OVIPOSITION DETERRENTS in LARVAL FRASS of FOUR Ostrinia SPECIES FED on an ARTIFICIAL DIET

P1: KEE Journal of Chemical Ecology [joec] pp1268-joec-489984 August 5, 2004 17:40 Style file version June 28th, 2002 中国科技论文在线 http://www.paper.edu.cn Journal of Chemical Ecology, Vol. 30, No. 7, July 2004 (C 2004) OVIPOSITION DETERRENTS IN LARVAL FRASS OF FOUR Ostrinia SPECIES FED ON AN ARTIFICIAL DIET GUOQING LI1,2 and YUKIO ISHIKAWA1,∗ 1Laboratory of Applied Entomology Graduate School of Agricultural and Life Sciences The University of Tokyo, Tokyo 113-8657, Japan 2Key Laboratory of Monitoring and Management of Plant Diseases and Pests Ministry of Agriculture, Nanjing Agricultural University, Nanjing, China (Received September 25, 2003; accepted March 16, 2004) Abstract—Behavioral bioassays have shown that volatile oviposition-deterring chemicals are present in the frass of Ostrinia zealis, O. furnacalis, O. scapulalis, and O. latipennis larvae fed on an artificial diet. These chemicals were extractable with acetone, and could be partitioned into a polar lipid fraction. This fraction mainly included palmitic, stearic, oleic, linoleic, and linolenic acids. No significant differences among the four Ostrinia species were found in the amount and composition of these free fatty acids. A mixture of the five authentic fatty acids of the composition found in the larval frass of O. zealis exhibited significant oviposition-deterring effects on all four species. Key Words—Ostrinia, larval frass, oviposition deterrents, fatty acids. INTRODUCTION In many insect species, chemicals contained in larval frass deter oviposition of conspecific females. The ecological significance of these deterrents is to avoid competition among conspecific larvae for food by claiming pre-occupation of the host. Deterring effects of larval frass have been verified in both phytophagous and entomophagous insects. The former includes lepidopterans such as yellow cutworm Agrotis segetum (Anderson and Lofqvist, 1996), pineapple borer Thecla basilides (Rhainds et al., 1996), Egyptian cotton leaf worm Spodoptera littoralis (Hilker and Klein, 1989; Klein et al., 1990; Anderson et al., 1993), fall armyworm S. frugiperda (Williams et al., 1986), cabbage looper Trichoplusia ni (Renwick and Radke, 1980), European corn borer Ostrinia nubilalis (Dittrick et al., 1983), ∗ To whom correspondence should be addressed. E-mail: [email protected] 1445 0098-0331/04/0700-1445/0 C 2004 Springer Science+Business Media, Inc. 转载 P1: KEE Journal of Chemical Ecology [joec] pp1268-joec-489984 August 5, 2004 17:40 Style file version June 28th, 2002 中国科技论文在线 http://www.paper.edu.cn 1446 LI AND ISHIKAWA and coleopterans such as Monochamus alternatus (Anbutsu and Togashi, 2002). The oviposition deterrence of larval frass has been reported recently in two entomophagous ladybird beetles, Harmonia axyridis and Propylea japonica (Agarwala et al., 2003). In all species listed above, larval frass decreased oviposition of conspecific females significantly, with the highest reduction rate of about 90% in A. segetum (Anderson and Lofqvist, 1996) and O. nubilalis (Dittrick et al., 1983). Chemical identification of oviposition deterrents is a primary step for further studies to explore the mode of communication, to infer the evolution of deterrents, and to develop applications in pest control. Reports on this aspect, however, are few. To the best of our knowledge, there has been only one paper on the chemical identification of deterrents in larval frass; a mixture of six compounds, benzaldehyde, carvacrol, eugenol, nerolidol, phytol, and thymol, was identified as an oviposition deterrent in frass of S. littoralis larvae fed on cotton leaves (Klein et al., 1990). In the genus Ostrinia (Crambidae), eight species, O. furnacalis, O. scapulalis, O. orientalis, O. zealis, O. zaguliaevi, O. palustralis, O. ovalipennis, and O. latipennis, currently inhabit Japan (Ishikawa et al., 1999; Ohno, 2003), and the majority of them are cultured in our laboratory. Among these species, O. latipennis (knotweed borer) and O. zealis (burdock borer) are both oligophagous, but their host plant ranges differ largely; O. latipennis usually feeds on knotweeds Reynoutria spp. (Polygonaceae), whereas O. zealis feeds on Compositae plants such as burdock Arctium lappa, and thistles Cirsium spp. (Ishikawa et al., 1999). O. furnacalis (Asian corn borer) is known as an important pest of maize in Asia, but this species is actually polyphagous and feeds on various plants such as ginger Zingiber officinalis (Zingiberaceae), docks Rumex (Polygonaceae), and cocklebur Xanthium (Compositae) (Hattori and Mutuura, 1987). O. scapulalis (adzuki bean borer) is also polyphagous and feeds on many plants such as leguminous crops, hop Humulus lupulus (Moraceae), cockleburs Xanthium, and docks Rumex,but O. scapulalis is distinct from O. furnacalis in that it does not feed on maize (Hattori and Mutuura, 1987; Ishikawa et al., 1999). As exemplified by these four species, host plant preference in Ostrinia is diverse. As mentioned above, European corn borer O. nubilalis, which is not found in the Far East, is known to produce oviposition deterrents in larval frass (Dittrick et al., 1983). Thus, Ostrinia species in Japan offer potential material for compar- ative studies on oviposition deterrents in larval frass. We addressed the following questions: (1) Do Japanese Ostrinia species produce oviposition deterrents in the frass? and (2) What are their interspecific effects? We report findings on oviposition deterrents from the larval frass of four Ostrinia species with different host plant preference, O. latipennis, O. zealis, O. furnacalis, and O. scapulalis. METHODS AND MATERIALS Insects and Frass Collection. Female adults of O. scapulalis and O. furnacalis were collected in the field at Matsudo (35.8◦N, 139.9◦E), Japan in June P1: KEE Journal of Chemical Ecology [joec] pp1268-joec-489984 August 5, 2004 17:40 Style file version June 28th, 2002 中国科技论文在线 http://www.paper.edu.cn OVIPOSITION DETERRENTS IN FRASS OF Ostrinia 1447 and August 2002, respectively. O. zealis females were netted at Kawaji (36.9◦N, 139.7◦E) in July 2002. Egg masses of O. latipennis were collected from leaves of the giant knotweed Reynoutria sachalinensis, at Towa (39.9◦N, C14:1.2◦E) in July 2002. Larvae were reared as broods in rearing jars (8-cm diam) filled with 200 g of commercial diet for insects (Insecta LF, Nosan Corp., Yokohama, Japan). The environmental conditions were 24 ± 1◦C, 15:9 hr L:D photocycle regime, and 60% RH. Females and males were separated during the pupal stage based on the morphology of the terminal abdominal segments. Mated females were obtained by introducing several pairs of 1- or 2-day-old males and females into a fabric screen cage (20 × 20 × 20 cm) and allowing them to mate for 2 nights. To minimize inbreeding, females from one family were put with males from another in each screen cage. Fifth-instar larvae were confined in plastic dishes (10-cm diam) containing pieces of fresh artificial diet (Insecta LF) and maintained in a rearing chamber under the conditions described above. Larval frass was collected daily and stored in 50-ml glass bottles at −20◦C until use. The common artificial diet was fed to the larvae of four species to facilitate comparison of deterrents among species; otherwise differences would be obscured by the natural variations in chemical compositions of the host plants. The precise composition of the artificial diet is not made public, but it is reported to contain mulberry leaf powder, soybean protein, starch, sugar, cereals powder, minerals, vitamins, citric acid, agar, and food antiseptics (water 72–76%; fiber <3.9%; protein >6.0%; ash <3.9%; fat <1.1%) (brochure from the supplier, Nosan Corp.). Chemicals and Preparation of Test Materials. n-Heptadecane (>99%) was purchased from Tokyo Kasei Kogyo Co., Japan. Hexadecanoic acid (palmitic acid, C16:0), octadecanoic acid (stearic acid, C18:0), (Z)-9-octadecenoic acid (oleic acid, C18:1), (Z,Z)-9,12-octadecadienoic acid (linoleic acid, C18:2), and (Z,Z,Z)- 9,12,15-octadecatrienoic acid (linolenic acid, C18:3) were purchased from Sigma (St. Louis, MO). Distilled water and analytical grade organic solvents were used to prepare the following test materials: (1) a water suspension of O. zealis larval frass, (2) a water suspension of fresh artificial diet, (3) an acetone extract of O. zealis larval frass, (4) a hexane-soluble fraction, which was obtained by drying the acetone extract under a stream of nitrogen, redissolving it in hexane, and filtration, and (5) a residual fraction, which was made by dissolving the residue on the filter in acetone. The hexane-soluble fraction was loaded on a Sep-Pak Plus NH2 cartridge (Waters, Milford, MA), and neutral (6) and polar (7) lipids were eluted with a 2:1 mixture of chloroform and 2-propanol (20 ml), and 2% acetic acid in diethyl ether (20 ml), respectively. Bioassay. Bioassays were carried out in the environmental conditions out- lined above. Before the test, about 20 pairs of newly emerged females and males were placed in a fabric screen cage (20 × 20 × 20 cm) for arbitrary mating for 2 nights to obtain mated females for the bioassay. After each experiment, the P1: KEE Journal of Chemical Ecology [joec] pp1268-joec-489984 August 5, 2004 17:40 Style file version June 28th, 2002 中国科技论文在线 http://www.paper.edu.cn 1448 LI AND ISHIKAWA bursa copulatrix was removed from each female and dissected to check for the presence of a spermatophore. Only those data obtained using mated females that had at least one spermatophore were used for statistical analyses. All bioassays were carried out using a dual-choice test in the fabric screen cage. Test solution/suspension equivalent to 100 mg of fresh frass and the same amount of solvent were, respectively, applied to cut halves of a filter paper circle (90-mm diam). After evaporation of the solvent, the halves were taped together and stapled on the ceiling of the fabric screen cage 2 hr before the start of scotophase.

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