Journal of Chemical Ecology, Vol. 15, No. 12, 1989

ALARM PHEROMONE OF PENTATOMID BUG, Erthesina fullo THUNBERG (HEMIPTERA: PENTATOMIDAE)

R. KOU, D.S. T A N G , and Y.S. C H O W

Institute of Zoology Academia Sinica Taipei, Taiwan 11529 Republic of China (Received November 14, 1988, accepted February 2, 1989) Abstract--In the pentatomid bug, Erthesina fullo Thunberg, the odor of male metathoracic scent gland elicits an alarm response, making the male individualsof the same species alert and disperse; the alarm responseof males is more obvious than that of females. Chemicalcompositionof the glandular secretion was identifiedby gas chromatography and mass spectrometry data in comparison with authentic compounds. No sexual dimorphism exists in the glandular composition in this species. A total of 9 compounds [(E)-2hexenal, (E)-4-keto-2-hexenal, (E)-2-hexenyl acetate, n-undecane, n-dodecane, (E)-2-decenal, n-tridecane, (E)-2-decenyl acetate, and n-pentadecane] are identified,among which n-tridecaneand (E)-4-keto-2-hexenalcomprised nearly 70% of the total secretion in both females and males. Key Words--Alarm pheromone, metathoracic scent gland, Hemiptera, Pentatomidae, Erthesina fullo.

INTRODUCTION In Hemiptera, the existence e,f an alarm pheromone was first reported by Calam and Youdeowei (1968); (E)-2-hexenal and some other aldehydic scent components of fifth instar larvae of Dysdercus intermedius Distant had an alaram effect to both larvae and adults. Ishiwatari (1974) also indicated that (E)-2hexenal had an alarm effect on three species of pentatomid bugs, Euryderna rugosa Motshulsky, E. pulchra (Westwood), and Nezara viridula (L.). Lockwood and Story (1985) reported that the first instar N. viridula use n-tridecane as a bifunctional pheromone which caused dispersal at high concentrations (10 ~ and 101 individual equivalent) and aggregation at low concentration (10 -2 indi2695 0093-0331/89/1200-2695506.00/0 9 1989 Plenum Publishing Corporation

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vidual equivalent). Recently, the metathoracic scent gland secretion of adult N. viridula was found to function as an alarm pheromone and two components, (E)-2-hexenal and (E)-2-hexenyl acetate, significantly increased movement during the first minute of exposure (Lockwood and Story, 1987). The Erthesina fullo Thunberg (Hemiptera: Pentatomidae) is a major pest of pine trees and nato trees (hardwood) in Taiwan. Both males and females of this species also produce a secretion from the metathoracic scent gland that causes conspecific adults to drop from a plant or scurry and fly away from the emission site. This phenomenon indicates that the secretion may function as an alarm pheromone in E. fullo. In this study, we examined the biological activity and chemistry of the male and female metathoracic scent gland secretions in pentatomid bugs in Taiwan.

METHODS AND MATERIALS

Insects and Gland Extracts. Adults and late fifth instars of the E. fullo were collected during summer from luchu pine trees, Pinus luchuensis Mayer, and Formosan nato trees, Palaquium formosanum Hayata, in a suburb of north Taiwan. In the laboratory, nato tree twigs and 10% honey solution were supplied. The insectory was kept at 27-28~ on a 12 : 12 hr light-dark cycle. The adults were disturbed by using a forceps to strike their heads, then a clean filter paper (5 • 5 mm) was placed near the orifice of the metathoracic scent gland on the ventral metathoracic surface. The discharge sprayed on the filter paper was extracted with methylene chloride. Because different numbers of filter papers of male or female extract were immersed in different volumes of methylene chloride, a total of 136 males and 108 females were extracted in 3500 tzl and 2400/~1 methylene chloride, respectively. The extract was stored under 0~ for later bioassay and chemical analysis. Bioassay of the Gland Extract. Two hours before the bioassay, one female or male was placed in each 17-cm • 9-cm glass jar with a piece of host twig. A total of 70 such glass jars of males or females were prepared for the six different concentrations and control. During bioassay, a small amount of female and male stock extract was taken out with a syringe and solvent was added to make the dilution equal to 1 individual equivalent (IE) per 30 /zl, and then serially diluted solutions of different IE (10 -5, 10 -4, 10 -3, 10 -2, 10 -1 , and 1 IE) were obtained. Each solution was injected with a syringe onto the inside wall of a pipet tip, and the air was expelled with a rubber bulb toward each test individual on the host plant in the glass jar. The number of individuals that dropped from the host plant or began to move away was the criterion of the biological activity. The experiment had three replicates, so a total of 30 insects

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were used at each concentration. The Kruskal-Wallis one-way analysis of variance by ranks (Siegel, 1956) was used to analyze the results. Chemical Analysis. Gas chromatography (GC) of the extract was performed on three capillary columns. A Varian 3700 GC equipped with a flame ionization detector was used with a 4-m fused silica capillary column coated with a 0.25-/xm film of DB-1 phase, a 25-m vitreous silica capillary column coated with 1.5-#m film of DB-5 phase, and a 35-m fused silica capillary column coated with 0.25-/zm film of Carbowax 20 M phase. Helium, at a flow rate of 30 cm/min, was the carrier gas. In the DB-1 and DB-5 column, the extract was run at 50~ for 2 min to 250~ at 5 ~ In Carbowax 20 M, the extract was run at 60~ for 2 min to 200~ at 5~ Gas chromatographic-mass spectrometric (GC-MS) analysis was conducted using a Finnigan 4600 mass spectrometer. The GC-MS data were obtained using a 30-m x 0.25-mm (ID) column of DB-5 phase. The column temperature was held at 50~ for 2 min to 300~ at 30 ~ C/min using helium as carrier gas. Electron impact (EI) mass spectra were collected at 70 eV with separator and source at 150~ Each compound was identified by comparison of its mass spectrum with the published mass spectrum or the mass spectrum of the authentic standards (Aldrich and Yonke, 1975; Cornu and Massot, 1979; Kitamura et al., 1984; Staddon et al., 1987). Subsequently, all the compounds identified by mass spectral data were cross-checked by comparison of the GC retention of the natural product to that of an authentic standard using the three capillary columns under the same GC conditions as described above. Standards of (E)-2-hexenal, (E)2-hexenyl acetate, n-undecane, n-dodecane, n-tridecane, and n-pentadecane were purchased from Sigma Chemical Co., (St. Louis, Missouri). (E)-4-Keto2-hexenal, (E)-2-decenal, and (E)-2-decenyl acetate were supplied by J.R. Aldrich (Insect Physiology Laboratory, USDA-ARS, Beltsville, Maryland). The relative proportions of these nine compounds in the scent secretion was ascertained from GC peak-area integration.

RESULTS

Bioassay of Gland Extract. The bioassay results showed that females are not as responsive as males to the metathoracic scent extraction (Figure 1). Although the male alarm response to 1 IE female scent extract was 85.0%, no significant difference was found at all tested female concentrations (Figure 1A), and the alarm response of females to female scent extract was not-significant at all tested female concentrations (Figure 1A). The alarm response of males to 1 IE male scent extract was 100.0%, significantly different from the alarm

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response to 10 .5 male concentration ( P < 0.05, Figure 1B); the alarm response of females to male scent extract was almost nonsignificant at all tested male concentrations (Figure 1B). Chemical Analysis. Gas chromatographs of the metathoracic scent gland secretions of male and female are qualitatively alike (Figure 2). The secretion was composed of straight-chain alkanes [n-undecane (4), n-dodecane (5), n-tridecane (7), and n-pentadecane (9)] and a, /3-unsaturated carbonyl compounds [(E)-2-hexenal (1), (E)-4-keto-2-hexenal (2), (E)-2-hexenyl acetate (3), (E)-2-decenal (6), and (E)-2-decenyl acetate (8)1. The mass spectral data of these compounds are listed in Table 1. The relative proportion of compounds in the scent secretion was the same in both females and males (Table 2), in which n-tridecane and (E)-4-keto-2-hexenal comprised nearly 70% of the total secretion. The other two unsaturated aldehydes, (E)-2-hexenal and (E)-2-decenal, comprised nearly 20% of the total secretion; the unsaturated acetates, (E)-2hexenyl acetate and (E)-2-decenyl acetate, comprised nearly 8% of the total secretion; the other straight-chain alkanes only comprised nearly 2 % of the total secretion.

DISCUSSION

Our work indicates that the male metathoracic scent gland secretion of E. fullo, functions as an alarm pheromone, which is concentration dependent, and induced obvious dispersal response at 1 IE in males. The females do not show significant alarm response to their own and male's secretion. Chemical defense against predation had been reported as the most important function of the scent glands of the pentatomids Hemiptera-Heteroptera (Blum, 1981), but in the laboratory, the phenomenon that E. fullo was preyed on by mantids without any defensive effect from the metathoracic gland secretion was usually observed. The biological activity of female metathoracic scent gland secretion and the reason why females are not as responsive as males still needs further study. The metathoracic scent gland secretions of male and female E. fullo are nearly identical. The composition of this glandular secretion is similar to that of other pentatomids (Gilby and Waterhouse, 1965, Staddon et al., 1987). Among all identified compounds, (E)-2-hexenal, n-tridecane, and (E)-2-hexenyl acetate had been reported as alarm pheromones as mentioned before. Calam and Youdeowei (1968) suggested that the saturated hydrocarbons such as n-undecane, n-dodecane, n-tridecane, and n-pentadecane may function as relatively nonvolatile solvents for the active alarm pheromone components and as spreading and wetting agents in the pyrrhocorid, D. intermedius. Kitamura et al. (1984) also indicated that in Pentatomidae and Plataspidae hydrocarbons act as the solvent for aldehydes such as the oxohexenal. Moreover, Remold (1963),

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TABLE 1. MASS SPECTRAOF NINE COMPOUNDSIDENTIFIEDIN METATHORACICSCENT GLAND SECRETIONOF Erthesina fullo Mass spectral data, M/Z (intensity, %)

Compound (E)-2-Hexenal (E)-4-Keto-2-Hexenal (E)-2-Hexenyl acetate n-Undecane n-Dodecane (E)-2-Decenal n-Tridecane (E)-2-Decenyl acetate n-Pentadecane

69(100), 55(88), 41(86), 83(80), 57(55), 42(53), 84(43), 70(42), 86(34), 43(27), 56(22), 53(16), 80(15) 83(100), 55(69), 84(24), 57(22), 43(18), 56(12), 54(8), 67(7) 67(100), 82(95), 84(93), 43(70), 55(58), 57(57), 86(54), 83(40), 49(34), 56(22), 100(18), 54(17), 51(16), 41(15) 43(100), 57(90), 55(56), 41(39), 49(38), 84(33), 71(32), 83(29), 85(26), 56(25), 42(22) 57(100), 43(94), 71(42), 41(36), 85(33), 55(29), 56(26), 84(19), 49(18), 86(10) 55(100), 43(70), 57(64), 41(62), 70(58), 83(48), 56(43), 69(35), 54(21), 97(20), 53(18), 67(17) 57(100), 43(98), 7i(97), 85(73), 41(69), 56(50), 79(41), 55(34), 42(29), 84(22), 69(20) 43(100), 54(43), 55(42), 67(35), 81(28), 68(25), 41(24), 57(23), 96(22), 82(21), 95(18), 69(17), 110(14) 57(100), 43(80), 71(54), 85(42), 41(36), 55(32), 56(22), 42(15), 70(13), 99(10)

Filshie and Waterhouse (1969), and Kitamura et al. (1984) reported that many bugs possess an area of precisely elaborated cuticles surrounding the orifices of the metathoracic glands, and these structures are supposed to be the evaporative area. The present results show that n-tridecane and the unsaturated compounds

TABLE 2. PERCENTAGESOF COMPOUNDSIN METATHORACICSCENT SECRETIONOF FEMALE AND MALE Erthesina fullo

Compound

Female

Male

(E)-2-Hexenal (E)-4-Keto-2-hexenal (E)-2-Hexenyl acetate n-Undecane n-Dodecane (E)-2-Decenal n-Tridecane (E)-2-Decenyl acetate n-Pentadecane

8.4 21.8 2.4 0.7 1.6 12.1 46.3 5.6 0.1

6.4 19.5 1.6 0.6 1.6 12.9 50.5 5.9 0.1

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such as ( E ) - 4 - k e t o - 2 - h e x e n a l are the m a j o r constituents o f the scent secretion o f E. f u l l o . T h e r e l a t i v e p r o p o r t i o n o f ( E ) - 2 - h e x e n a l was 8 . 4 % and 6 . 4 % in f e m a l e and m a l e , r e s p e c t i v e l y , and the relative p r o p o r t i o n o f ( E ) - 2 - h e x e n y l acetate was o n l y 2 . 4 % and 1.6% in f e m a l e and m a l e , respectively (Table 2). A l t h o u g h the c o m p o s i t i o n o f the g l a n d u l a r secretion has b e e n identified, the precise quantity and b i o l o g i c a l f u n c t i o n o f each c o m p o u n d in the scent secretion still n e e d further study. Acknowledgments--We thank the National Science Council, Taiwan, R.O.C., for financial support; Dr. J.R. Aldrich for supplying the authentic compounds; and Ms. Y.H. Chen for help during the experiment.

REFERENCES ALDRICH, J.R., and YONKE, T.R. 1975. Natural products of abdominal and metathoracic scent glands of coreid bugs. Ann. Entomol. Soc. Am. 68:955-959. BLUM, M.S. 1981. Chemical Defenses of Arthropods. Academic Press, New York. CALAM, D.H., and YOUDEOWEI,A. 1968. Identification and functions of secretion from the posterior scent gland of fifth instar larva of the bug Dysdercus intermedius. J. Insect Physiol. 14:1147-1158. CORNU, A., and MASSOT,R. 1979. Compilation of Mass Spectral Data, 2 ed. William Clowers & Sons Limited, Beccles and London. FmsmE, B.K., and WATERHOUSE,D.F. 1969. The structure and development of a surface pattern on cuticle of the green vegetable bug Nezara viridula. Tissue Cell 1:367-385. GmBY, A.R., and WATERHOUSE,D.F. 1965. The composition of the scent of the green vegetable bug, Nezara viridula. Proc. R. Entomol. Soc. 162B.~105-120. ISHIWATARI,T. 1974. Studies on the scent of stink bugs (Hemiptera: Pentatomidae). I. Alarm pheromone activity. Appl. Entomol. Zool. 9:153-158. KITAMURA,C., WAKAMURA,S., and TAKAHASHI,S. 1984. Identification and functions of ventral glands secretion of some Heteroptera. Appl. Entomol. Zool. 19:33-41. LOCKWOOD,J.A., and STORY,R.N. 1985. Bifunctional pheromone in the first instar of the southern green stink bug, Nezara viridula (L.) (Hemiptera: Pentatomidae): Its characterization and interaction with other stimuli. Ann. Entomol. Soc. Am. 78:474-479. LOCKWOOD, J.A., and STORY, R.N. 1987. Defensive secretion of the southern green stink bug (Hemiptera Pentatomidae) as an alarm pheromone. Ann. Entomol. Soc. Am.. 80:686-691. REMOLD, H. 1963. Scent-glands of land-bugs, their physiology and biological function. Nature 198:764-768. SIEGEL, S. 1956. Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York. STADDON,B.W., THORNE,M.J., and KNIGHT, D.W. 1987. The scent glands and their chemicals in the aposematic cotton harlequin bugs, Tectocoris diophthalmus (Heteroptera: Scutelleridae), Aust. J. ZooL 35:227-234.

Alarm pheromone of pentatomid bug,Erthesina fullo Thunberg (Hemiptera: Pentatomidae).

In the pentatomid bug,Erthesina fullo Thunberg, the odor of male metathoracic scent gland elicits an alarm response, making the male individuals of th...
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