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568

The Journal of Experimental 216, 568-572 © 2013. Published by The Company of Biologists Ltd doi:10.1242/jeb.073593

RESEARCH ARTICLE Species-specific recognition of the carrier by dauer larvae of the japonica

Etsuko Okumura1,2, Ryusei Tanaka1,3 and Toyoshi Yoshiga1,2,* 1Laboratory of Nematology, Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, Saga 840-8502, Japan, 2The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-8580, Japan and 3Forest Pathology Laboratory, Foresty and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan *Author for correspondence ([email protected])

SUMMARY recognition is crucial during the phoretic stage of because it facilitates their association with hosts. However, limited information is available on the direct cues used for host recognition and host specificity in nematodes. forms an intimate association with the burrower bug japonensis. Caenorhabditis japonica dauer larvae (DL), the phoretic stage of the nematode, are mainly found on adult P. japonensis females but no other species. To understand the mechanisms of species-specific and female carrier-biased ectophoresy in C. japonica, we investigated whether C. japonica DL could recognize their hosts using nematode loading and chemoattraction experiments. During the loading experiments, up to 300 C. japonica DL embarked on male and female P. japonensis, whereas none or very few utilized the other shield bugs Erthesina fullo and Macroscytus japonensis or the terrestrial isopod Armadillidium vulgare. In the chemoattraction experiments, hexane extracts containing the body surface components of nymphs and both adult P. japonensis attracted C. japonica DL, whereas those of other shield bugs did not. extracts also arrested the dispersal of C. japonica DL released at a site where hexane extracts were spotted on an ; i.e. >50% of DL remained at the site even 60min after nematode inoculation whereas M. japonensis extracts or hexane alone did not have the same effect. These results suggest that C. japonica DL recognize their host species using direct chemical attractants from their specific host to maintain their association. Key words: allelochemical, , kairomone, necromeny, phoresy. Received 9 April 2012; Accepted 6 October 2012

INTRODUCTION 1993); however, there is little information on whether DL are able Phoresy, in which nematodes use larger such as to recognize their host insects, and if so, the mechanism involved as vectors to transfer themselves to different sites for , in this recognition. Chemoattraction has been studied in many is a common phenomenon among many nematodes. It is a form of nematodes, and the chemotaxis of has been commensalism in which the growth, fecundity or survival of the clarified in depth at both the molecular and cellular levels nematode is enhanced while the host remains unaffected. Although (Bargmann, 2006). In addition, host-specific chemoattraction has some host preferences exist, the same nematodes have often been been reported in nematodes (Hong and Sommer, 2006). isolated from different types of invertebrates (Barrière and Félix, However, these studies in C. elegans and Pristionchus spp. used 2005; Kiontke and Sudhaus, 2006; Herrmann et al., 2006), indicating the adult stage, and thus no information is available on low specificity. In addition, information on species-specific phoretic chemoattraction of DL to host insects, which is the actual phoretic associations is limited (Kiontke, 1997; Herrmann et al., 2006), and stage, probably because DL are unresponsive to attractants in there are very few reports in which the specificity of association chemotaxis assays (Riddle, 1988). Recently, chemotaxis has been has been demonstrated experimentally (Baird, 1999). studied in DL of entomopathogenic nematodes (Rasmann et al., A key adaptation in that allows the development of 2005; Hiltpold et al., 2010; Ali et al., 2010; Ali et al., 2011). such a phoretic relationship is the formation of dauer larvae (DL) However, these studies focused on herbivore-induced volatiles and (Sudhaus, 2008). DL are non-feeding larvae that have an arrested used indirect host cues. CO2 acts as a direct host cue for nematodes, developmental stage specialized for dispersal and survival (Riddle, but very little information is available on the chemicals involved in 1988). In phoretic nematodes, DL exhibit a special type of behavior direct host recognition (Hallem et al., 2011). Moreover, no called waving, i.e. lifting the anterior part, or more, of their body off information is available on species-specific kairomones that are a moist substrate and waving it in the air (Croll and Matthews, 1977). released from host insects and directly attract DL. This behavior seems to improve the likelihood of phoresy or a host Caenorhabditis japonica is a bacterial-feeding nematode found on encounter (reviewed in Burr and Robinson, 2004). Although these the burrower bug Parastrachia japonensis (Kiontke et al., 2002). typical phoretic behaviors are well known, the mechanisms used by Caenorhabditis japonica DL are always detectable throughout the year DL to recognize their hosts have not been elucidated. as aggregates on the body surface, particularly under the scutellum, It is likely that DL use cues from their host insects, e.g. chemicals of almost all female adults of P. japonensis but rarely on male P. (volatile and water soluble) and physical contact (Grewal et al., japonensis. The biology and life history of C. japonica is currently

THE JOURNAL OF EXPERIMENTAL BIOLOGY Specific host recognition by a nematode 569 under investigation, but according to our observations, this nematode the campus of Saga University, Saga City, Saga Prefecture, Japan, appears to have a species-specific phoretic association because C. in autumn 2007. Armadillidium vulgare was collected on the japonica has thus far never been detected on other invertebrates. To campus of Saga University in July 2012. establish such an intimate association, C. japonica may have developed mechanisms to recognize and associate with its carrier bug P. Loading experiments japonensis. Our aim was to investigate whether C. japonica DL are Approximately 1000 DL were inoculated on a filter paper in a 3cm able to recognize and associate with their carrier bugs in a species- plastic Petri dish, and then a C. japonica-free insect (male or female specific manner. We used loading experiments in which C. japonica P. japonensis, M. japonensis, E. fullo or A. vulgare) or a pair of P. DL and a bug were incubated together in a small Petri-dish to japonensis (male and female) was released in the dish. Twenty-four demonstrate that C. japonica DL specifically associate with their carrier hours after inoculation at 25°C, the insects were dissected, their bugs. We also demonstrated the presence of kairomones, which are body parts were placed in water for 24h to release DL, and the host-specific attractants, on the host bug for C. japonica DL. nematodes were counted under the stereomicroscope. Nematode numbers on an insect were compared among insect species and MATERIALS AND METHODS between male and female P. japonensis. Nematodes Because C. japonica DL are usually associated with P. japonensis, Caenorhabditis japonica Kiontke, Hironaka and Sudhaus 2002 preparation of C. japonica-free P. japonensis was necessary before H1 was isolated from an adult female P. japonensis (Scott 1880) carrying out the loading experiments. To prepare nematode-free P. collected from Hinokuma Mountain Prefectural Park, Kanzaki japonensis samples, adult P. japonensis were partly soaked in tap City, Saga Prefecture, Japan. The nematode was maintained on dog water for 3days and rehydrated nematodes on the bugs were washed food medium (Hara et al., 1981) seeded with strain off (Tanaka et al., 2010). All DL were removed using this method OP50. Phoretically active DL were collected as described by (data not shown). The removal of nematodes was also confirmed Tanaka et al. (Tanaka et al., 2010). Briefly, a sterile yellow 200μl by observations made using a stereoscopic microscope. Other pipette tip (Watson Fukaekasei, Tokyo, Japan) was placed in the shield bugs and the isopod were used after rinsing with distilled dog food agar in a 100ml culture bottle, and DL waving on the top water. of the tip were collected using a worm picker (a 1cm piece of platinum wire mounted on the tip of a Pasteur pipette), washed with Chemoattraction experiments distilled water three times and used for the experiments. We modified the chemotaxis assay method developed for Caenorhabditis elegans to investigate the nematodes’ response to Insects the hexane extracts containing body surface components of shield Adult P. japonensis were collected from Hinokuma Mountain bugs (Matsuura, et al., 2005). Test and control spots (1cm circle) Prefectural Park during 2007–2009. Females and males in were set in a 6cm nematode growth medium (NGM) plate, and reproductive diapause were collected between autumn and spring 3µl of hexane extracts containing body surface components of from aggregations of bugs. Two other species of shield bugs, bugs and only hexane were spotted at the center of test and control Macroscytus japonensis Scott 1874 and Erthesina fullo (Thunberg spots, respectively. Next, ~20–30 DL in 2–3µl of water were 1783), were used for comparison. Macroscytus japonensis was placed at the center of the plate. Nematodes in the test and control chosen because it is a burrower bug that is present in the same habitat spots were counted at intervals of 10min for 60min. The as P. japonensis in Hinokuma Mountain Park and it was relatively chemoattraction index (CI) value [(number of nematodes in the easier to collect. A phoretic nematode species (not C. japonica) was test spot−number of nematodes in the control spot)/total number occasionally present on adult M. japonensis. Caenorhabditis of nematodes] was calculated as described in Bargmann et al. japonica has never been found on M. japonensis (Yoshiga et al., (Bargmann et al., 1993). Sodium azide, used in the original method 2013). Erthesina fullo was selected because it was a similar size to to keep nematodes on the spots, was omitted in these experiments that of P. japonensis and it was readily available. No nematodes because C. japonica DL actively moved on the plate and were have been detected in this bug species (data not shown). A species sometimes trapped by sodium azide during random movement. of terrestrial isopod, Armadillidium vulgare Latreille 1804, was also Hexane extracts containing body surface components of bugs were used in the loading experiment because isopods were often present prepared by soaking a bug in hexane in a glass tube with a lid for in the area where P. japonensis was found, and phoretic nematodes 10min. Bugs were weighed in advance, and only those with an (not C. japonica) were often found in terrestrial isopods. Adult M. average mass of 0.05±0.02g for M. japonensis, 0.13±0.03g for japonensis were collected in the autumn from below the litter near male P. japonensis, 0.17±0.03g for female P. japonensis and an aggregation of P. japonensis, whereas E. fullo were collected on 0.3±0.05g for E. fullo were used in the assays. Based on the

Table1. List of used in this study Experiments used Insecta/ Erthesina fullo (unsexed) Loading Chemoattraction Arrest of dispersal Macroscytus japonensis (unsexed) Loading Chemoattraction Arrest of dispersal Parastrachia japonensis Male (reproductive diapause) Loading Chemoattraction Arrest of dispersal Female (reproductive diapause) Loading Chemoattraction Arrest of dispersal Female (provisioning) Chemoattraction Arrest of dispersal Nymph (fifth instar) Chemoattraction Arrest of dispersal Malacostraca/ Armadillidium vulgare (unsexed) Loading

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** RESULTS *** Loading experiments 350 A * During the loading experiments with P. japonensis, we observed 300 DL crawling up the legs and abdominal parts of both male and female bugs. After dissection, aggregates of DL were found between 250 the body segments and under the wings, where naturally associating 200 DL are usually found. Up to 333 DL were found on P. japonensis. In contrast, nematodes were scarcely found on the body surfaces 150 of M. japonensis, E. fullo and A. vulgare (max.=3, min.=0, max.=21, min.=0, and max.=16, min.=0, respectively), and there were 100 significant differences between the nematode numbers on the 50 insects (Fig.1A). The number of DL associating with male and female P. japonensis 0 was not significantly different when a single male or female bug P. japonensis M. japonensis E. fullo A. vulgare was placed in a 3cm dish (Fig.1B) and when a pair of male and female bugs was placed in a 3cm dish (Fig.1C). 1cm1 cm 1 cm 1 cm 1 1cm 1 Chemoattraction experiments B C The P. japonensis-specific embarkment in the loading experiments 350 180

Number of nematodes may imply the presence of some specific attractant cues. To test 160 300 whether there were any differences in attraction toward the 140 250 chemicals from the different bug species, chemoattraction of C. 120 japonica DL to the hexane extracts containing body surface 200 100 components of bugs was compared. Hexane extracts containing 150 80 body surface components of P. japonensis moderately attracted 60 100 C. japonica DL, and the CI values reached a plateau (~0.3) within 40 60min of the start of experiments (Fig.2). In contrast, hexane 50 20 extracts containing body surface components of M. japonensis and 0 0 E. fullo did not affect nematode behavior and CI values were less Male Female Male Female than 0.03 (Fig.2). The mean response to body surface components of P. japonensis was significantly higher than that to the other Fig.1. Loading experiments of Caenorhabditis japonica dauer larvae (DL). species at every time point (ANOVA, F=33.297, P<0.0001). When (A) Comparison of the number of nematodes among the three bugs and attraction was compared among the different stages, sexes and the isopod. Significantly higher numbers of nematodes embarked on Parastrachia japonensis compared with Macroscytus japonensis, Erthesina physiological conditions of P. japonensis, the CI values for fullo and Armadillidium vulgare. (B) One female or male P. japonensis was nymphs were relatively high and were statistically higher than placed individually in a dish. No significant difference was observed those for males (ANOVA, F=3.024, P=0.0429). However, no between the number of nematodes that embarked. (C) A pair (female and significant differences were observed in CIs among males, females male) of P. japonensis was placed in the same dish. No significant in reproductive diapause (where C. japonica DL are usually difference was observed between the numbers on male and female P. associated) or provisioning females (where C. japonica DL are japonensis. ANOVA, *P>0.01, ***P>0.0001. N=17, 21, 5, 21 and 10 for P. japonensis males, P. japonensis females, M. japonensis, E. fullo and A. not found) (Fig.3). vulgare, respectively.

0.7 P. japonensis average insect sizes, 90, 300, 350 or 530µl of hexane was used 0.6 M. japonensis for M. japonensis, male P. japonensis, female P. japonensis or E. E. fullo fullo, respectively. For fifth instar nymphs of P. japonensis, 350µl 0.5 of hexane was used for extraction. 0.4 0.3 Test for the arrest of DL dispersal 0.2 We investigated the effects of hexane extracts containing body surface components of P. japonensis on the arrest of DL dispersal. 0.1 One circle (1cm diameter) was made at the center of an NGM plate, Chemoattraction index 0 and 3µl of hexane or hexane extracts was spotted at the center of –0.1 the circle. As soon as the hexane evaporated and/or was absorbed 10 20 30 40 50 60 on the plate, DL were inoculated directly onto the center of the Time after nematode inoculation (min) circle. The number of nematodes left in the circle was counted every 10min for 60min. Fig.2. Comparison of the chemoattraction response to the hexane extracts from three bugs. Caenorhabditis japonica DL were attracted to the extract Statistical analysis containing body surface components of P. japonensis, but not to the extracts from M. japonensis and E. fullo. The mean response to P. An ANOVA with Bonferroni/Dunn tests was used for statistical japonensis extracts was significantly higher than that to the other species analysis of chemoattraction (StatView Ver. 4.54; Abacus Concepts, at every time point. N=30 (P. japonensis), 10 (M. japonensis) and 2 (E. Piscataway, NJ, USA). fullo). ANOVA, F=33.297, P<0.0001. Error bars indicate s.d.

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0.7 * 100 90 ** 0.6 80 0.5 70 60 0.4 ** 50 0.3 40 0.2 circle (%) 30 20 0.1

Chemoattraction index 10

0 Nematodes in inoculation 0 Male Female Female Nymph Male Female Female Nymph (Reproductive diapause) (Provisioning) (Reproductive diapause) (Provisioning)

Fig.3. Chemoattraction response to four types of hexane extract containing Fig.5. Arrest of dispersal experiment: comparisons among the four types of body surface components of P. japonensis at 60min. Caenorhabditis extract containing body surface components of P. japonensis at 60min. japonica DL were attracted to four types of P. japonensis extracts. Males, Males, females, reproductive females and nymphs were tested (N=10, 11, females, reproductive females and nymphs were tested (N=10, 10, 10 and 10 and 8, respectively). There was no difference in arrest of dispersal 8, respectively). The mean for nymphs was significantly higher than that for among the P. japonensis developmental stages or sexes except between males. ANOVA, F=3.024, P=0.0429. *P<0.01. males and nymphs. ANOVA, F=5.858, P=0.0024. Error bars indicate s.d. **P<0.001.

Arrest of dispersal by hexane extracts with M. japonensis, E. fullo or A. vulgare. Hexane extracts We released DL directly on the site where the hexane extracts containing body surface components of P. japonensis significantly were spotted to evaluate the arresting effects of the hexane attracted C. japonica DL and arrested their dispersal after contact, extracts. When only hexane or hexane extracts containing body whereas DL were not attracted to hexane extracts containing body surface components of M. japonensis were used, DL rapidly surface components of other insects. These results indicate that dispersed on the plate (Fig.4). In contrast, when hexane extracts C. japonica DL recognize their carrier through some specific containing the body surface components of P. japonensis were chemicals from the carrier, thereby enabling them to associate with used, >50% of DL remained in the inoculation area even 60min the carrier. To the best of our knowledge, this is the first report after nematode inoculation, and the mean response to P. demonstrating the species-specific embarkment of DL onto their japonensis was significantly higher than that to any of the other host and the direct chemical recognition of a specific host by DL, species at any time point (ANOVA, F=78.083, P<0.0001). thereby suggesting the presence of species-specific kairomones in Hexane extracts containing the body surface components of E. nematodes. fullo paralyzed DL on the spot after nematode inoculation, Allelochemicals elicit a physiological or behavioral response possibly because of a toxin on its body surface, which resulted between members of different species (Huettel, 1986; Riga, 2004). in high percentages of arrest of dispersal (82 and 62% at 10 and A kairomone is an allelochemical that elicits a positive response 20min after nematode inoculation, respectively). Thus, these from the receiving organism. Although many nematodes form results were omitted from the analyses. When the effect of the phoretic or parasitic associations with insects, very little hexane extracts on arrest of dispersal was compared among information is available on kairomones other than CO2 with regard different P. japonensis stages, sexes and physiological conditions, to direct host recognition cues (Hallem et al., 2011). In the present no differences among the P. japonensis developmental stages or study, we found that C. japonica DL were attracted to hexane sexes, except between males and nymphs, were found (ANOVA, extracts containing only body surface components of their host F=5.858, P=0.0024; Fig.5). P. japonensis, indicating that the chemical components from their host appear to contain specific kairomones for the association of DISCUSSION C. japonica DL with their carrier bug. The ecology of C. japonica In the present study, we demonstrated that C. japonica DL is currently under investigation, but, to date, the nematode has specifically associate with their carrier bug P. japonensis, but not been isolated only from P. japonensis. Parastrachia japonensis

P. japonensis Fig.4. Arrest of dispersal experiments: comparisons between P. 100 M. japonensis japonensis and M. japonenesis. Caenorhabditis japonica DL 90 Control dispersal was arrested or they crawled on the extract containing body surface components of P. japonensis but not on the M. 80 japonensis or hexane alone. The mean response to P. japonensis 70 was significantly higher than that to M. japonensis or hexane alone 60 at any time point. N=31 (P. japonensis), 10 (M. japonensis) and 40 50 (control). ANOVA, F=78.083, P<0.0001. Error bars indicate s.d. 40 30 20 10 0 Nematodes in inoculation circle (%) 0 102030405060 Time after nematode inoculation (min)

THE JOURNAL OF EXPERIMENTAL BIOLOGY 572 The Journal of Experimental Biology 216 (4) feeds only on the drupes of Schoepfia jasminodora and is one of Ali, J. G., Alborn, H. T. and Stelinski, L. L. (2011). Constitutive and induced subterranean plant volatiles attract both entomopathogenic and plant parasitic only two species comprising the Parastrachia (Schaefer nematodes. J. Ecol. 99, 26-35. et al., 1991; Sweet and Schaefer, 2002). The specialized trophic Baird, S. E. (1999). Natural and experimental associations of with Trachelipus rathkii and other terrestrial isopods. Nematology 1, 471- ecology and evolutionary independence of P. japonensis may have 475. helped to develop the specific kairomone for C. japonica. Bargmann, C. I. (2006). Chemosensation in C. elegans. In WormBook (ed. The C. elegans Research Community), pp. 1-29. doi.org/10.1895/wormbook. However, further studies on the characterization and identification Bargmann, C. I., Hartwieg, E. and Horvitz, H. R. (1993). Odorant-selective of the kairomones are needed to understand the evolution of this and mediate olfaction in C. elegans. 74, 515-527. species-specific phoresy and the mechanisms of host recognition. Barrière, A. and Félix, M. A. (2005). High local genetic diversity and low outcrossing rate in Caenorhabditis elegans natural populations. Curr. Biol. 15, Although phoresy is commonly found in nematodes, there are 1176-1184. very few reports on -specific or sex-biased associations; Bedding, R. A. (1984). Nematode parasites of Hymenoptera. In Plant and Insect Nematodes (ed. W. R. Nickle), pp. 755-795. New York, NY: Marcel Dekker. female-biased association has been reported in the Fergusobia Burr, A. H. J. and Robinson, A. F. (2004). Locomotion behaviour. In Nematode nematode/Fergusonia fly mutualism (Currie, 1937; Davies et al., Behavior (ed. R. Gaugler and A. L. Bilgrami), pp. 25-62. Wallingford, UK: CABI Publishing. 2001) and Sphaerularia nematode/Bombus bee or Vespa hornet Croll, N. A. and Matthews, B. E. (1977). Biology of Nematodes. New York, NY: Wiley. parasitism (for reviews, see Bedding, 1984; Sayama et al., 2007). Currie, G. A. (1937). Galls on Eucalyptus trees. A new type of association between flies and nematodes. Proc. Linn. Soc. N. S. W. 62, 147-174. However, no information is available on the mechanisms of Davies, K. A., Makinson, J. and Purcell, M. F. (2001). Observations on the female-biased association. In the field, C. japonica DL are development and parasitoids of Fergusonina/Fergusobia galls on Melaleuca quinquenervia (Myrtaceae) in Australia. Trans. R. Soc. S. Aust. 125, 45-50. exclusively found on female P. japonensis. We expected that there Grewal, P. S., Gaugler, R. and Selvan, S. (1993). Host recognition by would be some difference in attraction to male and female P. entomopathogenic nematodes: behavioral response to contact with host feces. J. Chem. Ecol. 19, 1219-1231. japonensis, but no significant difference was observed in either Hallem, E. A., Dillman, A. R., Hong, A. V., Zhang, Y., Yano, J. M., DeMarco, S. F. the loading or chemoattraction experiments. These results suggest and Sternberg, P. W. (2011). A sensory code for host seeking in parasitic nematodes. Curr. Biol. 21, 377-383. that attraction of nematodes to both male and female bugs does Hara, A. H., Lindegren, J. E. and Kaya, H. K. (1981). Monoxenic mass production not differ. The differences in nematode association in the field of the entomogenous nematode, Neoaplectana carpocapsae Weiser, on dog food/agar medium. Adv. Agri. Tech. 16, 8. could be due to factors other than chemoattraction. One possibility Herrmann, M., Mayer, W. E. and Sommer, R. J. (2006). Nematodes of the genus is the difference in DL survival on male and female bugs. Male Pristionchus are closely associated with scarab beetles and the Colorado potato beetle in Western Europe. Zoology 109, 96-108. bugs are smaller than female bugs and DL on male bugs may Hiltpold, I., Baroni, M., Toepfer, S., Kuhlmann, U. and Turlings, T. C. (2010). face more severe desiccation than those on female bugs, resulting Selection of entomopathogenic nematodes for enhanced responsiveness to a volatile root signal helps to control a major root pest. J. Exp. Biol. 213, 2417-2423. in the death of DL; these dead DL would then fall off the bug. Hong, R. L. and Sommer, R. J. (2006). Chemoattraction in Pristionchus nematodes Another possible reason is behavioral differences between the and implications for insect recognition. Curr. Biol. 16, 2359-2365. Huettel, R. N. (1986). Chemical communicators in nematodes. J. Nematol. 18, 3-8. sexes. We observed grooming behavior in P. japonensis Kiontke, K. (1997). Description of (Caenorhabditis) drosophilae n. sp. and individuals in which they would open their wings and rub their R. (C.) sonorae n. sp. (Nematoda: Rhabditida) from saguaro cactus rot in Arizona. body, antennae and legs with their hind legs. Thus, DL on male Fundam. Appl. Nematol. 20, 305-315. Kiontke, K. and Sudhaus, W. (2006). Ecology of Caenorhabditis species. In bugs could be removed during grooming. The frequency of the WormBook (ed. The C. elegans Research Community), pp. 1-14. grooming behavior and/or the pattern of grooming may differ doi.org/10.1895/wormbook. Kiontke, K., Hironaka, M. and Sudhaus, W. (2002). Description of Caenorhabditis between sexes. Additional C. japonica and P. japonensis studies japonica n. sp. (Nematoda: Rhabditida) associated with the burrower bug in the field are necessary to understand the female-specific Parastrachia japonensis (: Cydnidae) in Japan. Nematology 4, 933-941. Matsuura, T., Sato, T. and Shingai, R. (2005). Interactions between Caenorhabditis association of DL, including studies on ecological and behavioral elegans individuals during chemotactic response. Zoolog. Sci. 22, 1095-1103. differences as well as on survivorship. Rasmann, S., Köllner, T. G., Degenhardt, J., Hiltpold, I., Toepfer, S., Kuhlmann, U., Gershenzon, J. and Turlings, T. C. (2005). Recruitment of entomopathogenic nematodes by insect-damaged roots. Nature 434, 732-737. LIST OF ABBREVIATIONS Riddle, D. L. (1988). The dauer lava. In The Nematode Caenorhabditis elegans (ed. W. B. Wood), pp. 393-412. New York, NY: Cold Spring Harbor Laboratory Press. CI chemoattraction index Riga, E. (2004). Orientation behaviour. In Nematode Behaviour (ed. R. Gaugler and A. DL dauer larvae L. Bilgrami), pp. 63-90. Wallingford, UK: CABI Publishing. NGM nematode growth medium Sayama, K., Kosaka, H. and Makino, S. (2007). The first record of infection and sterilization by the nematode Sphaerularia in hornets (Hymenoptera, Vespidae, Vespa). Insectes Soc. 54, 53-55. ACKNOWLEDGEMENTS Schaefer, C. W., Zheng, L. and Tachikawa, S. (1991). A review of Parastrachia C. elegans and E. coli OP50 strains were supplied by the Caenorhabditis (Hemiptera: Cydnidae: Parastrachiinae). Orient. Insects 25, 131-144. Center. Sudhaus, W. (2008). Evolution of insect parasitism and diplogasterid nematodes. In Advances in Arachnology and : Papers Dedicated to Professor Bozidař P. M. Curć ič ́ (Monographs, Vol. 12) (ed. S. E. Makarov and R. N. FUNDING Dimitrijevic),́ pp. 143-161. Belgrade, RS: Institute of Zoology, Faculty of Biology, University of Belgrade. This research received no specific grant from any funding agency in the public, Sweet, M. H. and Schaefer, C. W. (2002). Parastrachiinae (Hemiptera: Cydnidae) commercial, or not-for-profit sectors. raised to family level. Ann. Entomol. Soc. Am. 95, 441-448. Tanaka, R., Okumura, E. and Yoshiga, T. (2010). A simple method to collect phoretically active dauer larvae of Caenorhabditis japonica. Nihon Senchu Gakkai REFERENCES Shi 40, 7-12. Ali, J. G., Alborn, H. T. and Stelinski, L. L. (2010). Subterranean herbivore- Yoshiga, T., Ishikawa, Y., Tanaka, R., Hironaka, M. and Okumura, E. (2013). induced volatiles released by citrus roots upon feeding by Diaprepes abbreviatus Species-specific and female host-biased ectophoresy in the roundworm recruit entomopathogenic nematodes. J. Chem. Ecol. 36, 361-368. Caenorhabditis japonica. Naturwissenschaften (doi: 10.1007/s00114-013-1011-z).

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