bioRxiv preprint doi: https://doi.org/10.1101/2020.03.24.005017; this version posted June 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Persistence of a Wolbachia-driven sex ratio bias in an 2 island population of Eurema butterflies 3 4 Daisuke Kageyama,1,2 Satoko Narita,1,3 Tatsuro Konagaya,4 Mai N. Miyata,3 Jun Abe,5 5 Wataru Mitsuhashi,1 and Masashi Nomura3,6 6 7 1Institute of Agrobiological Sciences, National Agriculture a nd Food Research 8 Organization, 1-2, Owashi, Tsukuba, Ibaraki 305-0854, Japan 9 2E-mail: [email protected] 10 3Graduate School of Horticulture, Chiba University, Matsudo 648, Matsudo, Chiba 11 271-8510, Japan, 12 4Division of Evolutionary Developmental Biology, National Institute for Basic Biology, 13 Myodaiji, Okazaki 444-8585, Japan, 14 5Faculty of Liberal Arts, Meijigakuin University, Yokohama, Kanagawa 244-8539, Japan 15 6E-mail: [email protected] 16 17 18 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.24.005017; this version posted June 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 19 Abstract 20 It is generally believed that when maternally inherited sex ratio distorters become 21 predominant, either the host population goes extinct or nuclear suppressors evolve in the 22 host. Here, we show an empirical case where all-female-producing Wolbachia is likely to 23 be stably maintained at a high frequency. On an island population of the butterfly Eurema 24 mandarina, a Wolbachia strain wFem, which makes female hosts produce all-female 25 offspring without sibling lethality (female drive), is highly prevalent. We found that, with 26 some fluctuations, wFem appeared to be stably maintained for at least 12 years at a high 27 frequency, resulting in the existence of an abnormally high number of virgin females. 28 Interestingly, comparison between sex ratios of captive individuals and sex ratios deduced 29 from wFem frequencies suggested a plastic behavioral change of males and females in 30 response to the shift of sex ratios. wFem presence does not affect brood size but has a 31 slightly negative effect on body size. Stable coexistence of wFem-positive and -negative 32 females in the population may be explained via mate choice by males, which keeps wFem 33 in check. Taken together, this butterfly population is an attractive model for future studies 34 on the population dynamics of sex ratios and mating behavior. 35 36 Keywords: butterfly, female drive, Eurema mandarina, population sex ratio, Wolbachia 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.24.005017; this version posted June 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 37 Introduction 38 Arthropods are often influenced by selfish microbes, such as Wolbachia, which are 39 maternally transmitted through the cytoplasm and can skew the sex ratio toward females 40 (Hurst and Jiggins 2000; Werren et al. 2008; Kageyama et al. 2012). It is predicted that the 41 prevalence of cytoplasmic sex ratio distorters can lead to the extinction of the host 42 population (Hatcher et al. 1999), unless suppressors evolve in the host. To date, the 43 existence of host nuclear suppressors against cytoplasmic sex ratio distorters has been 44 described in various arthropod species (Jaenike 2007; Majerus and Majerus 2010), and the 45 rapid spread of suppressors in natural populations has been documented in two 46 species—i.e., the butterfly Hypolimnas bolina (Lepidoptera; Nymphalidae) (Mitsuhashi et 47 al. 2004; Hornett et al. 2006; Charlat et al. 2007a; Mitsuhashi et al. 2011) and the lacewing 48 Mallada desjardinsi (Neuroptera; Chrysopidae) (Hayashi et al. 2016, 2018)—which 49 suggests that an arms race concerning the sex ratio between the cytoplasmic and nuclear 50 genomes may be quite common in insects. 51 52 Here, we examined the sex ratio dynamics in an island population (Tanegashima Island, 53 Japan) of the butterfly Eurema mandarina (Lepidoptera: Pieridae), where 54 all-female-producing females and normal females coexist. In E. mandarina, cytoplasmic 55 incompatibility-inducing Wolbachia (wCI), which does not influence the sex ratio, is fixed 56 in most of the populations in Japan, including the Tanegashima Island population (Hiroki et 57 al. 2005; Narita et al. 2006). By contrast, all-female-producing females, which are infected 58 with another strain of Wolbachia, wFem, besides the wCI strain (Hiroki et al. 2002, 2004), 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.24.005017; this version posted June 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 59 are prevalent in the Tanegashima Island population (Narita et al. 2007a, b; Miyata et al. 60 2017). 61 62 In E. mandarina, females and males singly infected with wCI (referred to as C females and 63 C males) have the WZ karyotype and the ZZ karyotype, respectively, whereas females 64 doubly infected with wCI and wFem (referred to as CF females) have the Z0 karyotype 65 (Kern et al. 2015; Kageyama et al. 2017). By mating with C males, CF females produce 66 only Z0 embryos; all of which develop into females. Unlike the male killing effect induced 67 by many Wolbachia strains, wFem does not induce male-specific lethality. From a 68 population genetics perspective, this phenomenon can be regarded as female drive (Burt 69 and Trivers 2006), and thus, CF females will increase their frequency in the population if 70 the relative fitness of CF females is higher than that of 50% of C females. 71 72 If CF females continue to increase their frequency in the population, the population could 73 become extinct because the dwindling number of males would become too small to 74 maintain the population. Alternatively, nuclear suppressors against female drive, if they 75 arise, may rapidly increase in the population. With these possible scenarios in mind, we 76 monitored the island population of E. mandarina for 12 years. 77 78 79 Materials and Methods 80 COLLECTION OF BUTTERFLIES 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.24.005017; this version posted June 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 81 On Tanegashima Island (Kagoshima Prefecture, Japan), we patrolled the pavement and 82 made efforts to collect all E. mandarina adults that we encountered. Males were stored at 83 −30°C until DNA extraction began. After allowing females to lay eggs in the laboratory for 84 other experiments (Narita et al. 2007a; Kageyama et al. 2017), bursa copulatrixes were 85 dissected out from the female specimens; the remains were stored at −30°C until DNA 86 extraction began. 87 88 DISSECTION OF SPERMATOPHORES 89 As described in Konagaya and Watanabe (2015), the bursa copulatrixes were carefully 90 opened under a dissecting microscope, and the number of spermatophores that each 91 contained was recorded. 92 93 MEASUREMENT OF WING SIZE 94 For adult females, the distance between the tip of the forewing and the base of the forewing 95 jointed with the thorax was measured. 96 97 DNA EXTRACTION AND PCR 98 DNA was extracted from either specimen legs or thoraxes using a DNeasy Blood & Tissue 99 Kit (Qiagen, Tokyo, Japan). Wolbachia infections of either the wCI or wFem strain were 100 identified via specific PCR detection, which targeted the wsp gene (Hiroki et al. 2004; 101 Narita et al. 2007b; Kageyama et al. 2008). Specifically, wCI was detected using the 102 Wolbachia-specific forward primer wsp81F (5′-TGGTCCAATAAGTGATGAAGAAAC-3′) 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.24.005017; this version posted June 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 103 (Braig et al. 1998) and the wCI-specific reverse primer WHecFem1 104 (5′-ACTAACGTTTTTGTTTAG-3′) (Hiroki et al. 2004), which amplify a 232 bp DNA 105 fragment. The wFem strain was detected using the wFem-specific forward primer 106 WHecFem2 (5′-TTACTCACAATTGGCTAAAGAT-3′) (Hiroki et al. 2004) and the 107 Wolbachia-specific reverse primer wsp691R (5′-AAAAATTAAACGCTACTCCA-3′) 108 (Braig et al. 1998), which amplify a 398 bp DNA fragment. 109 110 STATISTICAL ANALYSES 111 Analyses of binary data (i.e., virgin or mated females) were conducted using generalized 112 linear mixed models (GLMM) with a binomial error distribution. Analyses of mating 113 frequencies (i.e., number of spermatophores per female) were conducted using GLMM with 114 a Poisson error distribution. The random effects of different visits were included in the 115 models. The GLMM were fitted using Laplace approximation.