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Page 1 of 24 1 Master manipulation continued: feminizing Wolbachia 2 endosymbiont distorts sex chromosome inheritance 3 4 Short title: Wolbachia distort sex chromosome inheritance 5 6 Daisuke Kageyamaa,1, Mizuki Ohnob, Tatsushi Sasakib, Atsuo Yoshidob, 7 Tatsuro Konagayac, Akiya Jourakua, Seigo Kuwazakia, Hiroyuki Kanamorid, 8 Yuichi Katayosed, Satoko Naritaa,e, Mai Miyataf, Markus Rieglerg, and Ken 9 Saharab,1 10 11 aInstitute of Agrobiological Sciences, National Agriculture and Food Research 12 Organization, 1-2, Owashi, Tsukuba, Ibaraki 305-0854, Japan; bLaboratory of Applied 13 Entomology, Faculty of Agriculture, Iwate University, 3-18-8, Ueda, Morioka 020-8550, 14 Japan; cGraduate School of Science, Kyoto University, Kitashirakawa Oiwake-cho, 15 Sakyo-ku, Kyoto 606-8502, Japan; dInstitute of Crop Science, National Agriculture and 16 Food Research Organization, 1-2, Owashi, Tsukuba, Ibaraki 305-0854, Japan; 17 eTsukuba Primate Research Center, National Institute of Biomedical Innovation, Health 18 and Nutrition, 1-1, Hachimandai, Tsukuba, Ibaraki 305-0843, Japan; fGraduate School 19 of Horticulture, Chiba University, Matsudo 648, Matsudo, Chiba 271-8510, Japan; and 20 gHawkesbury Institute for the Environment, Western Sydney University, Locked Bag 21 1797, Penrith, New South Wales 2751, Australia 22 23 Author contributions: D.K. and K.S. designed the research; D.K., M.O., T.S., A.Y., 24 T.K., S.K., H.K., Y.K., S.N., M.M., M.R., and K.S. performed the research; D.K., 25 A.J., and K.S. analyzed the data; and D.K., M.R., and K.S. wrote the paper. 26 The authors declare no conflict of interest. 27 1Correspondence should be addressed: [email protected] (D.K.), 28 [email protected] (K.S.) 29 30 Keywords: butterfly, feminization, meiotic drive, sex chromosome, sex 31 determination, Wolbachia bioRxiv preprint doi: https://doi.org/10.1101/115386; this version posted March 9, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Page 2 of 24 32 Abstract 33 Wolbachia is a maternally inherited ubiquitous endosymbiotic bacterium of 34 arthropods that displays a diverse repertoire of host reproductive manipulations. 35 For the first time, we demonstrate that Wolbachia manipulates sex chromosome 36 inheritance in a sexually reproducing insect. Eurema mandarina butterfly 37 females on Tanegashima Island, Japan, are infected with the wFem Wolbachia 38 strain and produce all-female offspring, while antibiotic treatment results in male 39 offspring. Fluorescence in situ hybridization (FISH) revealed that wFem-positive 40 and wFem-negative females have ZO and WZ sex chromosome sets, 41 respectively, demonstrating the predicted loss of the W chromosome from 42 wFem-infected lineages. Genomic quantitative polymerase chain reaction 43 (qPCR) analysis showed that wFem-positive females lay only ZO eggs that carry 44 a paternal Z, whereas females from lineages that are naturally wFem-negative 45 lay both WZ and ZZ eggs. In contrast, antibiotic treatment of adult wFem females 46 resulted in the production of ZO and ZZ eggs, suggesting that this Wolbachia 47 strain can induce meiotic drive. Moreover, most male offspring produced by 48 antibiotic-treated wFem females had a ZZ karyotype, implying reduced survival 49 of ZO individuals in the absence of feminizing effects of Wolbachia. Antibiotic 50 treatment of wFem-infected larvae induced male-specific splicing of the 51 doublesex (dsx) gene transcript, causing an intersex phenotype. Thus, the loss 52 of the female-determining W chromosome in ZO individuals is functionally 53 compensated by Wolbachia feminization. We discuss how Wolbachia may 54 manipulate oogenesis to cause meiotic drive and that Wolbachia may have 55 acquired this coordinated dual mode of reproductive manipulation first by the 56 evolution of feminization and then cytoplasmically induced meiotic drive. bioRxiv preprint doi: https://doi.org/10.1101/115386; this version posted March 9, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Page 3 of 24 57 Significance Statement 58 Genomes are vulnerable to selfish genetic elements that enhance their own 59 transmission often at the expense of host fitness. These include cytoplasmic 60 elements such as endosymbiotic bacteria that cause feminization, male-killing, 61 parthenogenesis and cytoplasmic incompatibility. We demonstrate, for the first 62 time, that meiotic drive, a phenomena so far seen only for nuclear genetic 63 elements, can also be caused by the ubiquitous endosymbiotic bacterium 64 Wolbachia. In female butterflies with a ZO sex chromosome constitution, 65 Wolbachia prevents the production of ZZ zygotes. Wolbachia also compensates 66 for the female-determining function of the W chromosome lost from infected 67 lineages, thereby causing the production of all-female progeny. Our findings 68 highlight that cytoplasmic elements play an important role in sex determination 69 systems and sex chromosome evolution. bioRxiv preprint doi: https://doi.org/10.1101/115386; this version posted March 9, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Page 4 of 24 70 Introduction 71 Genomes of sexually reproducing organisms are exposed to genetic conflicts. 72 For example, some genes bias reproduction towards male offspring while other 73 genes within the same genome may favor reproduction of more daughters. 74 Selfish genetic elements (SGEs), such as meiotic drivers, cytoplasmic sex ratio 75 distorters and transposons, are extreme examples, which enhance their own 76 transmission often at the expense of their hosts’ fitness (1, 2). There is growing 77 evidence that SGEs, and the resulting genetic conflict, trigger important 78 evolutionary change and innovation in eukaryotes (2). 79 Meiotic drive is a distortion of Mendelian inheritance as it leads to the 80 more frequent inheritance of one copy of a gene than the expected 50% (3, 4). A 81 meiotic drive factor that sits on a sex chromosome biases the sex ratio. For 82 example, X chromosome drive and Y chromosome drive in flies (Diptera), result 83 in female-biased and male-biased sex ratios, respectively (4). In 84 male-heterogametic species, meiotic drive factors are expected to be encoded in 85 the nuclear genome. In female-heterogametic species, however, W 86 chromosome and cytoplasm behave as a single linkage group and thus W 87 chromosome drive can theoretically also be caused by cytoplasmic elements. 88 Although this possibility has previously been proposed (5, 6), lack of empirical 89 evidence questions whether it is mechanistically possible for cytoplasmic 90 elements to cause meiotic drive. 91 Wolbachia pipientis (Alphaproteobacteria), a bacterium simply referred 92 to as Wolbachia, attracts significant interest in evolutionary and developmental 93 biology but also from applied perspectives such as pest management because it 94 can manipulate reproduction of arthropods in various ways such as cytoplasmic 95 incompatibility, parthenogenesis induction, feminization and male-killing (7). 96 Here we demonstrate for the first time that meiotic drive caused by Wolbachia 97 constitutes the underlying mechanism of all-female production in the butterfly 98 Eurema mandarina. In most populations, E. mandarina is infected with the 99 cytoplasmic-incompatibility (CI)-inducing Wolbachia strain wCI at a high 100 prevalence of close to 100%. Hiroki et al. (8, 9) first reported an all-female 101 production in E. mandarina (then known as Eurema hecabe yellow type), which bioRxiv preprint doi: https://doi.org/10.1101/115386; this version posted March 9, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Page 5 of 24 102 was considered to be due to the feminization of genetic males (ZZ) by 103 co-infections with the Wolbachia strain wFem (hereafter we refer to this double 104 infection status as CF and single infection with wCI as C). Three observations 105 about CF lineages supported this view, i.e., (a) antibiotic treatment of adult 106 females led to production of all-male offspring (8), (b) antibiotic treatment of 107 larvae resulted in intersexual adult phenotype (10) and (c) females did not have 108 the W chromatin body (8, 10). However, Kern et al. (11) challenged this notion by 109 demonstrating that CF females have only one Z chromosome and that this Z 110 chromosome always derived from their fathers. Therefore, two hypotheses were 111 formulated, namely that the CF females have either a ZO or a W’Z sex 112 chromosome set (whereby W’ cannot be visualized in W chromatin assays and 113 does not have a female-determining function), and that meiotic drive occurs in 114 CF lineages due to Wolbachia or another factor. 115 In a multifaceted approach, by combining chromosome FISH, genome 116 sequencing, quantitative PCR, reverse transcription PCR and antibiotic 117 treatment, we have tested these hypotheses and revealed that Wolbachia is the 118 cause for both the meiotic drive and the feminization of ZO individuals which 119 would otherwise be determined as males in the absence of Wolbachia. These 120 results highlight that cytoplasmic elements can have profound effects on 121 oogenesis, sex chromosome inheritance and sex determination – fundamental 122 biological processes of eukaryotes. 123 124 Results 125 All-female-producing CF females have a ZO karyotype 126 We performed FISH on E. mandarina chromosomes prepared from CF females, 127 C females, and C males collected on Tanegashima Island (Fig.