis, where sex was not selected as a variable to improve the model of
Spiroplasma infection in ticks (
Table 4). Similarly, two previous studies targeting wild populations of
R. decoloratus and wild and laboratory populations of
I. arboricola did not find any association between sex and
Spiroplasma infection [
27,
30].
In a previous study,
Spiroplasma was highly abundant in the salivary glands of
I. ovatus [
23]. It is known that
S. citri, a plant pathogenic
Spiroplasma, propagates in the salivary glands of arthropod hosts such as leafhoppers and is released along with the saliva into a new plant during feeding, which leads to transmission from an infected plant to new arthropod hosts [
57,
58]. Similarly, the presence of
Spiroplasma in the tick salivary glands may cause horizontal transmission via feeding to unidentified hosts. One recent study reported that the salivary protein components of
Wolbachia/Spiroplasma-infected spider mites differed from those of uninfected mites [
59]. Tick saliva is an important biological material for various processes such as combating host defences, accelerating blood-feeding processes, and facilitating the transmission of pathogens to hosts [
60]. Therefore, the effects of
Spiroplasma on tick physiology and pathogen transmission involving the tick salivary glands should be clarified in future experimental studies.