Conserved infections and reproductive phenotypes of Wolbachia symbionts in Asian tortrix moths

Abstract Wolbachia is a ubiquitous endosymbiotic bacterium that manipulates insect reproduction. A notable feature of Wolbachia is male killing (MK), whereby sons of infected females are killed during development; however, the evolutionary processes by which Wolbachia acquired the MK ability remain unclear. The tea tortrix moth Homona magnanima (Tortricidae) harbours three non‐MK Wolbachia strains (wHm‐a, wHm‐b and wHm‐c) and an MK strain wHm‐t. Although wHm‐t and wHm‐c are closely related, only wHm‐t has an MK‐associated prophage region. To understand the evolutionary processes underlying the emergence of MK wHm‐t, we examined Wolbachia infections and phenotypes in 62 tortricid species collected from 39 localities across Japan, Taiwan, Vietnam and Indonesia. PCR assays detected wHm‐c relatives in 51 species and triple infection of wHm‐a, wHm‐b and wHm‐c in 31 species. Apart from Taiwanese H. magnanima, no species exhibited the MK phenotype and were positive for the wHm‐t‐specific prophage. While wHm‐t infection was dominant in Taiwanese H. magnanima, wHm‐a, wHm‐b and wHm‐c were dominant in Japanese H. magnanima populations. These results suggest that wHm‐a, wHm‐b and wHm‐c strains descended from a common ancestor with repeated infection loss and that wHm‐t evolved from the wHm‐c acquiring MK ability in allopatric populations of H. magnanima.

For instance, MK directly skews the sex ratio of the host population towards females, allowing Wolbachia to spread the infection across the host population (Werren et al., 2008).
For decades, Wolbachia-induced MK has drawn the attention of basic and applied biologists.One unresolved question is how Wolbachia acquired the MK ability through evolutionary processes.Wolbachia occasionally transmits horizontally among arthropods but is primarily transmitted from female hosts to their offspring (Duron et al., 2008;Vavre et al., 1999;Werren et al., 2008).Previous studies have suggested that sister taxa of host species harbour closely related Wolbachia (Baldo et al., 2006;Kageyama et al., 2004;Watanabe et al., 2011;Watanabe et al., 2012).For instance, closely related MK-inducing Wolbachia strains are conserved among Ostrinia moths (Kageyama et al., 2004;Muro et al., 2023).In this case, an ancestral Wolbachia strain likely acquired MK properties and was descended from its host species.In contrast, closely related Wolbachia strains do not necessarily exhibit similar phenotypes (Arai et al., 2020;Metcalf et al., 2014).The phylogenetic relationship between MK Wolbachia strains and their host is not always concordant, suggesting that Wolbachia independently acquired their MK abilities multiple times (Baldo et al., 2006;Bleidorn & Gerth, 2018;Werren et al., 2008;Zhou et al., 1998).To understand the evolution of an MK Wolbachia strain, it is critical to determine whether MK Wolbachia descended from the ancestral host species or whether Wolbachia acquired an MK ability independently in the host.
Here, to clarify the evolutionary processes through which a Wolbachia strain wHm-t acquired MK abilities in the oriental tea tortrix moth Homona magnanima (Tortricidae, Lepidoptera), we report the comprehensive analysis of Wolbachia infections and phenotypes in Tortricidae, including H. magnanima and 61 tortricid species, collected from 39 populations across Japan, Taiwan, Vietnam and Indonesia.We previously reported that H. magnanima was co-infected with three Wolbachia strains in several Japanese populations (Arai et al., 2019;Takamatsu et al., 2021).Among them, wHm-a exhibited no apparent effects on the host; wHm-b induced CI that impaired the development of offspring of infected males and non-infected females, while wHm-c promoted host fecundity by increasing the pupal weight of the hosts (Arai et al., 2019;Ueda et al., 2023).In contrast, Taiwanese H. magnanima harbours an MK strain, wHm-t, which is closely related to the non-MK strain wHm-c (Arai et al., 2020).More recently, we identified an MK-associated prophage region that is present in wHm-t but absent in wHm-c (Arai et al., 2023).In the current study, we report (i) the conserved infections of Wolbachia wHm-a, wHm-b and wHm-c relatives among Asian tortricids and (ii) the restricted distribution of the MK wHm-t in Taiwanese H. magnanima.Our investigations provide insights into the evolutionary processes by which a non-MK Wolbachia acquired the MK ability and how Wolbachia strains have persisted in their host populations.

Insects
H. magnanima and other tortricids were collected from damaged tea leaves by hand or using UV light and sex pheromones (Sumitomo Chemical Co., Ltd., Tokyo, Japan) in Japan, Vietnam and Indonesia.Taiwanese H. magnanima were previously obtained by Arai et al. (2020).The species name of the collected tortricids was determined based on morphological characters described in Kishida (2011), Tortricid.net(http://www.tortricidae.com/identification.asp), and BOLD database (https://www.boldsystems.org/index.php/TaxBrowser_Home).Samples were stocked in ethanol at À35 C until analysis.
Larvae were reared individually until eclosion with an artificial diet INSECTA LF (Nosan Co. Ltd., Yokohama, Japan) at room temperature (20-26 C) under 14-16 L:8-10D conditions.Adults were mated in a plastic bag (20 Â 30 cm), and wax paper was placed to obtain egg masses.A collected egg mass was placed on sliced INSECTA LF in a plastic container (20 Â 30 Â 10 cm) for mass rearing.The sex ratio ([number of males/number of females] among enclosed adults) was determined.
PCR products were purified using the Qiaquick PCR purification kit (Qiagen, Hilden, Germany) and sequenced as described by Arai et al. (2019).Purified wsp amplicons of Wolbachia in Homona species were ligated with the pGEM-T easy vector (Promega, Madison, WI, USA) and transfected into JM109 competent cells.Plasmid DNAs extracted from eight JM109 clones using the Pure Yield Plasmid Miniprep System (Promega, Madison, WI, USA) were sequenced as described by Arai et al. (2019).

Constructions of the mitochondrial genome of H. magnanima
The mitochondrial genome of a tortricid, Choristoneura fumiferana (accession number: NC_037395.1),retrieved from the NCBI database, was used as a reference to identify mitochondrial reads from sequence reads of H. magnanima (DRA013595, BioProject: PRJDB13119) using BLASTn search.A complete mitochondrial genome was constructed through contig alignments and read mappings using minimap2 (Li, 2018), followed by manual cyclisation.Resequencing data of H. magnanima obtained from Japanese and Taiwanese populations (DRA013650, BioProject: PRJDB13119) were mapped to the mitochondrial genome of H. magnanima using minimap2, and consensus sequences were obtained using SAMtools (Li et al., 2009).

Phylogenetic analysis
Newly sequenced wsp fragments (Wolbachia) and mitochondrial genomes (H.magnanima) and COI fragments (tortricids) were aligned using ClustalW (Thompson et al., 1994).Wolbachia wsp sequences of Baldo et al. (2006) were retrieved to construct a Wolbachia phylogenetic tree.For phylogenetic analysis, the maximum likelihood method with bootstrap resampling of 1000 replications was performed in MEGA 7 (Kumar et al., 2016).Haplotype network analysis was conducted on mitochondrial COI sequences of H. magnanima using DnaSP6 (Rozas et al., 2017) and Network (Bandelt et al., 1999).

Statistical analysis
To examine whether Wolbachia infections correlate with host sex, GLMM was performed using the lme4 package (Bates et al., 2015) in R 4.0.0,assuming binomial error based on the presence/absence of the respective endosymbionts within an individual H. magnanima as shown by Takamatsu et al. (2021).In statistical modelling, we considered the infection status (i.e., Wolbachia presence or absence) as a fixed factor.Based on the GLMM, an analysis of variance (ANOVA) was conducted to evaluate the significance of the individual model terms.

RESULTS
Asian tortricids generally harboured wHm-a, wHm-b and wHm-c relatives but lacked a female-biased sex ratio We collected more than 1300 tortricid samples, which were classified based on morphological characters described by Kishida (2011) and mitochondrial COI sequences, if available, in the BOLD database.In addition to H. magnanima (991 individuals), 61 tortricid species (357 individuals) were identified, although we were not able to clarify the species' name in some samples (e.g., Archips sp. 1, Table S1).Among them, 58 species were positive for Wolbachia (with 50-100% frequency, Table S1).Of these, 48, 45 and 54 were positive for wHm-a, wHm-b and wHm-c, respectively (Table S1).Similar to H. magnanima (Takamatsu et al., 2021), triple infections predominated (33 species), followed by dual infections with wHm-a and wHm-c (16 species), wHm-a and wHm-b (4 species) and wHm-b and wHm-c (8 species).Although no apparent topological concordance was observed between the Wolbachia wsp phylogeny and the tortricid COI phylogeny, many of the Wolbachia strains in tortricids shared highly homologous wsp sequences with those of either wHm-a, wHm-b or wHm-c in H. magnanima (Figure 1).The genus Homona typically harboured wHm-c but frequently lost wHm-a and wHm-b strains (Table S1; Figure 1).Homona coffearia and Homona issikii were co-infected with wHm-c and other Wolbachia strains that were not identified from H. magnanima.Notably, males of 44 tortricid species harboured wHm-c (Table S1), and nine tortricid species harbouring wHm-c type Wolbachia did not exhibit a female-biased sex ratio (Figure 2).To clarify whether the tortricid species harbour wHm-t, we detected the Hm-oscar gene in the MK-associated prophage WOwHm-t76 region that is present in wHm-t but absent in wHm-c (Arai et al., 2023).Apart from the Taiwanese H. magnanima, no tortricid was positive for the Hm-oscar gene (Table S1).
Taiwanese and Japanese H. magnanima harboured distinct mitochondrial haplotypes Among 130 wild-caught individuals of H. magnanima, the mitochondrial COI (599 bp) was polymorphic at six nucleotide sites, forming six haplotypes (Figure 4A).Taiwanese H. magnanima (n = 30) and a male individual collected from Yonaguni Island, which is closer to Taiwan (111 km away) than to Okinawa Island (509 km away), had the haplotype T. Of the remaining five haplotypes, haplotype J1 (n = 71) dominated most of the F I G U R E 1 Phylogeny of Wolbachia strains identified from tortricids.The wsp sequences of Wolbachia strains in tortricids were aligned with those of other Wolbachia strains reported by Baldo et al. (2006).The mitochondrial COI sequences of tortricids were also shown.The phylogenetic tree was constructed using the maximum likelihood method based on the Tamura-Nei model with 1000 bootstrap replicates.

DISCUSSION
Evolutionary processes by which Wolbachia achieved MK in H. magnanima Here we showed that Wolbachia relatives of wHm-a, wHm-b and wHm-c were conserved among Asian tortricids, suggesting that these strains descended from a common tortricid ancestor.We also confirmed that wHm-c relatives in tortricid species were negative for the wHm-t-specific prophage gene and did not induce MK.Apart from H. magnanima, MK in tortricids has only been reported in a severe pear pest Epiphyas postvittana, which is distributed in Australasia (Geier et al., 1978, Figure 5A).Although MK factors in E. postvittana are currently unknown, the different distributions of H. magnanima (East Asia) and E. postvittana (Australia) suggest that they have acquired MK independently.In H. magnanima, Japanese and Taiwanese populations had different mitochondrial genotypes.Furthermore, there was no genetic divergence between the MK and non-MK Taiwanese host lines.These results suggest that the MK trait of wHm-t has emerged relatively recently, at least after the Taiwanese H. magnanima had diverged from the Japanese populations.The triply infected H. magnanima (ancestor of the Taiwanese population) had probably lost wHm-a and wHm-b, as is frequently observed in other species of the genus Homona.Thereafter, we suspect that the ancestral wHm-c type Wolbachia in H. magnanima acquired an MK ability (i.e., the MK-associated prophage region WOwHm-t76; Arai et al., 2023) through a bacteriophage integration, giving rise to the MK-inducing wHm-t (Figure 5B).

Sex-linked infection patterns of non-MK Wolbachia in H. magnanima
We found that wHm-a and wHm-b show distinct infection patterns between male and female H. magnanima.
Both strains possess the CI causative genes cifA and cifB (Arai et al., 2023), although wHm-a does not currently induce CI in contrast to wHm-b (Arai et al., 2019).Similar differences in Wolbachia infection patterns between the sexes have been reported in several insects, such as Diptera (Drosophila pseudotakahashii and Aedes albopictus), Coleoptera (Polygraphus proximus) and Siphonaptera (Synosternus cleopatrae) (Bykov et al., 2020;Cohen et al., 2015;Dobson et al., 2002;Dutton & Sinkins, 2004;Flatau et al., 2018;Richardson et al., 2019;Xi et al., 2005;Xi et al., 2006).For example, females of A. albopictus carry CI-inducing Wolbachia strains wAlbA and wAlbB, but males often lack wAlbA or carry it at very low densities (Dobson et al., 2002;Dutton & Sinkins, 2004;Xi et al., 2005;Xi et al., 2006).Although the evolutionary history of the distinct Wolbachia infection patterns between males and females remains unknown, it may reflect a host response to Wolbachia-induced reproductive manipulations such as CI.CI-inducing Wolbachia causes lethality in offspring when infected males mate with uninfected females (Werren et al., 2008).Furthermore, as observed in the A. albopictus-Wolbachia (wAlbA, wAlbB and wMel) system, multiple infections of CIinducing Wolbachia can reduce host fitness by leading to self-incompatibility (Ant & Sinkins, 2018).Theory suggests that mutation(s) can evolve to suppress the proliferation and/or functions of CI-inducing Wolbachia (Koehncke et al., 2009).If CI-inducing Wolbachia cannot be eliminated from the host, low frequencies of CI-inducing Wolbachia in males and high frequencies in females are assumed to be adaptive for host insects to produce viable offspring.We speculate that H. magnanima has developed mechanisms to control CI-inducing Wolbachia strains through evolutionary processes.Although the underlying mechanisms are unknown, we hypothesise that differences in the male and female environments (e.g., metabolites and/or tissues) regulate Wolbachia-Wolbachia interactions and their proliferation, resulting in distinct infection patterns between the sexes.
Alternatively, this phenomenon may be a strategy of Wolbachia rather than the host.It is possible that Wolbachia adopted a strategy of allocating resources to females rather than males.In D. pseudotakahashii, Wolbachia expresses strong CI (a long-lasting sperm modification effect) at extremely low titers in adult males (Richardson et al., 2019).The different infection patterns of Wolbachia between the sexes may be an evolutionarily acquired strategy of Wolbachia-low levels of Wolbachia being allocated to males only to induce CI, while high levels of Wolbachia to females to secure a stable infection.In H. magnanima, wHm-a and wHm-b may have systems to transmit to female siblings rather than males by recognising female-specific features such as the W chromosome.Further investigation is warranted to elucidate the underlying mechanisms and whether this phenomenon occurs in other host Wolbachia systems.

Formation of the current distribution of Wolbachia and H. magnanima
Wolbachia impacts genetic differentiation and distribution in insects (Kondo et al., 2005;Narita et al., 2007;Miyata et al., 2017;Miyata et al., 2020;Rokas, 2000; F I G U R E 5 Distribution and male killing (MK) acquisition scenarios of Wolbachia in Tortricidae.(A) Overview of Wolbachia infections, distributions, and MK in the Homona species examined in this study.The Wallace line (broken grey line) is a border of organisms between Asian and Australian regions.(B) Evolutionary scenarios of the MK wHm-t.While Wolbachia strains have descended from the ancestral host, wHm-t has evolved from wHm-c through phage infection in H. magnanima.Cytoplasmic incompatibility (CI) may have limited the distribution of wHm-t infected hosts, as crosses between wHm-t infected females (shown as T) and triply-infected males (shown as ABC) are lethal.JPN: Japan; VNM: Vietnam; IDN: Indonesia; TWN: Taiwan.(C) A putative evolutionary process of the Japanese and Taiwanese H. magnanima.Ancient H. magnanima diverged in mainland China, and triple-infected H. magnanima that entered the Japanese archipelago via the Korea-Tsushima route (shown as orange arrows) formed the Japanese population.H. magnanima infected with wHm-t was not expelled in Taiwan (Taoyuan).Turelli & Hoffmann, 1991).It is known that CI traits have led to the rapid spread of Wolbachia in Drosophila simulans, reducing the diversity of mitochondrial haplotypes in infected populations (Hale & Hoffmann, 1990;Ballad et al., 1996).In this study, we showed that a mitochondrial COI haplotype and three Wolbachia strains (wHm-a, wHm-b and wHm-c) were highly conserved among Japanese H. magnanima.We speculate that the CI-inducing wHm-b led to the spread of the triply infected H. magnanima and its mitochondria haplotype across Japan, followed by the emergence of the minor haplotypes in Japanese H. magnanima.In contrast to most Japanese populations, Taiwanese H. magnanima does not harbour wHm-a or wHm-b and has a homogeneous mitochondrial sequence.Notably, a Wolbachia-free male collected from Yonaguni Island had the Taiwanese mitochondrial haplotype.These findings suggest that the Taiwan-Yonaguni and mainland-Okinawa Island populations of H. magnanima have different origins and have been isolated over a long period.We speculate that the triply infected H. magnanima spread from mainland Japan across the Tokara Strait-a geographical boundary located between Yakushima Island and Amami-Oshima Island (Tojo et al., 2017)-to Okinawa Island.The Kerama Strait (Hachisuka Line, Figure 5C) has probably served as the geographical boundary between the H. magnanima populations of Taiwan-Yonaguni and mainland-Okinawa Island.Given that wHm-t does not confer any fitness advantages to H. magnanima (Arai et al., 2020), the wHm-t infected host struggles to coexist with the triply infected host that retains the fitness advantages of wHm-c and the CI traits of wHm-b (Arai et al., 2019).Therefore, wHm-t infected hosts are likely to be eliminated by competition from triply infected hosts when they coexist in the same population.We propose that the triple infection of wHm-a, wHm-b and wHm-c have determined the limited distribution of the MK strain wHm-t.

CONCLUSIONS
Our study demonstrated that Wolbachia relatives of wHm-a, wHm-b and wHm-c are widely conserved among Asian tortricids but do not induce MK in their hosts.Furthermore, Japanese and Taiwanese H. magnanima exhibit genetic divergence and distinct Wolbachia infection patterns.These results suggest that the Wolbachia strains are derived from a common ancestor, whereas the MK wHm-t has evolved from a non-MK wHm-c strain in the ancestral Taiwanese H. magnanima that lacked wHm-a and wHm-b infections.This work is relevant in that we presented the evolutionary process of Wolbachia by examining 62 tortricid species collected from 39 localities across East and Southeast Asia.However, we must note that the phylogeny and infection dynamics of Wolbachia may be more complicated because the genus Tortricidae is a large taxon with more than 10,000 species in diverse environments around the world (http://www.tortricidae.com/catalogue.asp).Further studies on Wolbachia in tortrix moths and other insects should clarify the dynamics of the acquisition process of the MK phenotype.

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I G U R E 2 Sex ratios of tortricid species harbouring Wolbachia strains.Wolbachia infections are shown as A (wHm-a), B (wHm-b), C (wHmc), and T (wHm-t).The sex ratio of each tortricid species is shown with red (female) and blue (male) bars.The numbers of female and male individuals are indicated within parentheses.F I G U R E 3 Distributions and infection status of Wolbachia strains in Japanese and Taiwanese H. magnanima.Pie charts of Wolbachia prevalence in males (A) and females (B).Taiwanese wHm-t strain is shown as a wHm-c type strain.ABC: wHm-a, wHm-b, and wHm-c triple infection; AB: wHm-a and wHm-b dual infection; BC: wHm-b and wHm-c dual infection; CA: wHm-a and wHm-c dual infection; A: wHm-a single infection; B: wHm-b single infection; C: wHm-c single infection; ND: not detected.Wolbachia prevalence data in Shimada and Taiwan were obtained from Takamatsu et al. (2021) and Arai et al. (2020), respectively.

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I G U R E 4 The mitochondrial haplotypes of Homona magnanima.(A) Distributions of the six H. magnanima mitochondrial COI haplotypes.Navy: haplotype J1; blue: haplotype J2; Navy with dots: haplotype J3; Navy with the horizontal line: haplotype J4; Navy with the diagonal line: haplotype J5; Red: haplotype T. Numbers of individuals are shown in parentheses.The H. magnanima COI haplotype network is highlighted by broken lines.(B) Haplotype network and phylogeny of H. magnanima mitochondria genome (16,529 bp).Red circle: Taiwanese individuals; blue circle: Japanese individuals; red letter: MK line; blue letter: non-MK line or male individual.The numbers of Mutated positions are shown as black bars.A phylogenetic tree was constructed using the maximum likelihood method (based on the Hasegawa-Kishino-Yano model) with 1000 bootstrap replicates.A: wHm-a, B: wHm-b, C: wHm-c; T: wHm-t; ND: undetected.