Two complete genomes of male-killing Wolbachia infecting Ostrinia moth species illuminate their evolutionary dynamics and association with hosts

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Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Two complete genomes of male-killing Wolbachia infecting Ostrinia moth species illuminate their evolutionary dynamics and association with hosts Tomohiro Muro , Hiroyuki Hikida , Takeshi Fujii , Takashi Kiuchi , Susumu Katsuma doi: https://doi.org/10.1101/2022.05.08.491107 Tomohiro Muro 1 Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo , Bunkyo-ku, Tokyo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hiroyuki Hikida 1 Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo , Bunkyo-ku, Tokyo, Japan 2 Institute for Chemical Research, Kyoto University , Uji, Kyoto, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Takeshi Fujii 3 Faculty of Agriculture, Setsunan University , Hirakata, Osaka, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Takashi Kiuchi 1 Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo , Bunkyo-ku, Tokyo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Susumu Katsuma 1 Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo , Bunkyo-ku, Tokyo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: skatsuma{at}g.ecc.u-tokyo.ac.jp Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Wolbachia is an extremely widespread endocellular symbiont which causes reproductive manipulation on various arthropod hosts. Male progenies are killed in Wolbachia -infected lineages of the Japanese Ostrinia moth population. While the mechanism of male killing and the evolutionary interaction between host and symbiont are significant concerns for this system, the absence of Wolbachia genomic information has limited approaches to these issues. We determined the complete genome sequences of w Fur and w Sca, the male-killing Wolbachia of O. furnacalis and O. scapulalis . The two genomes shared an extremely high degree of homology, with over 95% of the predicted protein sequences being identical. A comparison of these two genomes revealed nearly minimal genome evolution, with a strong emphasis on the frequent genome rearrangements and the rapid evolution of ankyrin repeat-containing proteins. Additionally, we determined the mitochondrial genomes of both species’ infected lineages and performed phylogenetic analyses to deduce the evolutionary dynamics of Wolbachia infection in the Ostrinia clade. According to the inferred phylogenetic relationship, Wolbachia infection was established in the Ostrinia clade prior to the speciation of related species such as O. furnacalis and O. scapulalis . Simultaneously, the relatively high homology of mitochondrial genomes suggested recent Wolbachia introgression between infected Ostrinia species. The findings of this study collectively shed light on the host-symbiont interaction from an evolutionary standpoint. Significance Despite the growing number of publicly available Wolbachia genome sequences, only a few high-quality male-killer genomes exist, particularly those found in lepidopteran hosts. The complete genomes of two male-killing Wolbachia of Ostrinia moth hosts were determined in this study. The genomic data obtained here will be used to elucidate the mechanism of reproductive manipulation and the origins of this endosymbiont’s extraordinary diversity. Additionally, phylogenetic analysis of mitochondria and Wolbachia revealed the evolutionary history of Ostrinia hosts and Wolbachia . The inferred dynamic pattern of infection adds to our understanding of evolution and ecology of Wolbachia endosymbiont, a promising agent for biological pest control. Introduction Endosymbionts have a variety of effects on host biology. While mutual symbionts are necessary for the growth of their hosts, there are numerous instances in which symbionts drastically impose parasitic phenotypes on their hosts ( Goryacheva & Andrianov 2021 ). Wolbachia is one of these bacteria, notable for its widespread distribution among insects ( Weinert et al. 2015 ). Wolbachia infection has been reported in a wide variety of insect lineages, as well as some other terrestrial arthropods and filarial nematodes. Wolbachia strains are classified into at least 17 supergroups, in which Supergroups A and B contain a majority of strains found in arthropod taxa ( Kaur et al. 2021 ). In many associations between Wolbachia and its hosts, Wolbachia induces reproductive manipulation on the hosts, which typically results in infected females having a higher fitness level than uninfected females ( Goryacheva & Andrianov 2021 ; Kaur et al. 2021 ). Due to the vertical transmission of Wolbachia from mothers to their offspring, such manipulation is thought to spread Wolbachia infection throughout the host population. Wolbachia has four modes of reproductive manipulation: cytoplasmic incompatibility, feminization of genetic males, induction of parthenogenesis, and male killing ( Kaur et al. 2021 ). Male killing is the process by which male offspring of infected mothers are killed during embryonic development or at a later stage. Although male killing has been observed in a variety of bacterial parasites, including at least four different groups of bacteria ( Goryacheva & Andrianov 2021 ), Wolbachia -induced male killing has ever been reported for relatively limited groups, including Drosophila flies ( Dyer & Jaenike 2004 ; Hurst et al. 2000 ; Sheeley & McAllister 2009 ), ladybugs ( Elnagdy et al. 2013 ; Hurst et al. 1999 ) and some lepidopteran insects such as Acraea species ( Hurst et al. 1999 ; Jiggins et al. 1998 , 2000 ), Hypolimnas bolina ( Dyson et al. 2002 ), Homona magnanima ( Arai et al. 2020 ), and Ostrinia species ( Fukui et al. 2015 ; Kageyama & Traut 2004 ). Several agricultural pests are found in the genus Ostrinia , which is a member of the Crambidae family of Lepidoptera. Historically, the genus was largely classified into three groups and are denoted by the letters I, II, and III, according to the morphology of male genitalia ( Mutuura & Munroe 1970 ). However, a recent phylogenomic study confirmed the monophyly of only group III and proposed a new classification system in which the genus is divided into three new species groups (Clade I, II, and III) ( Yang et al. 2021 ). All species of group III ( Mutuura & Munroe, 1970 ) were incorporated into Clade III, the Ostrinia nubilalis species group, along with some previously classified species in group II. Both O. furnacalis (Asian corn borer) and O. scapulalis (Adzuki bean borer), classified as group III or the new Clade III, are important agricultural pests. O. furnacalis is a significant maize pest in Asia, causing substantial economic damage, whereas O. scapulalis attacks some crops such as hops, hemp and legumes ( Ishikawa et al. 1999 ; Yang et al. 2021 ). Wolbachia causes male killing in Japanese populations of both O. furnacalis and O. scapulalis ( Fukui et al. 2015 ; Kageyama & Traut 2004 ). The infection rate of Wolbachia in O. furnacalis is comparatively low: a previous survey reported that 13 individuals out of 79 collected females were infected ( Kageyama et al. 2002 ). The striking feature of infected strains of Ostrinia moths is that when the infection is cured with antibiotics, cured females produce all-male broods due to female-specific death ( Kageyama & Traut 2004 ). This is in contrast to the situation with other male-killing Wolbachia , where antibiotic treatment restores a balanced sex ratio encompassing both sexes, as observed in Acraea butterflies ( Jiggins et al. 1998 , 2000 ), Hypolimnas bolina ( Charlat et al. 2007 ), and Homona magnanima ( Arai et al. 2020 ). When Wolbachia is present in O . furnacalis , the expression of Ostrinia furnacalis Masculinizer gene ( OfMasc ) is downregulated at the embryonic stage ( Fukui et al. 2015 ). OfMasc is an ortholog of Masculinizer gene, which was first discovered in the silkworm Bombyx mori and demonstrated to be required for both masculinization and dosage compensation ( Kiuchi et al. 2014 ). OfMasc is thought to have masculinizing activity in O. furnacalis because knockdown of OfMasc results in the expression of female-type splicing variants of Ostrinia furnacalis doublesex ( Ofdsx ) in male embryos ( Fukui et al. 2018 ). Males infected with Wolbachia were rescued from male killing by the injection of OfMasc cRNA, and the expression of Z-linked genes was increased in Wolbachia -infected embryos ( Fukui et al. 2015 ). Therefore, the depletion of OfMasc , which probably results in the failure of dosage compensation, may be the underlying mechanism of male-specific death in Wolbachia infection. Female-specific death in antibiotic treatment is also attributed to the aberrant expression of sex chromosome-linked genes ( Sugimoto et al. 2015 ). These studies highlight the Wolbachia ’s ingenious strategy for reproductive manipulation of Ostrinia hosts, generating increased interests in the evolutionary context of their association. Genomic information is a critical resource for characterizing host-symbiont interactions. Recent advances in sequencing technology have resulted in the accumulation of Wolbachia genome sequences, which have aided in the identification of factors responsible for reproductive manipulation ( Le Page et al. 2017 ; Perlmutter et al. 2019 ) as well as the characterization of host-symbiont association dynamics at an ecological scale ( Cooper et al. 2019 ; Turelli et al. 2018 ; Wolfe et al. 2021 ). In order to obtain reliable insights, high-quality genome sequences from a diverse set of Wolbachia strains representing both phenotypic and phylogenetic diversity are required. However, there are a few genome available for male-killing Wolbachia ( Duplouy et al. 2013 ; Hill et al. 2021 ; Metcalf et al. 2014 ). One is the complete genome sequence of w Inn strain (Supergroup A), a male-killer of Drosophila innubila ( Hill et al. 2021 ). The other two are draft genome assemblies. w Rec strain (Supergroup A), which causes cytoplasmic incompatibility in its native host D. recens and male killing in an introgressed host D. subquinaria , has the genome with reduced prophage regions compared to its close relative w Mel strain ( Metcalf et al. 2014 ). The only Lepidoptera-associated male-killing strain with a draft genome is w Bol1-b, a Supergroup B strain that infects H. bolina butterfly. The w Bol1-b genome assembly comprised 144 contigs, 91 of which were organized into 13 scaffolds ( Duplouy et al. 2013 ). Along with the scarcity of genomic data on male-killing Wolbachia , the number of Wolbachia genome assemblies infecting Lepidopteran hosts is also limited. Indeed, no complete genomes of Wolbachia associated with Lepidoptera have been published, although a few are available in public databases. Despite the low number of Lepidoptera-associated Wolbachia genomes, a significant number of Wolbachia strains are present in Lepidoptera, presumably at a higher rate than in other arthropods ( Ahmed, Araujo-Jnr, et al. 2015 ; Ilinsky & Kosterin 2017 ). Mitochondrial genomes are also useful for deciphering host-symbiont associations. Since both inherited symbionts and mitochondria are transmitted maternally in infected lineages, examining mitochondrial haplotypes and Wolbachia infection status enables us to infer the mode of Wolbachia acquisition among allied species ( Baldo et al. 2008 ; Charlat et al. 2009 ; Cooper et al. 2019 ; Sucháčková Bartoňová et al. 2021 ). Specifically, there are three distinct ways in which Wolbachia can be acquired by host species: cladogenic acquisition, introgression, and horizontal transmission ( Cooper et al. 2019 ; Raychoudhury et al. 2009 ; Turelli et al. 2018 ). Cladogenic acquisition occurs when related species codiverged (along cladogenesis) with Wolbachia derived from a common ancestor that was previously infected. Alternatively, Wolbachia infection can be introduced into a species via hybridization with infected sibling species and subsequent backcrossing. This is referred to as introgression. The relatively low level of genetic divergence of mitochondria and Wolbachia between host species can serve as an indicator in this case ( Meany et al. 2019 ; Raychoudhury et al. 2009 ). The third mode is horizontal transmission, which typically results in discordance of phylogenetic relationship between Wolbachia and the host’s mitochondria. Here, we report the complete genome sequences of male-killing Wolbachia infecting O. furnacalis and O. scapulalis (referred to as w Fur and w Sca, respectively), as well as the mitochondrial genomes of both hosts of infected lines. The comparison of these male-killing Wolbachia genomes revealed a trend in Wolbachia genome evolution. Along with mitochondrial genome analysis, the obtained genomic data enabled us to infer the evolutionary history of the Wolbachia infection in Ostrinia moths. Materials & Methods Insects w Fur-infected O. furnacalis and w Sca-infected O. scapulalis were used in this study. An O. furnacalis line infected with w Fur was previously described ( Fukui et al. 2015 ) and is maintained in our laboratory. w Sca-infected O. scapulalis line was newly established by collecting an infected female moth in Matsudo, Japan (35.8° N, 139.9° E) in September 2020. Sex pheromone analysis was used to determine the species of collected Ostrinia females. Pheromone glands (PG) of three females from each matriline were immersed in hexane to extract sex pheromone. The PG extracts were analyzed using a GC-MS unit (QP2010 SE GC- MS, Shimadzu) equipped with a capillary column (DB-Wax, 0.25 mm i.d. × 30 m; Agilent Technologies, Santa Clara, CA, USA). The initial column oven temperature of 80°C was held for 2 min and then increased at 8°C/min to 240°C. The final temperature was maintained for 2 min. The flow rate of the helium carrier gas was 1.0 mL/min. The diagnostic ion of the acetate ion at 61 ( m / z ) and its eliminated formation at 194 ( m / z ) were used for GC-MS analysis, in addition to the retention time. Both O. furnacalis and O. scapulalis lines were reared on an artificial diet (Insecta-LFS, Nosan Corp.) at 25°C under a photoperiod of 16L8D and maintained by crossing with males of Wolbachia -uninfected counterpart lines derived from females collected in Nishi-Tokyo, Japan (35.7° N, 139.5° E). The Infection was treated by adding 0.3 g tetracycline-HCl to a 500 g artificial diet. Reverse transcription-polymerase chain reaction (RT-PCR) RT-PCR was used to examine the sex-specific splicing patterns of O. scapulalis doublesex ( Osdsx ). Egg masses (96 hours post oviposition; hpo) from infected, uninfected, and cured lines were sampled. Total RNA and genomic DNA were prepared simultaneously from the egg masses using TRI Reagent (Molecular Research Center, Inc.) according to the manufacturer’s protocol. Total RNA was subjected to reverse transcription using AMV-RTase and an oligo-dT primer (TaKaRa). The resultant cDNA was used to perform PCR to examine the sex-specific splicing patterns of Osdsx . Genomic PCR of Wolbachia -specific wsp gene was used to examine the infection status. PCR was performed with KOD FX Neo (TOYOBO) for Osdsx and KOD One (TOYOBO) for wsp . Primer sequences were reported previously ( Sugimoto et al. 2010 ). Molecular sexing Ostrinia larvae were sexed using a previously reported method for O. furnacalis ( Fukui et al. 2015 ). Briefly, genomic DNA was purified using DNeasy Blood & Tissue Kit (QIAGEN), and the dosage of Z-linked genes ( Tpi and kettin ) normalized by the autosome-linked gene ( EF-1α ) was evaluated by genomic qPCR. Individual larvae were photographed prior to DNA extraction, and body lengths were measured using ImageJ software. Genome sequencing and assembly Genomic DNA was purified from the ovaries of w Fur-infected O. furnacalis and w Sca-infected O. scapulalis females using Genomic-tip 100/G (QIAGEN) according to the manufacturer’s protocol. Prepared DNA samples were subjected to genome sequencing on NovaSeq 6000 (Illumina) and Sequel (PacBio) platforms. For O. furnacalis , two DNA samples were prepared each from an infected female. One of them was used for Illumina sequencing, and subsequently the remainder was combined with the other sample for PacBio (Sequel system) sequencing. For O. scapulalis , one and four ovaries were used to prepare DNA samples for Illumina and PacBio (Sequel II system) sequencing, respectively. Long-reads obtained by PacBio sequencing were assembled using Canu v2.1 assembler ( Koren et al. 2017 ). An estimated genome size of 438.5 Mb was given as a parameter with reference to the deposited genome assembly of O. furnacalis (accession number: GCA_004193835.1). Construction of Wolbachia genomes Both w Fur and w Sca genomes were constructed through a following procedure. To identify contigs derived from Wolbachia in Canu assembly, the generated contigs were subjected to blastn search as a query against RefSeq representative prokaryotic genomes database (ref_prok_rep_genomes) with the following parameters: “evalue 1e-20”, “max_target_seqs 60”, and “max_hsps 10”. Illumina short-reads were mapped to the draft Wolbachia contig with BWA-MEM v.0.7.17 ( Li 2013 ), and the resultant SAM file was converted to BAM format using samtools v.1.9 ( Danecek et al. 2021 ). Pilon v.1.23 ( Walker et al. 2014 ) was utilized to polish the draft Wolbachia contig. In order to locate the overlap region at the beginning and end of the sequence, a blastn search was performed using the same polished contig as both a subject and a query. The overlap region was manually modified to construct a single-coverage genome (see Results). The oriC regions of w Fur and w Sca were identified by performing a homology search against the oriC sequence of w Mel strain. The oriC sequence of w Mel strain (DoriC ID: ORI10030016) was retrieved from the DoriC 5.0 database ( Gao et al. 2013 ). The binding sites in the oriC sequence were searched using the same criteria as previously described ( Ioannidis et al. 2007 ). For genome annotation, stand-alone PGAP v5.2 (2021-05-19.build5429) ( Tatusova et al. 2016 ) was utilized. Construction of mitochondrial genomes By performing a blastn search on mitochondrial gene sequences in the Canu assembly of w Fur- infected O. furnacalis , a contig corresponding to a mitochondrial genome (mitogenome) was identified. Publicly available COI (Gene ID 65331444), COII (65331445), and ND5 (65331454) sequences from an O. furnacalis mitogenome (accession number: NC_056248.1 ) were used as queries. Illumina short-reads were mapped to the identified mitogenome contig with BWA-MEM v.0.7.17 ( Li 2013 ). Visual inspection of mapped reads was performed using IGV v.2.8.7 ( Thorvaldsdóttir et al. 2013 ), and overlaps were manually modified to construct a single-coverage mitogenome. Subsequently, Illumina short-reads from w Sca-infected O. scapulalis were mapped to the constructed mitogenome of w Fur-infected O. furnacalis with BWA-MEM v.0.7.17 and visualized using IGV v.2.8.7. Detected mutations were manually modified to construct a mitogenome of w Sca-infected O. scapulalis . Genome analysis Some Wolbachia genes were searched in the w Fur and w Sca genomes using tblastn. The deduced amino acid sequences of w Mel strain cifA (AAS14330.1), cifB (AAS14331.1), wmk (AAS14326.1), and TomO (AAS14922.1) were downloaded from GenBank and used as queries. To calculate the average nucleotide identity (ANI) between Wolbachia or mitochondrial genomes, FastANI v1.33 ( Jain et al. 2018 ) was used. In order to conduct syntenic analysis, the w Fur and w Sca genomes were aligned using MUMmer v4.0.0rc1 ( Marçais et al. 2018 ) in the “nucmer” mode, and a dot plot was generated with the packaged program, mummerplot. A comparison of the gene repertoires of w Fur and w Sca was performed. To obtain non-redundant sequences for each strain’s annotated protein sequences, duplicated identical sequences were removed using SeqKit ( Shen et al. 2016 ) in “rmdup” mode. Reciprocal blastp searches were used to identify orthologous gene pairs between the non-redundant w Fur and w Sca protein sequences. The hmmscan program was used in conjunction with HmmerWeb v.2.41.2 ( Potter et al. 2018 ) to conduct a domain search against the Pfam 35.0 database using an E-value threshold of 0.05. Phylogenetic analysis For Wolbachia , annotated protein sequences derived from 136 Wolbachia genome assemblies found in NCBI RefSeq as of December 2021 were downloaded. Additionally, two complete genome assemblies included in the analysis were not registered in RefSeq but were found in GenBank (GCA_000953315 and GCA_020995475). BUSCO v.5.2.2 ( Simão et al. 2015 ) was used to determine the completeness of each genome assembly using rickettsiales_odb10. Two assemblies (GCF_000174095.1 and GCF_000167475.1) with low complete BUSCO percentage were excluded from the subsequent analysis. Using OrthoFinder v2.5.4 ( Emms & Kelly 2018 ) with the following options: “-M msa” and “-os”, single-copy orthologs were identified across the aforementioned assemblies and the w Fur and w Sca genomes (140 in total; Supplementary table S1 ). Each of the 63 predicted single-copy orthologous groups was aligned using MAFFT v.7.490 ( Katoh & Standley 2013 ) with default parameters, and the resultant alignments were trimmed using trimAl v.1.4.1 ( Capella-Gutiérrez et al. 2009 ) in the “automated1” mode. The trimmed alignments for each strain were concatenated using SeqKit v2.1.0 ( Shen et al. 2016 ), and were used for phylogenetic analysis. View this table: View inline View popup Supplementary table S1. The list of Wolbachia genome assemblies used in this study. The column “Strain name2” indicates special notes on strain names of Wolbachia provided in each Genbank entry. View this table: View inline View popup Download powerpoint Supplementary table S2. The list of loci which exhibit homology with host-manipulating Wolbachia genes in w Fur and w Sca genomes. Gene loci with E value < 1E-05 (except for wmk) in the results of tblastn search are shown. For wmk, a threshold is set as 1E-40 due to many fragmented hits. The columns “nt length” show the length of the loci (not the alignment length of tblastn). View this table: View inline View popup Download powerpoint Supplementary table S3. The list of protein sequences which do not have identical counterparts between w Fur and w Sca. For one strain-specific sequences, the subject sequences of BLASTp searc are described. We assigned “low similarity in the other’s genome” when E value is bigger than 1E-10. View this table: View inline View popup Download powerpoint Table 1. General characteristics of w Fur, w Sca, and representative Wolbachia genomes. A maximum likelihood (ML) tree was constructed using IQ-TREE v.1.6.12 ( Nguyen et al. 2015 ), and node support was estimated with 1000 ultrafast bootstrap replicates ( Hoang et al. 2018 ). ModelFinder ( Kalyaanamoorthy et al. 2017 ) was used to determine the best-fitting substitution model, and the HIVw+F+R4 model was chosen. Additionally, we conducted phylogenetic analysis using only Wolbachia strains belonging to Supergroup B. Wolbachia genomes belonging to Supergroup B were chosen based on the constructed ML tree of all genome assemblies, and subjected to the following analysis. w Mel (GCF_000008025.1) and w Ri (GCF_000022285.1) genomes (Supergroup A) were also included as outgroups. The phylogenetic analysis was performed in the manner described previously. A total of 339 orthologs were used, with the HIVw+F+R3 model being the best-fitting. In order to conduct phylogenetic analysis on mitochondrial genomes, we used publicly available sequences from GenBank (accession numbers are shown in Fig. 6 ). Mitogenomes were annotated de novo using MitoZ v2.4 ( Meng et al. 2019 ) in the “annotate” mode with the following parameters: “--genetic_code 5” and “--clade Arthropoda”. Phylogenetic trees were constructed using the amino acid sequences of 13 protein-coding genes (PCGs) and the nucleotide sequences of 13 PCGs, rRNAs, and tRNAs. In both cases, ML trees were constructed using a partitioned model with partitioning scheme selection (“-m MFP+MERGE” option) in IQ-TREE v.1.6.12 ( Chernomor et al. 2016 ). Alignments of each deduced amino acid sequence were generated using MAFFT v.7.471, trimmed with trimAl v1.4.rev22, and subsequently concatenated. Each protein was assigned a partition. For nucleotide sequences, mitogenomes were aligned directly using MAFFT v.7.471, and partitions for each gene were defined based on annotated positions in a single strain (MN793323.1). The optimal partition scheme and substitution model for each partition were selected based on Bayesian information criterion. Results 1. Characterization of w Sca-induced male killing in O. scapulalis We used w Fur-infected O. furnacalis ( Fukui et al. 2015 ) and w Sca-infected O. scapulalis lines for genome sequencing. A w Sca-infected O. scapulalis line was newly established from a field- collected female moth in this study. We used GC-MS to identify species using hexane extract of pheromone glands. The extract of the collected line contained (E)-11- and (Z)-11- tetradecenyl acetates (retention times were 16.87 min and 16.99 min, respectively), confirming that it is O. scapulalis ( Ishikawa et al. 1999 ). This w Sca-infected line exhibited a nearly entirely female-biased sex ratio through at least 11 generations, indicating the presence of male killing ( Fig. 1A and B ). Antibiotics treatment of infection resulted in the occurrence of only male progeny. In order to ascertain the period during which males are killed in w Sca-infected O. scapulalis , molecular sexing using qPCR was performed on 4- and 14-days post-hatch (dph) larvae. As a result, both females and males were detected in 4 dph larvae, but only females were observed in 14 dph larvae ( Fig. 1C ). In addition, w Sca-infected males were significantly smaller than females in 4 dph larvae, unlike uninfected larvae, which showed no difference in body length between sexes ( Fig. 1D ). The splicing pattern of Osdsx , which represents the phenotype of sexual differentiation, was female in the infected line, male in the cured progeny, and both female and male in the uninfected line ( Fig. 1E ). These observations demonstrate that w Sca disrupts the host’s sex determination cascade from embryogenesis, and that w Sca-infected males die during the larval stage of O. scapulalis . Download figure Open in new tab Fig. 1. Characterization of male killing in w Sca-infected O. scapulalis . (A) Brood sex ratios in the w Sca-infected matriline. The female:male ratio of each mating is shown. Tetracycline treatment was conducted to remove w Sca from a subpopulation of the second generation. Sexing was conducted based on the morphology of the pupal abdominal tips. (B) An adult female moth infected with w Sca. Bar, 10 mm. (C) The sex ratios of w Sca-infected larvae at 4 and 14 dph. The number indicates the sample size of each group. (D) The body length of w Sca- infected and uninfected larvae at 4 dph. Data presented are mean ± standard error of the mean. Dot plots show all data points individually. An asterisk denotes statistical significance ( P 0.1; Wilcoxon rank-sum test). (E) Splicing patterns of Osdsx in uninfected, w Sca-infected, and infection-cured embryos at 96 hpo. The results of technical duplicates of the RT-PCR assay are presented for each sample. The numbers indicate individual samples (biological duplicates for each condition). Adult female and male moths were used as positive controls. The letters F and M indicate female- and male-type splicing variants, respectively. AF: adult female; AM: adult male; NT: no template. 2. The construction of the w Fur and w Sca genomes We conducted both long-read (PacBio) and short-read (Illumina) sequencing. First, long-reads were assembled by Canu assembler ( Koren et al. 2017 ). The resultant assembly of O. furnacalis and O. scapulalis contained 4,890 and 3,535 contigs, respectively. For each assembly of O. furnacalis and O. scapulalis , the blastn search using each contig as a query resulted in only one contig that frequently aligned to publicly available Wolbachia sequences. Given that both of these two contigs were 1.3–1.4 Mbp in length, which is within the range of typical Wolbachia genome sizes ( Kaur et al. 2021 ), and their coverage was among the highest in each assembly (Supplementary Fig. S1), these two contigs were most likely the genomes of w Fur and w Sca. The polishing process using Illumina reads by Pilon ( Walker et al. 2014 ) identified a few errors in initial contigs (2 changes for w Fur and no changes for w Sca; all of them were numbers of the same sequential nucleotides, e.g., AAAAA to AAAAAA). Since the polished genomes had an overlap region of approximately 50 kb ( w Fur) and 80 kb ( w Sca) at the head and tail of the contigs, duplicated sequences were removed to make the genomes single coverage. When there was inconsistency between the head and tail overlapping regions, we consulted the mapped short-reads to determine which one should be adopted. Due to the circular nature of the Wolbachia genome, we manually set the starting points of the sequences to the estimated oriC regions. The oriC region was identified using a blastn search with the w Mel oriC sequence as a query. w Fur and w Sca had identical estimated oriC sequences (405 bp). It contained three DnaA-, four CtrA-, and two IHF-binding sites, and was flanked by hemE and CBS domain protein genes, all of which are characteristic of Wolbachia ( Ioannidis et al. 2007 ). BUSCO program ( Simão et al. 2015 ) was used to assess the completeness of the genomes. This program was routinely used to determine the presence/absence of benchmarking single-copy orthologs expected to exist among specific taxa. The BUSCO profiles of the w Fur and w Sca genomes against the Rickettsiales database were identical, with 362/364 single-copy orthologs detected, comparable to the profiles of complete Wolbachia genome assemblies ( Table 1 and Supplementary Fig. S2). Thus, we ascertained that our assemblies are of sufficient quality to conduct further analyses. 3. The properties of the w Fur and w Sca genomes The genome of w Fur and w Sca are 1,321,828 and 1,320,340 bp in length, respectively ( Fig. 2 ). The w Fur genome contained 1,040 protein-coding genes, 158 pseudogenes, 34 tRNAs, 3 rRNAs, and 4 ncRNAs, while the w Sca genome contained 1,050 protein-coding genes, 153 pseudogenes, 34 tRNAs, 3 rRNAs, and 4 ncRNAs. Some of host-manipulating Wolbachia genes were screened individually (Supplementary table S2). Each of the two genomes encodes a putatively pseudogenized copy of cifA , one of the two factors responsible for cytoplasmic incompatibility ( Le Page et al. 2017 ). Regarding cifB , one gene shares partial homology with the w Mel cifB sequence, but there do not appear to be any full-length copies. Two intact copies of wmk , a candidate gene for male killing ( Perlmutter et al. 2019 ), were found in each of the two genomes. Three genes (including two putative pseudogenes) share homology with TomO gene, a growth-inhibitory and RNA-interacting factor of w Mel ( Ote et al. 2016 ). However, two of them might be a single ORF misinferred during the automated annotation process. Download figure Open in new tab Fig. 2. Circular map of the w Fur and w Sca genomes. Circles arranged in order from outer to inner indicate the following: The locations of annotated coding sequences (CDS) on the positive (outermost) and the negative (second outermost) strands, the position of annotated RNAs, GC content, and GC skew. The GC content and GC skew are calculated in 10 kb windows and expressed as deviations from an average of the whole sequence. Subsequently, phylogenetic analysis using 138 Wolbachia genome assemblies was performed to ascertain the phylogenetic relationship of w Fur and w Sca within Wolbachia ( Fig. 3 and Supplementary Fig. S3). As a result, w Fur and w Sca were assigned to Supergroup B collectively. The most closely related strain was that which infects the Noctuid moth, Spodoptera picta . Notably, the second closest strain was w Tpre, which is found in a parasitoid wasp Trichogramma pretiosum and is responsible for parthenogenesis ( Lindsey et al. 2016 ). Download figure Open in new tab Fig. 3. Phylogenetic relationship of Supergroup B Wolbachia genomes. The maximum likelihood tree constructed from concatenated protein sequences of 339 single-copy orthologs is shown. w Mel and w Ri (Supergroup A) were used as outgroups. The strain names and their hosts are labeled. If no suitable strain name is available, it is denoted by “NA”. Clades composed of almost the same strains are collapsed, and the number of contained strains is labeled. Branch support calculated using 1000 replicates of ultrafast bootstrap is shown on the nodes. The sequences determined in this study are highlighted in red and bold, while strains associated with other lepidopterans are in blue. Intriguingly, the w Fur and w Sca strains did not cluster phylogenetically with the w Bol1-b strain, which induces male killing in the H. bolina butterfly ( Duplouy et al. 2013 ). 4. Comparative analysis of the w Fur and w Sca genomes The ANI calculated by fastANI program ( Jain et al. 2018 ) was 99.9755%, indicating extremely high similarity between these two Wolbachia . At the genome structure level, however, we found several large inversions between them ( Fig. 4 ). To compare gene repertoires, we first removed duplicated, identical protein sequences from each genome. We then used blastp to compare the strains’ unique sequences (referred to as “non-redundant sequences”). As a result, 976 out of 982 non-redundant w Fur sequences have reciprocal best hit counterparts in 993 non-redundant w Sca sequences. Out of these 976 sequences, 952 were identical between two strains. The remaining 24 sequences differed between the strains to a varying degree; whereas 18 have a single amino acid substitution, one of the remaining six had 24 amino acid substitutions and seven gaps (Supplementary table S3). Most of the 6 w Fur- and 17 w Sca-specific sequences were hypothetical proteins or transposase variants (Supplementary table S3). However, w Sca uniquely has three ankyrin repeat-containing proteins. Download figure Open in new tab Fig. 4. Synteny conservation between the w Fur and w Sca genomes. Dots and lines represent the alignments generated by nucmer program. Forward matches are shown in red, while reverse matches are shown in blue. 5. Analysis of the mitochondrial genome The genome assemblies included information about both the hosts and Wolbachia . Mitochondria, in particular, are maternally inherited elements that are descended together with Wolbachia in infected lineages. Therefore, we analyzed mitochondrial genomes to gain insight into an evolutionary perspective of the host and symbiont. The mitochondrial genome of w Fur- infected O. furnacalis was successfully extracted from the assembly using blastn, and constructed as a single-coverage complete contig. In contrast, since the mitochondrial genome of O. scapulalis was not found in the Canu assembly, it was reconstructed by mapping the short- reads to the mitochondrial genome of w Fur-infected O. furnacalis . The resultant mitochondrial genomes are 15,266 bp for w Fur-infected O. furnacalis and 15,249 bp for w Sca-infected O. scapulalis . The ANI between these two mitogenomes was 99.90%, which was greater than the ANI between normal O. furnacalis and O. scapulalis (98.77%–98.78%) ( Fig. 5 ). Between the mitogenomes of w Fur-infected O. furnacalis and w Sca-infected O. scapulalis , we detected 14 SNPs (all of which were transitions; no transversions were detected) and several indels (1 base deletion, 2 base insertion, and 18 base deletion in the O. scapulalis mitochondrial genome compared to that of O. furnacalis , of which the last two were in AT-rich region). At the protein level, we found only two amino acid substitutions in the entire mitochondrial protein sequence. On the other hand, the mitochondrial haplotypes of infected Ostrinia differed substantially from those of uninfected counterparts. For example, 45 substitutions in whole protein sequences deduced from the mitochondrial genomes exist between normal (Genbank accession: MN793323 ) and w Fur-infected O. furnacalis . These findings indicate that Wolbachia -infected Ostrinia species have a distinct mitochondrial haplotype from uninfected individuals, which likely reflects a long period of maternal lineage separation. Download figure Open in new tab Fig. 5. Graphical representation of ANI values for mitochondrial genomes of Ostrinia species. ANI values between 11 mitochondrial genomes of Ostrinia moths are calculated by fastANI. The sequence determined in this study are indicated in bold. Phylogenetic analysis was performed using mitochondrial genomes of related species obtained from NCBI Genbank. Both infected lineages formed a cluster, which was located outside Clade III Ostrinia species including O. furnacalis , O. scapulalis , O. nubilalis , O. zealis and O. kasmirica ( Fig. 6 ). We conducted a phylogenetic analysis using either nucleotide or protein sequences, and found that the topology of resultant ML trees was identical except for the phylogenetic relationship between infected lineages and O. kasmirica and the detailed intraclade relationship within Clade III Ostrinia species (Supplementary Fig. S4). Essentially, the inferred trees were consistent with previous reports ( Gschloessl et al. 2020 ; Luo et al. 2021 ). According to these results, we conclude that the mitochondrial haplotype associated with Wolbachia infection originated in an ancestral population of Clade III Ostrinia species (probably excluding O. kasmirica based on phylogeny estimated by nucleotide sequences; Supplementary Fig. S4). Given that Wolbachia and mitochondria are maternally related, we can infer that Wolbachia infection was established in the ancestral population prior to the speciation in Clade III Ostrinia . Download figure Open in new tab Fig. 6. Phylogenetic relationship of mitochondrial genomes of Ostrinia and allied moth species. The maximum likelihood tree constructed from concatenated sequences of 13 proteins with partitions for each protein is shown. Two pyralid species ( Lista haraldusalis and Ephestia kuehniella ) were used as outgroups. Branch support calculated using 1000 replicates of ultrafast bootstrap is shown on the nodes. The sequence determined in this study are highlighted in red and bold. Discussion We successfully sequenced the genomes of two closely related Wolbachia strains, w Fur and w Sca, which represent the first complete genomes of male-killing Wolbachia in lepidopteran hosts. The two genomes shared remarkably high homology, with over 95% of protein sequences identical. On the other hand, there are substantial differences between the genomes, most notably some large inversions ( Fig. 4 ). It is well established that Wolbachia genomes poorly retain synteny between distant strains, particularly parasitic strains found in arthropod hosts ( Comandatore et al. 2015 ; Newton et al. 2016 ). We observed nearly minimal transition of genome structure in two male-killing Wolbachia from Ostrinia . The w Fur and w Sca genomes encode at least 29 and 32 ankyrin repeat-containing proteins, respectively (confirmed by hmmscan). The ankyrin repeat motif is a 33 amino acid sequence that plays a role in protein-protein interaction. Although ankyrin repeat-containing proteins are found predominantly in eukaryotes, they are also used as effectors by various pathogenic and symbiotic microbes ( Al-Khodor et al. 2010 ). Certain genes encoding ankyrin repeat-containing proteins are found only in one of the two strains, implying that ankyrin repeat-containing proteins evolved rapidly in Wolbachia . The features identified by comparing closely related Wolbachia strains ( w Fur and w Sca), namely rampant genome structure rearrangement and rapid evolution of ankyrin repeat-containing proteins, most likely represent a trend of minute genome evolution in Wolbachia . The phylogenetic analysis identified Wolbachia of S. picta (the lily caterpillar) as the closest relative of w Fur and w Sca ( Fig. 3 ). This implies the presence of Wolbachia host shifts between these lepidopteran hosts in evolutionary time scale. The second most closely related strain was w Tpre, a Wolbachia strain found in the parasitoid wasp T. pretiosum . Given the close ecological relationship between parasitic wasps and their hosts, including moths, the high sequence homology detected among Wolbachia species possibly indicate Wolbachia transmission among lepidopteran insects and their parasites. Some studies showed that horizontal transmission of Wolbachia is possible with the involvement of parasitic wasps ( Ahmed, Li, et al. 2015 ; Huigens et al. 2000 ). Thus, wasps may be a major driver of macro-scale dynamics of Wolbachia , even though interspecies transmission is less well understood. Mitochondrial analysis of Wolbachia -infected lineages revealed an evolutionary history of symbiotic association in the Ostrinia clade. Because mitochondria are inherited maternally, infected lineages’ mitochondrial haplotypes inevitably descended with Wolbachia . Thus, the mitochondrial phylogeny of infected lineages provides information about the time of infection establishment, from which the association of a particular mitochondrial haplotype with Wolbachia began. Particularly in Ostrinia , the removal of Wolbachia results in female-specific death ( Kageyama & Traut 2004 ; also confirmed in this study), which limits the emergence of Wolbachia -free matrilines that retain the original Wolbachia -associated mitochondrial haplotype. This unique feature likely underlies the distinctiveness of mitochondrial phylogeny in infected Ostrinia moths. Therefore, as a result of the phylogenetic tree, we can infer that the infection was established before the speciation of Clade III Ostrinia , which includes O. furnacalis and O. scapulalis . The ANI between mitochondrial haplotypes of w Fur-infected O. furnacalis and w Sca-infected O. scapulalis was greater than that between uninfected counterparts ( Fig. 5 ). This demonstrates that Wolbachia -infected O. furnacalis and O. scapulalis have unusually similar mitochondrial haplotypes compared to their divergence time. Among the possible modes of Wolbachia dynamics ( Cooper et al. 2019 ; Raychoudhury et al. 2009 ), the situation is best explained by the introgressive transmission of Wolbachia . The cross between O. furnacalis and O. scapulalis produces fertile hybrids that exhibit both parents’ sex pheromone types in a laboratory setting ( Sakai et al. 2009 ). Besides, in natural populations in China, presumed hybrid individuals and introgressed genotypes have been detected ( Bourguet et al. 2014 ). These reports demonstrate the incompleteness of reproductive isolation between these two species, which supports the possibility of Wolbachia introgression. We now propose an evolutionary history model of the symbiotic association between Ostrinia species and Wolbachia based on the preceding discussion ( Fig. 7 ): (i) Wolbachia infection was established in the ancestral population prior to Clade III Ostrinia speciation. (ii) Subsequently, Clade III Ostrinia species such as O. furnacalis , O. scapulalis , and others diversified, and at least one species retained Wolbachia infection. (iii) Wolbachia was recently transmitted via introgression between O. furnacalis and O. scapulalis . Download figure Open in new tab Fig. 7. A proposed model for evolutionary history of Wolbachia infection in Ostrinia moths. Each circle represents an assumed individual species in evolutionary time scale. Wolbachia - infected subpopulations are depicted in a dark color. While the outline of an evolutionary scenario was inferred, there are still unanswered questions that should be addressed in future work. First, what is the real origin of Wolbachia in Ostrinia moths? The phylogenetic tree clearly depicts the evolutionary relationship between Wolbachia associated with Ostrinia and other moth- and parasitoid wasp-associated Wolbachia , implying the possibility of interspecies transfer. However, since Wolbachia was estimated to be present in roughly half of all terrestrial arthropods ( Weinert et al. 2015 ), the current analysis contained only a fraction of Wolbachia ’s total diversity. A thorough survey of Wolbachia diversity within the Lepidoptera and wasp clades, as well as possibly other clades, is required to characterize interspecies dynamics of Wolbachia . Ostrinia -associated strains with a different origin than w Fur and w Sca may provide insight into interspecies dynamics. For instance, Wolbachia strains belonging to Supergroup A have been reported from O. furnacalis populations in China ( Li et al. 2013 ). Second, we sought to determine the direction of O. furnacalis and O. scapulalis introgression. Unidirectional mitochondrial introgression from O. furnacalis into O. scapulalis has been observed in Chinese populations ( Bourguet et al. 2014 ). This indicates that the direction’s introgressive Wolbachia transmission was more likely. Given the high degree of homology between w Fur and w Sca or the hosts’ mitogenomes, and the resulting assumption of recent introgression, such transmission and hybridization may frequently occur among Ostrinia species. Indeed, it is unclear whether infected Ostrinia populations are genetically isolated clearly along with classical species such as O. furnacalis and O. scapulalis or they are spread across multiple species with incomplete reproductive isolation. In conclusion, we determined two complete genomes of male-killing Wolbachia in two closely related lepidopteran hosts. These sequences were used to characterize nearly minimal genome evolution of Wolbachia . The mitochondrial genome analysis facilitated our investigation of the evolutionary relationship between Ostrinia moths and Wolbachia . It revealed Wolbachia ’s complex dynamics across multiple hosts, including the infection establishment before allied species diversification and the introgressive transmission following host speciation. The genomic data on male-killing Wolbachia obtained in this study will serve as a foundation for future research on host-symbiont interaction. Author contributions T.M., T.K., and S.K. designed and performed the experiments. T.M. and H.H. performed bioinformatics analysis. T.F. performed sex pheromone analysis. T.M. and S.K. wrote the manuscript with intellectual input from all authors. S.K. supervised the project. Data availability All data are available in the main text or the supplemental materials. The genome sequences of w Fur and w Sca have been deposited in GenBank under accession numbers CP096925 and CP096926 , respectively. Download figure Open in new tab Fig. S1. Coverage distribution of long-read assembly. Dots represent individual contigs in the genome assembly for (A) w Fur-infected O. furnacalis and (B) w Sca-infected O. scapulalis constructed by Canu. Note that a contig corresponding to the mitochondrial genome (shown in blue) is approximately 2.8 times longer than the actual mitochondrial genome due to repetitive assembly of the genome. Download figure Open in new tab Fig. S2. Quality evaluation of Wolbachia genome assemblies. A graphical representation of the BUSCO analysis in protein mode for 140 assemblies is shown. Each assembly is labeled with the identification number found in supplementary table S1 . Download figure Open in new tab Fig. S3. Phylogenetic relationship of 138 Wolbachia genomes. The maximum likelihood tree constructed from concatenated protein sequences of 63 single-copy orthologs is shown. The identification numbers, strain names, and host species are labeled. If no suitable strain name is available, it is denoted by “NA”. Branch support calculated using 1000 replicates of ultrafast bootstrap is shown on the nodes. Download figure Open in new tab Fig. S4. Phylogenetic relationship of mitochondrial genomes of Ostrinia and allied moth species based on nucleotide sequences. The maximum likelihood tree constructed from concatenated nucleotide sequences of 13 protein-coding genes, tRNAs and rRNAs is shown. Two pyralid species ( Lista haraldusalis and Ephestia kuehniella ) were used as outgroups. Branch support calculated using 1000 replicates of ultrafast bootstrap is shown on the nodes. Acknowledgements We thank the Institute for Sustainable Agro-ecosystem Services, The University of Tokyo, for collecting Ostrinia moths. Computations were partially performed on the NIG supercomputer at ROIS National Institute of Genetics. 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Share Two complete genomes of male-killing Wolbachia infecting Ostrinia moth species illuminate their evolutionary dynamics and association with hosts Tomohiro Muro , Hiroyuki Hikida , Takeshi Fujii , Takashi Kiuchi , Susumu Katsuma bioRxiv 2022.05.08.491107; doi: https://doi.org/10.1101/2022.05.08.491107 Share This Article: Copy Citation Tools Two complete genomes of male-killing Wolbachia infecting Ostrinia moth species illuminate their evolutionary dynamics and association with hosts Tomohiro Muro , Hiroyuki Hikida , Takeshi Fujii , Takashi Kiuchi , Susumu Katsuma bioRxiv 2022.05.08.491107; doi: https://doi.org/10.1101/2022.05.08.491107 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (8023) Biochemistry (18792) Bioengineering (14930) Bioinformatics (44506) Biophysics (22634) Cancer Biology (19775) Cell Biology (26954) Clinical Trials (138) Developmental Biology (13999) Ecology (21050) Epidemiology (2067) Evolutionary Biology (25475) Genetics (16187) Genomics (23537) Immunology (18736) Microbiology (42581) Molecular Biology (18106) Neuroscience (93635) Paleontology (701) Pathology (2989) Pharmacology and Toxicology (5105) Physiology (8133) Plant Biology (16029) Scientific Communication and Education (2098) Synthetic Biology (4573) Systems Biology (10255) Zoology (2393) window.__CF$cv$params={r:'a4011a25dc8bbc14',t:'MTc5MDI0NTcwNA==',u:'01a0d2f59cd170c4aed89c0bb565d6dc',ut:'XxL8Maliw770CRVdWXfZH9c_PJ7ofC_vbBI7vegDMHM-1790245706-1.2.1.1-x1jr07f2V7R7gvFFk6LLSrbgv8WKAe_5x67X7Ku_IgWvo7RF4cTXBuDmpInS6j2wsbypvHCZWWTfB6RVMMkXB.SWtZarSn8UjYVugV5m3XQ',i:60};(function(){if(!document.body)return;var s=document.createElement('script');s.src='/cdn-cgi/challenge-platform/scripts/precursor/main.js';document.head.appendChild(s);})();