Ecological Entomology (2011), 36, 309–317 DOI: 10.1111/j.1365-2311.2011.01274.x Wolbachia-induced feminisation newly found in Eurema hecabe, a sibling species of Eurema mandarina (Lepidoptera: Pieridae) SATOKO NARITA, 1 DAISUKE KAGEYAMA, 1 MASATO HIROKI, 2 TAKESHI SANPEI, 2 SAORI HASHIMOTO, 2 TAKEHIKO KAMITOH 2 and Y O S H I O M I K A T O 2∗ 1 Insect-Microbe Research Unit, National Institute of Agrobiological Sciences (NIAS), Ibaraki, Japan and 2 Department of Life Science, International Christian University, Tokyo, Japan Abstract. 1. Complete feminisation of genetic males into functional females, a unique case among insects, is known in Eurema mandarina (former Eurema hecabe Y type) that are infected with two strains of Wolbachia, w CI Em and w Fem Em . 2. Here, we newly found that a proportion of wild-caught E. hecabe (former E. hecabe B type) produced only female offspring. Cytogenetic observations indicated that individuals of E. hecabe displaying the all-female trait were genetically male (i.e. feminisation). 3. Multilocus sequence typing analyses demonstrated that the feminised individuals of E. hecabe were infected with two Wolbachia strains, w CI Eh and w Fem Eh , that were indistinguishable from w CI Em and w Fem Em , respectively. 4. Even identical strains of Wolbachia can be regulated differently depending on the host genetic background. Therefore, we compared the infection densities and vertical transmission efficiencies of Wolbachia between feminised E. mandarina and E. hecabe, but detected no significant differences in these traits. 5. The possible routes by which the two Wolbachia strains have transferred between E. mandarina and E. hecabe are discussed. Key words. Eurema hecabe, Eurema mandarina, feminisation, multilocus sequence typing, sibling species, Wolbachia. Introduction It was previously considered that, in Japan, the butterfly Eurema hecabe L. (Lepidoptera: Pieridae) has two biotypes, yellow type (Y type) and brown type (B type), that inhabit temperate regions and subtropical regions, respectively (Kato, 1999, 2000a). The two biotypes differ in their expression of polyphenism in response to the photoperiod and temperature (Kato & Handa, 1992), host plants (Kato et al., 1992), coloration of the wing fringe (Kato, 1999), allelic frequencies of allozymes (Nomura & Kato, 1993), and mate choice by females (Kobayashi et al., 2001; Kato, 2000c). Moreover, it was recently shown that the nucleotide sequences of a Correspondence: Satoko Narita, Insect-Microbe Research Unit, National Institute of Agrobiological Sciences (NIAS), 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan. E-mail: naritas@affrc.go.jp ∗ Current address: Laboratory of Biodiversity Science, Graduate School, The University of Tokyo, Tokyo, Japan. nuclear gene, Tpi, are distinct between the two biotypes (Narita et al., 2006). These data consistently and strongly suggest that the Y type and B type constitute two closely related, but distinct, biological species, and have led us to revise the taxonomic statuses of these two biotypes. At present, E. hecabe has been assigned as the former E. hecabe B type while Eurema mandarina de l’Orza has been assigned as the former E. hecabe Y type (Kato & Yata, 2005; Shirozu, 2006). Eurema hecabe (former E. hecabe B type) mainly inhab- its the southwestern islands of Japan while E. mandarina mainly inhabits the mainland of Japan. However, E. mandarina and E. hecabe sympatrically inhabit Okinawa Island, one of the southwestern islands, although they feed on differ- ent host plants (Kato, 2000b; Kato & Yata, 2005) and are behaviourally and reproductively isolated (Kato, 2000c; Kobayashi et al., 2001). Previous studies have shown that E. hecabe and E. mandarina are infected with bacteria belonging to the © 2011 The Authors Ecological Entomology © 2011 The Royal Entomological Society 309