Screening Mollusks for Wolbachia Infection Menno Schilthuizen and Edmund Gittenberger Institute for Evolutionary and Ecological Sciences, Leiden University, P.O. Box 9516, NL-2300 RA Leiden, The Netherlands Received September 2, 1997; accepted November 14, 1997 We screened 38 species of mollusks for infection by Wolbachia, a bacterium that is a common endosymbi- ont in arthropods, where it induces alterations in reproduction. Using a PCR assay, we could not detect the symbiont in any of the samples, indicating that, in mollusks, it might be absent.  1998 Academic Press Key Words: Wolbachia; Mollusca; reproductive alter- ation; PCR; ftsZ. INTRODUCTION The -proteobacterium Wolbachia is a well-known endosymbiont in various arthropods, including insects, isopods, and mites. It has figured prominently in the evolutionary literature over the past decade for its ability to interfere with host reproduction (see, for a review, Werren, 1997). Its most common manifestation is the induction of ‘‘cytoplasmic incompatibility’’ (CI), which is exhibited in many insects (Hoffmann and Turelli, 1997), 11 species of mites (Johanowicz and Hoy, 1996; Breeuwer and Jacobs 1996; Tsagkarakou et al., 1996), and an isopod (Legrand et al., 1986). In CI, the bacterium manipulates its host’s sperm in such a way that it can only fertilize egg cells that have already been infected with Wolbachia. If the sperm enter an uninfected egg, the zygote dies (Barr, 1980; Breeuwer and Werren, 1990). In many Hymenoptera, the symbi- ont has a different effect: it changes normally sexual individuals into parthenogens, which only produce female offspring. This feat is achieved by diploidisation of egg cells (Stouthamer et al., 1990, 1993). In isopods, Wolbachia can turn males into functional ‘‘neofemales’’ (Rousset et al., 1992; Juchault et al., 1994; Martin et al., 1994). These three effects (CI, parthenogenesis, and feminization) all result in an increased transmission of the symbiont. Surveys have shown these bacteria to be quite com- mon. In insects, circa 15% of all species appear to be infected (Werren et al., 1995a), and in isopods even higher infection rates have been found (Bouchon, per- sonal communication). Phylogenetic studies have also shown that, over evolutionary time, frequent horizon- tal transmissions have taken place from one arthropod lineage to another (Stouthamer et al., 1993; Werren et al., 1995b; Schilthuizen and Stouthamer, 1997). However, although many authors have hinted at the possibility, Wolbachia has not been reported from non- arthropod hosts, with possibly one exception: a Wolba- chia-like organism has recently been found in a nema- tode (Sironi et al., 1995). In this study, we report an initial survey for Wolba- chia infection in a nonarthropod group, the Mollusca. Molluscan life histories offer good opportunities for endosymbiotic manipulation. Most are sexual, which would allow the action of CI. In the pulmonates, outcrossing hermaphrodites are the rule, which would even double the rate at which CI-inducing bacteria can spread. Also, like crustaceans, many gastropods show sex reversal from male to female (protandry) at some stage in their lives. This apparent flexibility in their sex determination might offer opportunities to feminizing Wolbachia. Parthenogenesis is also known, for ex- ample, in prosobranchs (Tompa et al., 1984). Proso- branchs appear to have homogametic females (Tompa et al., 1984), and diploidisation of unfertilized eggs would render the individual parthenogenetic. So, in theory, parthenogenesis in snails might be induced by a symbiont also. MATERIAL AND METHODS We obtained live material of 38 species of land and freshwater mollusks from The Netherlands, Greece, and Italy (see Table 1), including both bivalves and gastropods. We focused on terrestrial and limnic spe- cies since, in isopods at least, Wolbachia appears to be absent from marine habitats (Bouchon, personal com- munication). The material was stored at -80°C until further use. We extracted DNA from three complete individuals for the smallest species (largest dimension less than 2 mm) and one complete individual for species of interme- diate size (3–5 mm), while for the largest species, we used only fragments of the reproductive tissue (i.e., the albumen gland). In each extraction series, we included five specimens of the Wolbachia-infected parasitic wasp JOURNAL OF INVERTEBRATE PATHOLOGY 71, 268–270 (1998) ARTICLE NO. IN974739 268 0022-2011/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.