Journal of Chemical Ecology, Vol. 19, No. 4, 1993
BEHAVIORAL RESPONSES TO FOOD VOLATILES BY TWO SPECIES OF STORED-PRODUCT COLEOPTERA, Sitophilus oryzae (CURCULIONIDAE) and Tribolium castaneum (TENEBRIONIDAE)
T.W. PHILLIPS,* X.-L. JIANG, W.E. BURKHOLDER, J.K. PHILLIPS, and H.Q. TRAN USDA ARS, Stored-Product Insect Research Unit Department of Entomology, University of Wisconsin Madison, Wisconsin 53706
(Received September 3, 1992; accepted November 23, 1992)
Abstract--Laboratory experiments were conducted to study the behavioral activity of grain-derived volatiles as attractants and pheromone synergists for Sitophilus oryzae, an internal-feeding pest of sound grain, and Tribolium castaneum, an external-feeding pest of damaged grains and flour. Behavioral studies with two-choice pitfall bioassays determined that the fresh grain volatiles valeraldehyde, maltol, and vanillin were attractive to S. oryzae at various doses, hut T. castaneum were not attracted to any dose of any of these three compounds. When oils from pressed grains were bioassayed, sesame oil was significantly repellent and oat and wheat germ oils were attractive to S. oryzae. However, rice, soybean, oat, wheat germ, and corn oils were all attractive to T. castaneum. A commercial food product composed primarily of soybean oil and wheat germ was highly attractive to T. castaneum, but elicited no response from S. oryzae. A combination of the three grain volatiles valeraldehyde, maltol, and vanillin with the synthetic pheromone sitophinone was more attractive to S. oryzae than either the pheromone alone or the tripartite grain volatile mix. Similarly, a combination of the commercial food product with the pheromone 4,8-dimethyldecanal was more attractive to 7". castaneum than either food alone or pheromone alone. Behavioral responses to grain volatiles may reflect the ecological niche of the granivore: S. oryzae colonizes sound grain and is attracted to volatiles characteristic of fresh grain, while T. castaneum utilizes damaged or deteriorated grains and responds best to oils char-
* TO whom correspondence should be addressed. 723 0098-0331/93/0400-0723507.00/09 1993PlenumPublishingCorporation
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PHILIAPSET AL. acteristic of damaged or fungus-infestedgrain. Synergismof food odors and pheromonessuggeststhat moreeffectivetraps can be devisedfor management of these pest insects. Key Words--Aggregation pheromone, stored groin, synergism, Sitophilus oryzae, rice weevil, Tribolium castaneum, red flour beetle, bioassay, sitophinone, 4,8-dimethyldecanal. INTRODUCTION
Phytophagous insects generally utilize volatile semiochemical cues from host plants during one or more phases of the host selection process. Plant semiochemicals may act as direct attractants for insects or they may synergistically enhance the activity of pheromones produced by insects that have contacted the host plant. These phenomena are known in scolytid bark beetles in which some species respond to terpenes released by stressed or injured host trees and others require that a particular host odor be present to synergize the activity of beetleproduced pheromones (Borden, 1982; Wood, 1982). Various studies on weevils (e.g., Phillips et al., 1984; Dickens, 1989) found that host plant volatiles significantly enhance field responses to male-produced aggregation pheromones. Recent work on certain moth species determined that females and males can orient to host plants in response to odors (Landolt, 1989; Liu et al., 1988) and that plant volatiles can have profound effects on sex pheromone biology of both males and females (Landolt and Heath, 1990; Raina et al., 1992). The use of sex-specific pheromones by phytophagous insects seems so closely tied to the host plant for a variety of functions (e.g., host selection, pheromone biosynthesis, assembly, mating, and oviposition) that the influence of plant odors on orientation and response to pheromones may be a general theme throughout these species (e.g., Dickens et al., 1990). Stored-product insects present a special situation for research on host plant volatiles because nearly all species are intimately adapted to human-stored grains and grain products, truly nonanthropogenic populations may not exist, and prehistoric host plants and habitats are not well known (Lindsley, 1944). The guilds of insects that infest stored grains are viewed as part of a unique ecosystem (Sinha, 1973). Species can be assigned to ecological niches in which, for example, either fresh, sound grain may be the breeding substrate, or broken, fungusinfected material is used. Volatile attractants for several beetle species that infest broken grain have been identified from cereal grains and their products (e.g., Mikolajczak et al., 1984; Nara et al., 1981; Pierce et al., 1990) and from the fungi that infect stored grains (Pierce et al., 1991). Work on the maize weevil, Sitophilus zeamais Motschulsky, a species that attacks sound grain, demonstrated that odors from cracked wheat synergistically enhanced responses to male-produced pheromone (Walgenbach et al., 1987). Trapping technology for
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the red flour beetle, Tribolium castaneum Herbst, utilizes wheat germ oil as a food attractant in combination with a synthetic male-produced pheromone (Barak and Burkholder, 1985), but synergistic activity was not experimentally addressed. Identification of key host attractants and pheromone synergists in both primary and secondary insect pests could greatly increase efficacy of pheromone-baited traps and aid in pest management. The objective of this study was to examine the behavioral activity of several grain-derived volatiles as attractants and pheromone synergists for two species of stored-product beetles that occupy different niches in the stored-grain ecosystem. We studied Sitophilus oryzae (L), the rice weevil, because it represents internal-feeding insects that infest sound grain. Female S. oryzae oviposit in kernels of cereal grain, and the larvae and pupae develop entirely within the kernel. A male-produced aggregation pheromone, (4S,5R)-5-hydroxy4-methyl-3-heptanone, sitophinone, was isolated and identified in studies by Phillips and Burkholder (1981) and Phillips et al. (1985), but specific grain volatiles that may enhance response to pheromone have not been investigated. The second species studied, T. castaneum, the red flour beetle, can not penetrate sound grain, but represents species that feed and reproduce on damaged grain, fine material, and milled products. T. castaneum also uses a male-produced aggregation pheromone, 4,8-dimethyldecanal (Suzuki, 1980), for which the natural stereochemistry is not known. The grain volatiles we tested fall into two general classes: low-molecular-weight or aromatic flavor constituents common to many cereal grains, particularly when freshly broken (e.g., Maga, 1978), and pressed oils from several species of grains and one legume that contain a complex of compounds (predominantly fatty acids) and represent volatiles typical of older, damaged, and insect- or fungus-infected grains. In addition to these materials, we also tested a commercial food product for activity against the two beetle species.
METHODS AND MATERIALS
Experimental Insects. All experiments were conducted in the laboratory using established colonies of insects. S. oryzae were reared on soft spring wheat kernels at 12 % moisture content in a growth chamber maintained at 27 ~ 60 % relative humidity, and 16: 8 (light-dark) photoperiod. T. castaneum were reared on a mixture of whole wheat flour and brewer's yeast (95 : 5) under the same environmental conditions as S. oryzae. Parent beetles of both species were sifted from cultures one week after inoculation, and new adult progeny were removed for bioassays one week after emergence. Chemical Compounds. Synthetic grain volatiles and pheromones were 95 + % pure and were diluted in hexane at various concentrations prior to bioas-
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say. The single compounds we studied were valeraldehyde (1-pentanal), maltol (3-methoxy-2-methyl-4-pyrone), and vanillin (3-methoxy-4-hydroxybenzaldehyde); all were obtained commercially (Aldrich Chemical Company, Milwaukee, Wisconsin). We chose these three grain volatiles because they are common in several species of grain (Maga, 1978) and because preliminary screening revealed some behavioral activity against S. oryzae and other beetle species. Cereal oils were 100% pure pressed oils and were used undiluted from newly opened containers obtained from local commercial suppliers. Oils included those from sesame, rice, soy beaus, oats, wheat germ, and corn. The commercial food product we used, referred to here as WGN, was studied because of its reputation for becoming infested by various species of secondary beetles while in stored bulk packages. WGN is a prepared, extruded product containing, in descending order of relative amounts, hydrogenated soybean oil, wheat germ, sugar, sodium caseinate (milk protein), soy protein, natural and artificial flavors, and artificial color. The synthetic sitophinone, 5-hydroxy-4-methyl-3-heptanone, was a mix of the R ' S * isomers (Phillips et al., 1985). We used a mixture of 4R,8R and 4R,8S diastereomers of 4,8-dimethyldecanal (Zoecon Corporation, Palo Alto, California), which was reported to have optimal activity for T. castaneum (Levinson and Mori, 1983). Bioassays. The majority of experiments employed a two-choice pitfall bioassay in which beetles oriented to one of two holes in the floor of an arena, below which were placed stimulus or control materials in a glass collection dish. Test arenas were open steel cans, 25 cm diam. • 20 cm high; 3-cm-diam. pitfall holes were located directly opposite from each other, 4 cm from the side wall. Glass collection dishes were the bottoms of 15 • 60-mm Petri dishes. The inside top edges of the collection dishes were coated with liquid Teflon to prevent responding beetles from returning to the arena. One experiment utilized a four-choice arena of the same dimensions as the one described above, but in which four pitfall holes were located equidistant from each other in a square arrangement, each 2 cm from the side wall. A single layer of wheat kernels was placed on the floors of all test arenas. We chose wheat to provide a familiar substrate for footing and to approximate conditions in stored grain. Wheat kernels were prevented from falling into the pitfall holes by a 12-mesh screen soldered in place over the holes. The screen allowed easy passage of the insects into the collection dishes. Bioassays were conducted for 2 hr in complete darkness at 27~ and 60 + 10% relative humidity. Twenty test insects were used as mixed-sex adults in each replicate and were placed under an inverted glass funnel (3 cm diam. at widest point) at the center of the arena for 20 min prior to release to allow for acclimation to the experimental conditions. A response index (RI) for beetles in the two-choice bioassay was calculated as R / = (T C/Tot)lO0, for which T is the number responding to the treatment, C is the number responding to the control, and Tot is the total number of insects released.
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Positive RIs indicate attraction to the treatment and negative RIs indicate repellency; values could theoretically range from - 1 0 0 for complete repellency, to + 100 for complete attraction. Numbers responding to treatment and control were subjected to Student's t test for paired comparisons. Mean RIs are reported in all cases for results of two-choice bioassays. In the one four-choice experiment for T. castaneum, 50 beetles were released in the arena and the mean number of beetles responding to each treatment was determined. Ten replicates (i.e., 10 separate arena bioassays) were performed for each test material or combination of materials studied. The first series of bioassays assessed the responses of S. oryzae and T. castaneum to doses ranging from 1.0 ng to 100 /zg of valeraldehyde, maltol, and vanillin. Ten-microliter aliquots of hexane solutions were applied to l-cm-diam, filter-paper disks, while control disks received 10/~1 of hexane only. Treatment and control disks were randomly assigned to collection dishes in the two-choice pitfall arenas. A second series of two-choice bioassays examined the responses of the two beetle species to individual oils. One milliliter of the test oil was placed directly into each treatment collection dish, and 1 ml of mineral oil was added to control dishes; responding beetles were trapped in the oils. A third series of experiments assessed the responses of beetles to 3.0 g of WGN compared to an empty dish as a control. A fourth series of bioassays compared a mixture of grain volatiles only (100 #g valeraldehyde, 100 ng maltol, 100/zg vanillin), synthetic sitophinone only (100 ng), and a combination of the grain volatiles with sitophinone for activity against S. oryzae. The last experiment utilized the four-choice bioassay for T. castaneum in which 50 beetles were released and could choose among an empty dish (control), 0.5 g of WGN only, 1.0 #g of 4,8-dimethyldecanal only, and a combination of the WGN and 4,8-dimethyldecanal.
RESULTS
In experiments with valeraldehyde (Figure 1), maltol (Figure 2), and vanillin (Figure 3), S. oryzae displayed attraction at some doses of all three compounds, but T. castaneum were not attracted to any treatments and responded at levels that were not significantly different from responses to controls. S. oryzae responded significantly to all doses of valeraldehyde tested (Figure 1), and regression analysis found that there was no significant effect of dose on response ( e 2 = 0 . 0 0 3 , P = 0.703). Response of S. oryzae to maltol was highest at 0.1 /~g, and there was a significant negative effect of dose on response (R2 = 0.152, P = 0.005). Responses of S. oryzae to vanillin were generally low compared with responses to the other two compounds, resulting in significant attraction only at the 10-/~g and 100-/~g levels, and there was no significant
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PHILLIPS ET AL. VALERALDEHYDE S. oryzae
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effect of dose on response (R2 = 0.017, P = 0.363). When response indices of S. oryzae to the three flavor compounds were compared within dosage levels using analysis of variance, response to maltol was higher than to vanillin at 0.1 /zg (F2,1s = 3.819, P = 0.041) and response to valeraldehyde was higher than to maltol at 100.0/zg (F2,ts = 4.614, P = 0.024), but responses did not differ significantly at other doses. Responses to oils varied among treatments and also differed between the two beetle species (Figure 4). S. oryzae was significantly repelled by sesame oil, displayed no significant response to rice, soy bean, or corn oils, but was significantly attracted to oat and wheat germ oils. T. castaneum displayed significant attraction to all oils except sesame oil and expressed the highest mean attractive responses to oat, wheat germ, and corn oils. In two sets of bioassays we found that S. oryzae did not respond to the W G N food product (mean R I = - 0 . 5 -t- 0.89 SE, P > > 0.05), but that T. castaneum were highly attracted to W G N (mean R I = 89.5 _+ 4.62 SE, P < < 0.01). Both S. oryzae and T. castaneum displayed increased responses to their respective pheromones when the pheromones were combined with food odors. A comparison of response indices from three sets of two-choice experiments with S. oryzae found that the combination of the grain volatiles valeraldehyde, maltol, and vanillin with the pheromone sitophinone elicited a higher relative response by weevils than did the three grain volatiles alone or sitophinone alone (Figure 5). In a four-choice experiment with T. castaneum, more beetles
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FIG. 4. Response of S. oryzae and T. castaneum to different oils in two-choice bioassays. Response index was calculated as in Figure 1. Significant differences between treatment and control responses (t test) for each oil within each species indicated by * (P < 0.05) and ** (P < 0.01); N = 10 bioassays for each species with each oil. Mean responses to different oils followed by different letters for S. oryzae (left side) and T. castaneum (right side) are significantly different (ANOVA, P < 0.05, means comparison by Fisher's LSD)~
responded to the combination of 4,8-dimethyldecanal and W G N than to either component alone or a blank control (Figure 6). DISCUSSION
Our results indicate that S. o r y z a e and T. c a s t a n e u m may respond quite differently to the same grain-related volatiles. Orientation of S. o r y z a e to volatile flavor constituents may be correlated to the ecological tendency of this species to breed in fresh grain. Fresh grains contain a number of volatiles, including valeraldehyde, maltol, and vanillin (Maga, 1978). The lack of response by T. c a s t a n e u m to valeraldehyde, maltol, and vanillin suggests that these compounds do not signal a food source for this species. Older grains, particularly if infested by fungi, are known to have a higher fatty acid content than fresh grain (Christensen and Kaufman, 1969). Volatiles from grain oils may signify the quality (i.e., age or level of deterioration) of a food source, or they may facilitate discrimination of suitable species of grain by beetles. The greater response of T. c a s t a n e u m to various oils may reflect the habitat preference for this species of breeding in older and damaged grain substrates. The overall lower responses,
RESPONSES
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MEAN RESPONSEINDEX (+/-SE) F~c. 5. Response of S. oryzae to pheromone and flavor volatiles in three sets of twochoice bioassays in which the controls were solvent only on filter paper. FLAVORS is a combination of 100 /~g valeraldehyde, 100 ng maltol, and 100 t~g vanillin on filter paper; PHEROMONE is 100 ng of sitophinone on filter paper. Response index = (T C/Tot) x 100, for which T is the number responding to the treatment, C is the number responding to the control, and Tot is the total number released. Mean response indices followed by different letters are significantly different (P < 0.05, ANOVA, with StudentNeuman-Keuls comparison among means; N = 10 for each set of bioassays).
and even repellency (e.g., as with sesame oil, Figure 4), of S. oryzae to the different oils demonstrates that not all grain oils are attractive for weevils. The very high attractive response of T. castaneum to W G N indicates that the processing and combined ingredients of this food product yield a stimulus that may represent an optimal food source for this species. Lack of response by S. oryzae to W G N contrasts sharply with attraction of T. castaneum to this material and again demonstrates a profound species difference in response to food volatiles. Both S. oryzae and T. castaneum exhibited increased responses to their aggregation pheromones when these pheromones were associated with grain odors. More work will need to be done to determine if these combinations can truly act synergistically (e.g., in which the response to the combination is greater than the combined responses to the individual components), but the present data indicate a clear effect of food volatiles. Walgenbach et al. (1987) reported the first record of enhanced response to a combination of natural grain odors and pheromone in S. oryzae, and Trematerra and Girgenti (1989) demonstrated a similar phenomenon with S. oryzae using intact and broken kernels o f rice,
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PHILLIPS ET AL.
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FIG. 6. Response of T. castaneum in a four-choice bioassay with food volatiles and pheromone. Control = empty dish, WGN = 0.5 g of a commercial food product (see text for explanation), DMD = 1.0/zg of 4,8-dimethyldecanal. Means followed by different letters are significantly different (P < 0.05, ANOVA, with Student-Neuman-Keuls comparison among means; N = l0 for each set of bioassays). corn, and wheat. Our data here support those earlier studies and show that particular synthetic compounds from grain volatiles can increase response of S. o r y z a e to its pheromone. Our result with T. c a s t a n e u m demonstrates enhancement of pheromone activity by addition of food volatiles for this species. Results for both S. o r y z a e and T. c a s t a n e u m suggest that efficacy of pheromone-baited traps used for detecting these species could be increased by the addition of a proper food volatile formulation. Current use of certain food volatiles (e.g., oil mixtures) in commercially available traps may require reevaluation as more information on pheromone and food volatile interactions is collected. Stored grains can vary in quality as a function of biotic and abiotic factors (Christensen and Kaufman, 1969), and volatiles that come from grain can be indicative of the current grain quality (e.g., Sinha et al., 1988). This study demonstrates that grain volatiles can have significant effects on host selection behavior in granivores and that these effects may differ substantially between species sharing the same resource. Niche partitioning in the stored-grain ecosystem may therefore be facilitated by semiochemicals originating from a heterogeneous food substrate. More research is needed with stored-product insects in which natural volatile extracts from food sources are studied and key semiochemicals isolated that affect various species. Studies of food volatiles will
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advance a better understanding of interactions in the stored-product ecosystem and will yield more effective applications of semiochemicals in pest management (Burkholder, 1990) REFERENCES BARAK, A.V., and BURKHOLDER,W.E., 1985. A versatile and effective trap for detecting and monitoring stored-product Coleoptera. Agric. Ecosyst. Environ. 12:207-218 BORDEN,J.H. 1982. Aggregation pheromones, pp. 74-139, in J.B. Mitton and K.B. Sturgeon (eds.). Bark Beetles in North American Conifers: A System for the Study of Evolutionary Biology. University of Texas Press, Austin. BURKHOLDER, W.E. 1990. Practical use of pheromones and other attractants for stored-product insects, pp. 497-516, in R,L. Ridgway, R.M. Silverstein, and M.N. Inscoe (eds.). BehaviorModifying Chemicals for Insect Management: Applications of Pheromones and Other Attractants. Marcel Dekker, New York. CHRISTENSEN,C.M., and KAUFMAN,H.H. 1969. Grain Storage, The Role of Fungi in Quality Loss. University of Minnesota Press, Minneapolis, 153 pp. DICKENS, J.C. 1989. Green leaf volatiles enhance aggregation pheromone of boll weevil, Anthonomus grandis. EntomoL Exp. Appl. 52:191-203. DICKENS, J.C., JANG, E.B., LIGHT, D.M., and ALFORD,A.R. 1990. Enhancement of insect pheromone responses by green leaf volatiles. Naturwissenschafien 77:29-31. LANDOLT, P.J. 1989. Attraction of the cabbage looper to host plants and host plant odor in the laboratory. Entomol. Exp. Appl. 53:117-124. LANDOLT,P.J., and HEATH,R.R. 1990. Sexual role reversal in mate-finding strategies of the cabbage looper moth. Science 249:1026-1028. LEVINSON, H.Z., and MORI, K. 1983. Chirality determines pheromone activity for flour beetles. Naturwissenschafien 70:190-192. LINDSLEY, E.G. 1944. Natural sources, habitats, and reservoirs of insects associated with stored food products. Hilgardia 16:187-224. LIu, S.-H., NORRIS,D.M., and MART1, E. 1988. Behavioral responses of female adult Trichoplusia ni to volatiles from soybeans versus a preferred host, lima bean. Entomol. Exp. Appl. 49:99109. MAGA, J.A. 1978. Cereal volatiles, a review. J. Agric. Food Chem. 26:175-178. MIKOLAJCZAK,K.L., ZILKOWSKI,B.W., SMITH,C.R. JR., and BURKHOLDER,W.E. 1984. Volatile food attractants for Oryzaephilus surinamensis (L.) from oats. J. Chem. Ecol. 10:301-309. NARA, J.M., LINDSAY, R.C., and BORKHOLDER,W.E. 1981. Analysis of volatile compounds in wheat germ oil responsible for an aggregation response in Trogoderma glabrum larvae. Agric. Food Chem. 29:68-72. PHILLIPS, J.K., and BURKHOLDER,W.E. 1981. Evidence for a male-produced aggregation pheromone in the rice weevil. J. Econ. Entomol. 74:539-542. PHILLIPS,J.K., WALGENBACH,C.A., KLEIN,J.A., BURKHOLDER,W.E,, SCHMUFF,N.R., and FALES, H.M. 1985. (R*S*)-5-Hydroxy-4-methyl-3-heptanone: Male-produced aggregation pheromone of Sitophilus oryzae (L.) and S. zeamais Motsch. J. Chem Ecol. 11:1263-1274. PHILLIPS, T.W., WEST, J.R., FOLTZ, J.L., SILVERSTEIN,R.M., and LANIER, G.N. 1984. Aggregation pheromone of the deodar weevil, Pissodes nemorensis (Coleoptera: Curculionidae): Isolation and activity of grandisol and grandisal. J. Chem. Ecol, 10:1417-1423. PIERCE, A.M., PIERCE, H.D., OEHLSCHLAGER,A.C., and BORDEN, J.H. 1990. Attraction of Oryzaephilus surinamensis (L.) and Oryzaephilus mercator (Fauvel) (Coleoptera: Cucujidae) to some common volatiles of food. J. Chem. Ecol. 16:465-475.
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PIERCE, A.M., PIERCE, H.D., BORDEN, J.H., and OEHLSCHLAGER, A.C. 1991. Fungal volatiles: Semiochemicals for stored-product beetles (Coleoptera: Cucujidae). J. Chem. Ecol. 17:581597. RAINA, A.K., KINGAN,T.G., and MATTOO, A.K. 1992. Chemical signals from host plant and sexual behavior in a moth. Science 255:592-594. SINHA, R.N. 1973. Ecology of storage. Ann. Tech. Agric. 22:351-369. SINHA, R.N., TUMA, D., ABRAMSON, D., and MUIR, W.E. 1988. Fungal volatiles associated with moldy grain in ventilated and non-ventilated bin-stored wheat. Mycopathologia 101:53-60. SUZUKI, T. 1980. 4,8-dimethyldecanal: the aggregation pheromone of the flour beetles T. castaneum and T. confususm (Coleoptera: Tenebrionidae). Agric. Biol. Chem. 44:2519-2520. TREMATERRA, P., and GIRGENTI, P. 1989. Influence of pheromone and food attractants on trapping of Sitophilus oryzae (L.) (Col., Curculionidae): A new trap. J. Appl. Entomol. 108:12-20. WALGENBACH, C.A., BURKHOLDER,W.E., CURTIS, M.J., and KHAN, Z.A. 1987. Laboratory trapping studies with Sitophilus zeamais (Coleoptera: Curculionidae). J. Econ. Entomol. 80:763767. WOOD, D.L. 1982. The role of pheromones, kairomones, and allomones in the host selection and colonization behavior of bark beetles. Annu. Rev. Entomol. 27:411-446.
BEHAVIORAL RESPONSES TO FOOD VOLATILES BY TWO SPECIES OF STORED-PRODUCT COLEOPTERA, Sitophilus oryzae (CURCULIONIDAE) and Tribolium castaneum (TENEBRIONIDAE)
T.W. PHILLIPS,* X.-L. JIANG, W.E. BURKHOLDER, J.K. PHILLIPS, and H.Q. TRAN USDA ARS, Stored-Product Insect Research Unit Department of Entomology, University of Wisconsin Madison, Wisconsin 53706
(Received September 3, 1992; accepted November 23, 1992)
Abstract--Laboratory experiments were conducted to study the behavioral activity of grain-derived volatiles as attractants and pheromone synergists for Sitophilus oryzae, an internal-feeding pest of sound grain, and Tribolium castaneum, an external-feeding pest of damaged grains and flour. Behavioral studies with two-choice pitfall bioassays determined that the fresh grain volatiles valeraldehyde, maltol, and vanillin were attractive to S. oryzae at various doses, hut T. castaneum were not attracted to any dose of any of these three compounds. When oils from pressed grains were bioassayed, sesame oil was significantly repellent and oat and wheat germ oils were attractive to S. oryzae. However, rice, soybean, oat, wheat germ, and corn oils were all attractive to T. castaneum. A commercial food product composed primarily of soybean oil and wheat germ was highly attractive to T. castaneum, but elicited no response from S. oryzae. A combination of the three grain volatiles valeraldehyde, maltol, and vanillin with the synthetic pheromone sitophinone was more attractive to S. oryzae than either the pheromone alone or the tripartite grain volatile mix. Similarly, a combination of the commercial food product with the pheromone 4,8-dimethyldecanal was more attractive to 7". castaneum than either food alone or pheromone alone. Behavioral responses to grain volatiles may reflect the ecological niche of the granivore: S. oryzae colonizes sound grain and is attracted to volatiles characteristic of fresh grain, while T. castaneum utilizes damaged or deteriorated grains and responds best to oils char-
* TO whom correspondence should be addressed. 723 0098-0331/93/0400-0723507.00/09 1993PlenumPublishingCorporation
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PHILIAPSET AL. acteristic of damaged or fungus-infestedgrain. Synergismof food odors and pheromonessuggeststhat moreeffectivetraps can be devisedfor management of these pest insects. Key Words--Aggregation pheromone, stored groin, synergism, Sitophilus oryzae, rice weevil, Tribolium castaneum, red flour beetle, bioassay, sitophinone, 4,8-dimethyldecanal. INTRODUCTION
Phytophagous insects generally utilize volatile semiochemical cues from host plants during one or more phases of the host selection process. Plant semiochemicals may act as direct attractants for insects or they may synergistically enhance the activity of pheromones produced by insects that have contacted the host plant. These phenomena are known in scolytid bark beetles in which some species respond to terpenes released by stressed or injured host trees and others require that a particular host odor be present to synergize the activity of beetleproduced pheromones (Borden, 1982; Wood, 1982). Various studies on weevils (e.g., Phillips et al., 1984; Dickens, 1989) found that host plant volatiles significantly enhance field responses to male-produced aggregation pheromones. Recent work on certain moth species determined that females and males can orient to host plants in response to odors (Landolt, 1989; Liu et al., 1988) and that plant volatiles can have profound effects on sex pheromone biology of both males and females (Landolt and Heath, 1990; Raina et al., 1992). The use of sex-specific pheromones by phytophagous insects seems so closely tied to the host plant for a variety of functions (e.g., host selection, pheromone biosynthesis, assembly, mating, and oviposition) that the influence of plant odors on orientation and response to pheromones may be a general theme throughout these species (e.g., Dickens et al., 1990). Stored-product insects present a special situation for research on host plant volatiles because nearly all species are intimately adapted to human-stored grains and grain products, truly nonanthropogenic populations may not exist, and prehistoric host plants and habitats are not well known (Lindsley, 1944). The guilds of insects that infest stored grains are viewed as part of a unique ecosystem (Sinha, 1973). Species can be assigned to ecological niches in which, for example, either fresh, sound grain may be the breeding substrate, or broken, fungusinfected material is used. Volatile attractants for several beetle species that infest broken grain have been identified from cereal grains and their products (e.g., Mikolajczak et al., 1984; Nara et al., 1981; Pierce et al., 1990) and from the fungi that infect stored grains (Pierce et al., 1991). Work on the maize weevil, Sitophilus zeamais Motschulsky, a species that attacks sound grain, demonstrated that odors from cracked wheat synergistically enhanced responses to male-produced pheromone (Walgenbach et al., 1987). Trapping technology for
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the red flour beetle, Tribolium castaneum Herbst, utilizes wheat germ oil as a food attractant in combination with a synthetic male-produced pheromone (Barak and Burkholder, 1985), but synergistic activity was not experimentally addressed. Identification of key host attractants and pheromone synergists in both primary and secondary insect pests could greatly increase efficacy of pheromone-baited traps and aid in pest management. The objective of this study was to examine the behavioral activity of several grain-derived volatiles as attractants and pheromone synergists for two species of stored-product beetles that occupy different niches in the stored-grain ecosystem. We studied Sitophilus oryzae (L), the rice weevil, because it represents internal-feeding insects that infest sound grain. Female S. oryzae oviposit in kernels of cereal grain, and the larvae and pupae develop entirely within the kernel. A male-produced aggregation pheromone, (4S,5R)-5-hydroxy4-methyl-3-heptanone, sitophinone, was isolated and identified in studies by Phillips and Burkholder (1981) and Phillips et al. (1985), but specific grain volatiles that may enhance response to pheromone have not been investigated. The second species studied, T. castaneum, the red flour beetle, can not penetrate sound grain, but represents species that feed and reproduce on damaged grain, fine material, and milled products. T. castaneum also uses a male-produced aggregation pheromone, 4,8-dimethyldecanal (Suzuki, 1980), for which the natural stereochemistry is not known. The grain volatiles we tested fall into two general classes: low-molecular-weight or aromatic flavor constituents common to many cereal grains, particularly when freshly broken (e.g., Maga, 1978), and pressed oils from several species of grains and one legume that contain a complex of compounds (predominantly fatty acids) and represent volatiles typical of older, damaged, and insect- or fungus-infected grains. In addition to these materials, we also tested a commercial food product for activity against the two beetle species.
METHODS AND MATERIALS
Experimental Insects. All experiments were conducted in the laboratory using established colonies of insects. S. oryzae were reared on soft spring wheat kernels at 12 % moisture content in a growth chamber maintained at 27 ~ 60 % relative humidity, and 16: 8 (light-dark) photoperiod. T. castaneum were reared on a mixture of whole wheat flour and brewer's yeast (95 : 5) under the same environmental conditions as S. oryzae. Parent beetles of both species were sifted from cultures one week after inoculation, and new adult progeny were removed for bioassays one week after emergence. Chemical Compounds. Synthetic grain volatiles and pheromones were 95 + % pure and were diluted in hexane at various concentrations prior to bioas-
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say. The single compounds we studied were valeraldehyde (1-pentanal), maltol (3-methoxy-2-methyl-4-pyrone), and vanillin (3-methoxy-4-hydroxybenzaldehyde); all were obtained commercially (Aldrich Chemical Company, Milwaukee, Wisconsin). We chose these three grain volatiles because they are common in several species of grain (Maga, 1978) and because preliminary screening revealed some behavioral activity against S. oryzae and other beetle species. Cereal oils were 100% pure pressed oils and were used undiluted from newly opened containers obtained from local commercial suppliers. Oils included those from sesame, rice, soy beaus, oats, wheat germ, and corn. The commercial food product we used, referred to here as WGN, was studied because of its reputation for becoming infested by various species of secondary beetles while in stored bulk packages. WGN is a prepared, extruded product containing, in descending order of relative amounts, hydrogenated soybean oil, wheat germ, sugar, sodium caseinate (milk protein), soy protein, natural and artificial flavors, and artificial color. The synthetic sitophinone, 5-hydroxy-4-methyl-3-heptanone, was a mix of the R ' S * isomers (Phillips et al., 1985). We used a mixture of 4R,8R and 4R,8S diastereomers of 4,8-dimethyldecanal (Zoecon Corporation, Palo Alto, California), which was reported to have optimal activity for T. castaneum (Levinson and Mori, 1983). Bioassays. The majority of experiments employed a two-choice pitfall bioassay in which beetles oriented to one of two holes in the floor of an arena, below which were placed stimulus or control materials in a glass collection dish. Test arenas were open steel cans, 25 cm diam. • 20 cm high; 3-cm-diam. pitfall holes were located directly opposite from each other, 4 cm from the side wall. Glass collection dishes were the bottoms of 15 • 60-mm Petri dishes. The inside top edges of the collection dishes were coated with liquid Teflon to prevent responding beetles from returning to the arena. One experiment utilized a four-choice arena of the same dimensions as the one described above, but in which four pitfall holes were located equidistant from each other in a square arrangement, each 2 cm from the side wall. A single layer of wheat kernels was placed on the floors of all test arenas. We chose wheat to provide a familiar substrate for footing and to approximate conditions in stored grain. Wheat kernels were prevented from falling into the pitfall holes by a 12-mesh screen soldered in place over the holes. The screen allowed easy passage of the insects into the collection dishes. Bioassays were conducted for 2 hr in complete darkness at 27~ and 60 + 10% relative humidity. Twenty test insects were used as mixed-sex adults in each replicate and were placed under an inverted glass funnel (3 cm diam. at widest point) at the center of the arena for 20 min prior to release to allow for acclimation to the experimental conditions. A response index (RI) for beetles in the two-choice bioassay was calculated as R / = (T C/Tot)lO0, for which T is the number responding to the treatment, C is the number responding to the control, and Tot is the total number of insects released.
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Positive RIs indicate attraction to the treatment and negative RIs indicate repellency; values could theoretically range from - 1 0 0 for complete repellency, to + 100 for complete attraction. Numbers responding to treatment and control were subjected to Student's t test for paired comparisons. Mean RIs are reported in all cases for results of two-choice bioassays. In the one four-choice experiment for T. castaneum, 50 beetles were released in the arena and the mean number of beetles responding to each treatment was determined. Ten replicates (i.e., 10 separate arena bioassays) were performed for each test material or combination of materials studied. The first series of bioassays assessed the responses of S. oryzae and T. castaneum to doses ranging from 1.0 ng to 100 /zg of valeraldehyde, maltol, and vanillin. Ten-microliter aliquots of hexane solutions were applied to l-cm-diam, filter-paper disks, while control disks received 10/~1 of hexane only. Treatment and control disks were randomly assigned to collection dishes in the two-choice pitfall arenas. A second series of two-choice bioassays examined the responses of the two beetle species to individual oils. One milliliter of the test oil was placed directly into each treatment collection dish, and 1 ml of mineral oil was added to control dishes; responding beetles were trapped in the oils. A third series of experiments assessed the responses of beetles to 3.0 g of WGN compared to an empty dish as a control. A fourth series of bioassays compared a mixture of grain volatiles only (100 #g valeraldehyde, 100 ng maltol, 100/zg vanillin), synthetic sitophinone only (100 ng), and a combination of the grain volatiles with sitophinone for activity against S. oryzae. The last experiment utilized the four-choice bioassay for T. castaneum in which 50 beetles were released and could choose among an empty dish (control), 0.5 g of WGN only, 1.0 #g of 4,8-dimethyldecanal only, and a combination of the WGN and 4,8-dimethyldecanal.
RESULTS
In experiments with valeraldehyde (Figure 1), maltol (Figure 2), and vanillin (Figure 3), S. oryzae displayed attraction at some doses of all three compounds, but T. castaneum were not attracted to any treatments and responded at levels that were not significantly different from responses to controls. S. oryzae responded significantly to all doses of valeraldehyde tested (Figure 1), and regression analysis found that there was no significant effect of dose on response ( e 2 = 0 . 0 0 3 , P = 0.703). Response of S. oryzae to maltol was highest at 0.1 /~g, and there was a significant negative effect of dose on response (R2 = 0.152, P = 0.005). Responses of S. oryzae to vanillin were generally low compared with responses to the other two compounds, resulting in significant attraction only at the 10-/~g and 100-/~g levels, and there was no significant
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PHILLIPS ET AL. VALERALDEHYDE S. oryzae
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effect of dose on response (R2 = 0.017, P = 0.363). When response indices of S. oryzae to the three flavor compounds were compared within dosage levels using analysis of variance, response to maltol was higher than to vanillin at 0.1 /zg (F2,1s = 3.819, P = 0.041) and response to valeraldehyde was higher than to maltol at 100.0/zg (F2,ts = 4.614, P = 0.024), but responses did not differ significantly at other doses. Responses to oils varied among treatments and also differed between the two beetle species (Figure 4). S. oryzae was significantly repelled by sesame oil, displayed no significant response to rice, soy bean, or corn oils, but was significantly attracted to oat and wheat germ oils. T. castaneum displayed significant attraction to all oils except sesame oil and expressed the highest mean attractive responses to oat, wheat germ, and corn oils. In two sets of bioassays we found that S. oryzae did not respond to the W G N food product (mean R I = - 0 . 5 -t- 0.89 SE, P > > 0.05), but that T. castaneum were highly attracted to W G N (mean R I = 89.5 _+ 4.62 SE, P < < 0.01). Both S. oryzae and T. castaneum displayed increased responses to their respective pheromones when the pheromones were combined with food odors. A comparison of response indices from three sets of two-choice experiments with S. oryzae found that the combination of the grain volatiles valeraldehyde, maltol, and vanillin with the pheromone sitophinone elicited a higher relative response by weevils than did the three grain volatiles alone or sitophinone alone (Figure 5). In a four-choice experiment with T. castaneum, more beetles
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FIG. 4. Response of S. oryzae and T. castaneum to different oils in two-choice bioassays. Response index was calculated as in Figure 1. Significant differences between treatment and control responses (t test) for each oil within each species indicated by * (P < 0.05) and ** (P < 0.01); N = 10 bioassays for each species with each oil. Mean responses to different oils followed by different letters for S. oryzae (left side) and T. castaneum (right side) are significantly different (ANOVA, P < 0.05, means comparison by Fisher's LSD)~
responded to the combination of 4,8-dimethyldecanal and W G N than to either component alone or a blank control (Figure 6). DISCUSSION
Our results indicate that S. o r y z a e and T. c a s t a n e u m may respond quite differently to the same grain-related volatiles. Orientation of S. o r y z a e to volatile flavor constituents may be correlated to the ecological tendency of this species to breed in fresh grain. Fresh grains contain a number of volatiles, including valeraldehyde, maltol, and vanillin (Maga, 1978). The lack of response by T. c a s t a n e u m to valeraldehyde, maltol, and vanillin suggests that these compounds do not signal a food source for this species. Older grains, particularly if infested by fungi, are known to have a higher fatty acid content than fresh grain (Christensen and Kaufman, 1969). Volatiles from grain oils may signify the quality (i.e., age or level of deterioration) of a food source, or they may facilitate discrimination of suitable species of grain by beetles. The greater response of T. c a s t a n e u m to various oils may reflect the habitat preference for this species of breeding in older and damaged grain substrates. The overall lower responses,
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and even repellency (e.g., as with sesame oil, Figure 4), of S. oryzae to the different oils demonstrates that not all grain oils are attractive for weevils. The very high attractive response of T. castaneum to W G N indicates that the processing and combined ingredients of this food product yield a stimulus that may represent an optimal food source for this species. Lack of response by S. oryzae to W G N contrasts sharply with attraction of T. castaneum to this material and again demonstrates a profound species difference in response to food volatiles. Both S. oryzae and T. castaneum exhibited increased responses to their aggregation pheromones when these pheromones were associated with grain odors. More work will need to be done to determine if these combinations can truly act synergistically (e.g., in which the response to the combination is greater than the combined responses to the individual components), but the present data indicate a clear effect of food volatiles. Walgenbach et al. (1987) reported the first record of enhanced response to a combination of natural grain odors and pheromone in S. oryzae, and Trematerra and Girgenti (1989) demonstrated a similar phenomenon with S. oryzae using intact and broken kernels o f rice,
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FIG. 6. Response of T. castaneum in a four-choice bioassay with food volatiles and pheromone. Control = empty dish, WGN = 0.5 g of a commercial food product (see text for explanation), DMD = 1.0/zg of 4,8-dimethyldecanal. Means followed by different letters are significantly different (P < 0.05, ANOVA, with Student-Neuman-Keuls comparison among means; N = l0 for each set of bioassays). corn, and wheat. Our data here support those earlier studies and show that particular synthetic compounds from grain volatiles can increase response of S. o r y z a e to its pheromone. Our result with T. c a s t a n e u m demonstrates enhancement of pheromone activity by addition of food volatiles for this species. Results for both S. o r y z a e and T. c a s t a n e u m suggest that efficacy of pheromone-baited traps used for detecting these species could be increased by the addition of a proper food volatile formulation. Current use of certain food volatiles (e.g., oil mixtures) in commercially available traps may require reevaluation as more information on pheromone and food volatile interactions is collected. Stored grains can vary in quality as a function of biotic and abiotic factors (Christensen and Kaufman, 1969), and volatiles that come from grain can be indicative of the current grain quality (e.g., Sinha et al., 1988). This study demonstrates that grain volatiles can have significant effects on host selection behavior in granivores and that these effects may differ substantially between species sharing the same resource. Niche partitioning in the stored-grain ecosystem may therefore be facilitated by semiochemicals originating from a heterogeneous food substrate. More research is needed with stored-product insects in which natural volatile extracts from food sources are studied and key semiochemicals isolated that affect various species. Studies of food volatiles will
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advance a better understanding of interactions in the stored-product ecosystem and will yield more effective applications of semiochemicals in pest management (Burkholder, 1990) REFERENCES BARAK, A.V., and BURKHOLDER,W.E., 1985. A versatile and effective trap for detecting and monitoring stored-product Coleoptera. Agric. Ecosyst. Environ. 12:207-218 BORDEN,J.H. 1982. Aggregation pheromones, pp. 74-139, in J.B. Mitton and K.B. Sturgeon (eds.). Bark Beetles in North American Conifers: A System for the Study of Evolutionary Biology. University of Texas Press, Austin. BURKHOLDER, W.E. 1990. Practical use of pheromones and other attractants for stored-product insects, pp. 497-516, in R,L. Ridgway, R.M. Silverstein, and M.N. Inscoe (eds.). BehaviorModifying Chemicals for Insect Management: Applications of Pheromones and Other Attractants. Marcel Dekker, New York. CHRISTENSEN,C.M., and KAUFMAN,H.H. 1969. Grain Storage, The Role of Fungi in Quality Loss. University of Minnesota Press, Minneapolis, 153 pp. DICKENS, J.C. 1989. Green leaf volatiles enhance aggregation pheromone of boll weevil, Anthonomus grandis. EntomoL Exp. Appl. 52:191-203. DICKENS, J.C., JANG, E.B., LIGHT, D.M., and ALFORD,A.R. 1990. Enhancement of insect pheromone responses by green leaf volatiles. Naturwissenschafien 77:29-31. LANDOLT, P.J. 1989. Attraction of the cabbage looper to host plants and host plant odor in the laboratory. Entomol. Exp. Appl. 53:117-124. LANDOLT,P.J., and HEATH,R.R. 1990. Sexual role reversal in mate-finding strategies of the cabbage looper moth. Science 249:1026-1028. LEVINSON, H.Z., and MORI, K. 1983. Chirality determines pheromone activity for flour beetles. Naturwissenschafien 70:190-192. LINDSLEY, E.G. 1944. Natural sources, habitats, and reservoirs of insects associated with stored food products. Hilgardia 16:187-224. LIu, S.-H., NORRIS,D.M., and MART1, E. 1988. Behavioral responses of female adult Trichoplusia ni to volatiles from soybeans versus a preferred host, lima bean. Entomol. Exp. Appl. 49:99109. MAGA, J.A. 1978. Cereal volatiles, a review. J. Agric. Food Chem. 26:175-178. MIKOLAJCZAK,K.L., ZILKOWSKI,B.W., SMITH,C.R. JR., and BURKHOLDER,W.E. 1984. Volatile food attractants for Oryzaephilus surinamensis (L.) from oats. J. Chem. Ecol. 10:301-309. NARA, J.M., LINDSAY, R.C., and BORKHOLDER,W.E. 1981. Analysis of volatile compounds in wheat germ oil responsible for an aggregation response in Trogoderma glabrum larvae. Agric. Food Chem. 29:68-72. PHILLIPS, J.K., and BURKHOLDER,W.E. 1981. Evidence for a male-produced aggregation pheromone in the rice weevil. J. Econ. Entomol. 74:539-542. PHILLIPS,J.K., WALGENBACH,C.A., KLEIN,J.A., BURKHOLDER,W.E,, SCHMUFF,N.R., and FALES, H.M. 1985. (R*S*)-5-Hydroxy-4-methyl-3-heptanone: Male-produced aggregation pheromone of Sitophilus oryzae (L.) and S. zeamais Motsch. J. Chem Ecol. 11:1263-1274. PHILLIPS, T.W., WEST, J.R., FOLTZ, J.L., SILVERSTEIN,R.M., and LANIER, G.N. 1984. Aggregation pheromone of the deodar weevil, Pissodes nemorensis (Coleoptera: Curculionidae): Isolation and activity of grandisol and grandisal. J. Chem. Ecol, 10:1417-1423. PIERCE, A.M., PIERCE, H.D., OEHLSCHLAGER,A.C., and BORDEN, J.H. 1990. Attraction of Oryzaephilus surinamensis (L.) and Oryzaephilus mercator (Fauvel) (Coleoptera: Cucujidae) to some common volatiles of food. J. Chem. Ecol. 16:465-475.
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PIERCE, A.M., PIERCE, H.D., BORDEN, J.H., and OEHLSCHLAGER, A.C. 1991. Fungal volatiles: Semiochemicals for stored-product beetles (Coleoptera: Cucujidae). J. Chem. Ecol. 17:581597. RAINA, A.K., KINGAN,T.G., and MATTOO, A.K. 1992. Chemical signals from host plant and sexual behavior in a moth. Science 255:592-594. SINHA, R.N. 1973. Ecology of storage. Ann. Tech. Agric. 22:351-369. SINHA, R.N., TUMA, D., ABRAMSON, D., and MUIR, W.E. 1988. Fungal volatiles associated with moldy grain in ventilated and non-ventilated bin-stored wheat. Mycopathologia 101:53-60. SUZUKI, T. 1980. 4,8-dimethyldecanal: the aggregation pheromone of the flour beetles T. castaneum and T. confususm (Coleoptera: Tenebrionidae). Agric. Biol. Chem. 44:2519-2520. TREMATERRA, P., and GIRGENTI, P. 1989. Influence of pheromone and food attractants on trapping of Sitophilus oryzae (L.) (Col., Curculionidae): A new trap. J. Appl. Entomol. 108:12-20. WALGENBACH, C.A., BURKHOLDER,W.E., CURTIS, M.J., and KHAN, Z.A. 1987. Laboratory trapping studies with Sitophilus zeamais (Coleoptera: Curculionidae). J. Econ. Entomol. 80:763767. WOOD, D.L. 1982. The role of pheromones, kairomones, and allomones in the host selection and colonization behavior of bark beetles. Annu. Rev. Entomol. 27:411-446.
