The Journal of Experimental Biology © 2014. Published by The Company of Biologists Ltd | The Journal of Experimental Biology (2014) 217, 3197-3199 doi:10.1242/jeb.105353 3197 ABSTRACT Host orientation is the most important step in host-searching nematodes; however, information on direct cues from hosts to evoke this behaviour is limited. Caenorhabditis japonica establishes a species-specific phoresy with Parastrachia japonensis. Dauer larvae (DL), the non-feeding and phoretic stage of C. japonica, are predominantly found on female phoretic hosts, but the mechanisms underlying the establishment of this phoresy remain unknown. To determine whether C. japonica DL are able to recognize and orient themselves to a host using a volatile cue from the host, we developed a Y-tube olfactory assay system in which C. japonica DL were significantly attracted to the air from P. japonensis but not to the air from three other insects or to CO 2 . These results demonstrated that C. japonica DL utilize volatiles for host recognition and orientation and that the presence of a specific volatile kairomone released by the host attracts C. japonica DL. KEY WORDS: Dauer, Kairomone, Nictation, Olfactometer, Navigation INTRODUCTION Finding a host is the most critical step for parasitic and phoretic nematodes. Host-searching nematodes respond to different types of host-related stimuli such as vibrations (Torr et al., 2004), temperature (Byers and Poinar, 1982), electric fields (Shapiro-Ilan et al., 2009) and chemical compounds (Pye and Burman, 1981; Grewal et al., 1993; Shapiro et al., 2000). However, host-searching strategies vary among species or ecological niches, and mechanisms of host finding, including host recognition and orientation, are poorly understood. Volatiles could be a useful cue for nematodes to orient, particularly those from distant locations. The recognition and importance of volatiles in host searching have been reported in entomopathogenic nematodes (EPNs). The infective juveniles (IJs) of steinernematid and heterorhabditid EPNs respond to volatiles from insects and CO 2 (Lewis et al., 1993; Campbell and Kaya, 1999; Hallem et al., 2011; Dillman et al., 2012). IJs are also attracted to herbivore-induced plant volatiles (HIPVs), which facilitates host detection, resulting in higher infection rates (Rasmann et al., 2005; Hiltpold et al., 2010; Ali et al., 2010; Ali et al., 2011). Caenorhabditis japonica Kiontke, Hironaka and Sudhaus is a bacterial-feeding nematode that forms a species-specific and female SHORT COMMUNICATION 1 Laboratory of Nematology, Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, Saga 840-8502, Japan. 2 The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-8580, Japan. *Present address: Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake, Sakyo, Kyoto 606-8502, Japan. ‡ Author for correspondence (tyoshiga@cc.saga-u.ac.jp) Received 13 March 2014; Accepted 3 July 2014 host-biased phoresy with the burrower bug Parastrachia japonensis Scott (Kiontke et al., 2002; Yoshiga et al., 2013). Dauer larvae (DL), a developmentally arrested and phoretic stage of the nematode and a developmental analogue of IJs in EPNs, are predominantly found on P. japonensis adult females throughout the year in fields. Only the nematode benefits from this phoresy. The nematode uses the host insect to prolong its own survival (Tanaka et al., 2012) and for transport to food resources, such as the nest of the host insect where the mother insect stores fruits of Schoepfia jasminodora Siebold & Zuccarini for her nymphs, as well as eggs and nymphal carcasses (Okumura et al., 2013b; Yoshiga et al., 2013). Thus, the phoresy is essential only for the nematode, but the mechanisms of forming the species-specific phoresy are not well understood. In our previous study, we demonstrated that C. japonica DL specifically embark on P. japonensis and are attracted to the hexane extracts containing body surface components of P. japonensis (Okumura et al., 2013a). These studies indicate the presence of a species-specific kairomone that is directly released by the host insect and attracts DL. However, hexane extracts contain not only volatiles but also non-volatiles, and it is difficult to distinguish whether C. japonica DL are attracted to volatiles or non-volatiles in the agar plate assay, which is commonly used in nematode chemoattraction studies (Bargmann, 2006). Thus, a new method to evaluate volatile attraction is necessary. Because C. japonica DL embark on a host insect that wanders on the soil surface, DL have to detect the presence of a host on the soil surface and quickly orient themselves for embarkation on the host. Thus, it is possible that C. japonica DL recognize volatiles from a host and orient themselves to the host. To examine this possibility, we developed an assay system for C. japonica DL using a Y-tube olfactometer and demonstrated their recognition of and orientation to a host insect. RESULTS AND DISCUSSION Y-tube olfactometers are commonly used in experiments involving insect olfactory responses (Smith et al., 1994). Volatiles pass through one arm of the Y-tube and clean air passes through the other; this gives the possibility of choosing between two different stimuli. However, Y-tube olfactometers are not commonly used in nematode research because of some technical difficulties such as conditioning of humidity and locomotion activity. Thus, we modified a Y-tube olfactometer so that C. japonica DL could freely move in it (Fig. 1A,B). Unlike arthropods, nematodes require high humidity for their movements; thus, using water agar in the assay arena improved nematode movement. In addition, the assay arena was positioned vertically because C. japonica DL have a tendency to move upward during host searching (Okumura et al., 2013c); this improved nematode locomotion and response to volatiles (data not shown). In the olfactory assay using P. japonensis as the test insect, C. japonica DL moved towards the direction of air flow from P. japonensis soon after the initiation of the experiments, and 43% Host orientation using volatiles in the phoretic nematode Caenorhabditis japonica Etsuko Okumura 1,2, * and Toyoshi Yoshiga 1,3,‡