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

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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]

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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

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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

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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. Solid larval frass (100 mg) of the four Ostrinia species was bioassayed directly by sandwiching it between the layers of a piece of cotton (4 × 4 × 0.1 cm). A blank piece of cotton was used as the control. Only one mated female was allowed to oviposit in each fabric screen cage to facilitate the checking of mating status, and also to avoid any possible influence on oviposition by other females. The following morning, the eggs (O. zealis)oregg masses (O. furnacalis, O. scapulalis, and O. latipennis) laid on each half of filter paper or piece of cotton were counted. O. zealis females usually laid 30–60 eggs, and females of the other species laid 3–6 egg masses on the following night after copulation. Chromatography and Chemical Identification. Polar lipid fractions from larval frass of the four Ostrinia species and from fresh diet were dried under a stream of nitrogen and redissolved, respectively, in hexane containing 1 ng/µl of n-heptadecane as an internal standard. An aliquot (2 µl) of solution was in- jected into a gas chromatograph (GC-17A, Shimadzu, Kyoto, Japan) fitted with an FID, a split/splitless injector and a fused silica capillary column (DB-Wax or DB-35, 30 m × 0.25 mm i.d., film thickness 0.25 µm, J&W Scientific, Folsom, CA). The oven temperature was controlled as follows. DB-Wax: maintained at 100◦C for 2 min, programmed to increase at 15◦C/min to 220◦C, then 5◦C/min to 230◦C, and held at 230◦C for 40 min; DB-35: 100◦C for 2 min, increased at 20◦C/min to 310◦C, then at 2◦C/min to 330◦C, and held at 330◦C until all components eluted. The carrier gas was nitrogen. The quantity of each compound was calculated on the basis of the peak area and calibrated by comparing it with that of heptadecane. The polar lipid fractions were analyzed by GC–mass spectrometry (GC–MS) directly or after derivatization with N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) according to the method of Shepherd et al. (1995). A QP-5050A GC–MS (Shimadzu) system equipped with a DB-Wax or DB-35 column was run under the same temperature conditions as described for GC analysis. Helium was used as carrier gas. The mass spectrometer was used in the electron impact mode (70 eV) and scanned over the mass range 40–600 m/z. The interface and ion source tem- peratures were 250 and 270◦C, respectively. Fatty acids were tentatively identified by matching their mass spectra with those in the NIST Library using the software CLASS-5000 (Shimadzu), and further P1: KEE Journal of Chemical Ecology [joec] pp1268-joec-489984 August 5, 2004 17:40 Style file version June 28th, 2002

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veriﬁed by comparison of the diagnostic ions and the GC retention time with those of the respective authentic standard. Statistical Analysis. In all tests, the number of eggs or egg masses for control (C) and treatment (T) were summed respectively for each replicate. The result is presented as an avoidance index (Ai): Ai = (C − T )/(C + T ) (Renwick and Radke, 1980). Ai = 1 indicates complete rejection of the test material. The null hypothesis that equal number of eggs or egg masses were laid for control and treatment (Ai = 0) was examined using the t test at α = 0.05. Differences in the total amount of fatty acids in the frass of four Ostrinia species were examined with a one-way ANOVA.

RESULTS

Oviposition-Deterring Effect of Larval Frass. The water suspensions of O. zealis larval frass significantly deterred oviposition of conspecific females (FS in Figure 1). The water suspension of fresh artificial diet (DS) had a similar effect but the avoidance index was substantially lower. When exposed to the halves of filter paper treated with larval frass and fresh diet simultaneously, ovipositing females tended to select the latter significantly (Figure 1). Larval frass from the four Ostrinia species sandwiched between the layers of a piece of cotton significantly deterred conspecific females from ovipositing

FIG. 1. Oviposition-deterring effect of water suspensions of O. zealis larval frass (FS) and artificial diet (DS) in dual-choice experiments. One milliliter of suspension (100 mg/ml) and water (W) was applied to a pair of halves of filter paper. See text for oviposition avoidance index. Values represent the mean ± SD. Numbers of replicates are shown inside the bars. ∗significantly different from 0 (t test, P < 0.05). P1: KEE Journal of Chemical Ecology [joec] pp1268-joec-489984 August 5, 2004 17:40 Style file version June 28th, 2002

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FIG. 2. Oviposition-deterring effect of larval frass from four Ostrinia species on respective females in dual-choice experiments using a pair of square cotton pieces (4 × 4 × 0.1 cm): a piece sandwiching 100 mg of larval frass was tested against a blank piece. See text and the legend to Figure 1 for further details.

(Figure 2). The avoidance index of the frass was similar among the four species (0.39–0.43). The avoidance indexes of the fresh and stored (at −20◦C) frass were also not significantly different (data not shown). Fractionation of Active Chemicals. The acetone extract of O. zealis larval frass deterred oviposition of conspecifics (AE in Figure 3). Both hexane-soluble and residual fractions had significant oviposition-deterring effects. However, when the two fractions were offered simultaneously, females preferred ovipositing on the residual fraction (HF and RF in Figure 3). When the hexane-soluble fraction was further fractionated into neutral and polar lipid fractions, the latter deterred oviposition more strongly (NL and PL in Figure 3). GC-MS Analyses. The polar lipid fraction from O. zealis larval frass and that from the artificial diet were analyzed by GC and GC-MS (Table 1). The main components in larval frass were five free aliphatic fatty acids, palmitic, stearic, oleic, linoleic, and linolenic acids. Although the fresh artificial diet also contained these acids, their total amount was 3.3 times less than that in the frass (Table 1). Compositions of these fatty acids were similar among the four Ostrinia species (Figure 4). Linoleic acid was most abundant, followed by palmitic, linolenic, and oleic acid. Stearic acid was the least abundant. To- tal amounts of the fatty acids produced by the four Ostrinia species were 110– 180 µg/100 mg fresh frass, and did not vary significantly among species (ANOVA, P > 0.05). Effects of Authentic Free Fatty Acids. The oviposition-deterring effect of a synthetic mixture of the five fatty acids was tested at the ratio found in O. zealis larval frass. The mixture significantly deterred oviposition in the four Ostrinia species (Figure 5). P1: KEE Journal of Chemical Ecology [joec] pp1268-joec-489984 August 5, 2004 17:40 Style file version June 28th, 2002

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FIG. 3. Oviposition-deterring effect of fractions of O. zealis larval frass extract in dual- choice experiments. Extracts or fractions (equivalent to 100 mg of fresh frass) and cor- responding solvents were, respectively, applied to a pair of halves of ﬁlter paper. AE, A, HF, H, RF, NL, and PL indicate acetone extract, acetone, hexane-soluble fraction, hexane, residual fraction, neutral lipids, and polar lipids, respectively. See text for hexane-soluble fraction, residual fraction, neutral lipids, and polar lipids. See the legend to Figure 1 for further explanations.

DISCUSSION

The present study has shown that larval frass from four Ostrinia species fed on an artiﬁcial diet exhibited signiﬁcant oviposition-deterring effects. Compared with the avoidance index exhibited in A. segetum (Anderson and Lofqvist, 1996) and O. nubilalis (Dittrick et al., 1983), which was as high as 0.8, the values in our tests were low, 0.28–0.55. However, the lower avoidance index is partly attributable to the smaller amount of larval frass used for the bioassay in the present study (0.1 g

TABLE 1. AMOUNT OF CHEMICALS IN THE POLAR LIPID FRACTION FROM O. zealis FRASS AND IN FRESH DIET

µg/100 mg Chemical Diagnostic ions of trimethylsilyl derivative Frass of zealis Fresh diet

Palmitic acid 328(M), 313, 145,132, 129, 117, 75, 73 27.8 12.9 Stearic acid 356(M), 341, 145,132, 129, 117, 75, 73 13.9 1.3 Oleic acid 354(M), 339, 145,132, 129, 117, 75, 73 20.9 3.3 Linoleic acid 352(M), 337, 145,132, 129, 117, 75, 73 52.8 19.8 Linolenic acid 350(M), 335, 145,132, 129, 117, 75, 73 23.6 4.4 Total 139.0 41.7 P1: KEE Journal of Chemical Ecology [joec] pp1268-joec-489984 August 5, 2004 17:40 Style ﬁle version June 28th, 2002

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FIG. 4. The compositions of fatty acids in the polar lipid fraction from larval frass of four Ostrinia species analyzed by GC. C16:0,C18:0,C18:1,C18:2, and C18:3 represent palmitic, stearic, oleic, linoleic, and linolenic acids, respectively. Values represent the mean ± SD for four replicates (100% = total of the ﬁve fatty acids). P1: KEE Journal of Chemical Ecology [joec] pp1268-joec-489984 August 5, 2004 17:40 Style ﬁle version June 28th, 2002

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FIG. 5. Oviposition-deterring effect of a mixture of authentic fatty acids of the composition found in larval frass of O. zealis on four Ostrinia species. A hexane solution of fatty acids (150 µg) or hexane was applied to a pair of halves of ﬁlter paper. See text and legend to Figure 1 for further explanations.

fresh weight equivalent). In O. nubilalis and A. segetum, the highest reduction rate was obtained when 0.3 g and 0.6 g dry weight frass equivalent was used, respectively. When an ovipositing female of O. zealis was exposed to the filter paper treated with the water suspension of larval frass, it may have perceived the frass powder visually, or perceived its texture and/or the chemicals in it after making contact. However, the obvious deterring effects of sandwiched frass demonstrated that the deterrence is mostly attributable to volatile chemicals. Smaller amounts of oviposition-deterring fatty acids were found in fresh artificial diet. This accounts for the significant but lower oviposition deterrence of the fresh diet as compared with the larval frass. Larger amounts of the rel- evant fatty acids in frass as compared with fresh diet suggest that these acids are actively secreted by the larval gut. Analysis of the frass of larvae fed on an artificial diet free of these fatty acids should be useful in verifying this possibility. The avoidance index of the mixture of the five fatty acids was lower than that of the acetone extract, hexane-soluble fraction, or polar lipid fraction, suggesting that some unidentified chemicals have oviposition-deterring or synergistic effects. Further effort is necessary to identify these deterrents and/or synergists. In parallel, the relative deterrence of the five fatty acids must be determined to elucidate structure–activity relationships. Interestingly, similar aliphatic fatty acids have been identified as oviposition deterrents on the surface of egg masses of O. scapulalis and O. furnacalis (Li and P1: KEE Journal of Chemical Ecology [joec] pp1268-joec-489984 August 5, 2004 17:40 Style file version June 28th, 2002

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Ishikawa, unpublished). Moreover, oviposition-deterring effects of aliphatic fatty acids or their methyl esters have been reported in several species in Lepidoptera, Coleoptera, and Diptera. Gabel and Thiery (1996) demonstrated that C14:0 (myristic acid), C16:0,C16:1 (palmitoleic acid), C18:0,C18:1,C18:2, and C18:3 were the main deterrents in Lobesia botrana eggs. From Cydia pomonella eggs, C14:0,C16:0,C16:1, C18:0,C18:1,C18:2, and C18:3 were identified as oviposition deterrents (Thiery et al., 1995). In Helicoverpa armigera,C16:0 and C18:1 were identified as oviposition deterrents (Li et al., 2001). Methyl esters of C16:0,C16:1,C18:0,C18:1, and C18:2 were reported to be oviposition deterrents from eggs of O. nubilalis (Thiery and Le Quere, 1991). In Coleoptera, the main oviposition deterrent from Callosobruchus maculates was C18:1 (Sakai et al., 1986). Similarly, C16:0,C18:0,C18:1,C18:2, and C18:3 were identified in extract of the seventh urotergite gland, the secretory organ of oviposition deterrents, of Ceutorhynchus assimilis (Mudd et al., 1997). Moreover, C18:1 was the most effective repellent to ovipositing Culex quinquefasciatus among Z-9-alkenoic acids (Hwang et al., 1984). Thus, straight chain fatty acids, especially C16:0,C16:1,C18:0,C18:1,C18:2, and C18:3, seem to have broad repelling and deterring effects on oviposition. Why do so many insect species belonging to different orders use similar fatty acids as oviposition deterrents? There should be some causality behind this phenomenon. Further studies are necessary to shed light on this issue. A question that must be addressed is the effectiveness of these fatty acids in the field. Although the larvae of Ostrinia usually feed inside the stem of host plants, most frass produced is conveyed outside through a hole made on the stem and often built up near it. Therefore, it is reasonable to assume that female adults perceive the oviposition deterrents emanating from the frass when they fly around the host plants for egg laying. However, this needs to be verified with field experiments. Common usage of the same fatty acids as oviposition deterrents among the four Ostrinia species suggests that this trait was acquired in a common ancestor. It would be interesting to investigate whether all extant Ostrinia (21 species world- wide; Mutuura and Munroe, 1970; Ohno, 2003) have similar fatty acids in their larval frass. Ecologically, since pre-occupation of a host plant by larvae, includ- ing different species, suggests strong competition for food, avoiding oviposition in response to common chemical signals is probably adaptive for an Ostrinia female. In fact, several host plants are commonly utilized by multiple Ostrinia species (Ishikawa et al., 1999; S. Ohno and Y. Ishikawa, unpublished), and, thus, competition between heterospecific larvae can occur in nature.

Acknowledgments—A research fellowship to G. Li from the Japanese Society for the Promotion of Science (No. 1402209) is acknowledged. We thank Drs. S. Tatsuki and S. Hoshizaki of our laboratory for useful discussions during the course of this research, as well as Mr. J. Tabata and Ms. M. Fukuzawa for helping with GC and GC-MS analyses and insect rearing. P1: KEE Journal of Chemical Ecology [joec] pp1268-joec-489984 August 5, 2004 17:40 Style ﬁle version June 28th, 2002

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