Journal of Chemical Ecology, Vol. 7, No. 2, 1981

O N I O N F L Y 1 A N D LITTLE H O U S E F L Y 2 H O S T F I N D I N G S E L E C T I V E L Y M E D I A T E D BY DECOMPOSING ONION AND MICROBIAL VOLATILES 3

LIENE L. DINDONIS and JAMES R. MILLER Department of Entomology and Pesticide Research Center, Michigan State University East Lansing, Michigan 48824 (Received May 23, 1980; revised July 19, 1980)

Abstract--Responses of onion flies, Hylemya antiqua (Meigen), to various synthetic onion and microbial volatiles as well as volatiles from microbial cultures and decomposing onions were tested to characterize the most effective host-finding stimuli. Of nine onion and microbial volatiles tested individually, only the known attractant, n-dipropyl disulfide, caught significant numbers of flies. However, a blend of these volatiles attracted more flies than any single chemical, including n-dipropyl disulfide. In another experiment, agar plates inoculated with microorganisms from decomposing onions did not attract onion flies. However, cut onions inoculated with microorganisms and conditioned 4 days caught more onion flies than freshly cut onions and n-dipropyl disulfide. These results suggest that a blend of chemicals, rather than a single key chemical, is the more effective host-finding stimulus, and that microbial activity enhances the attractancy of a blend of onion volatiles. Large numbers of Fannia canicularis (L.), the little house fly, responded to the microbial cultures, demonstrating the existence of a potent attractant for this muscid.

Key Words--Onion fly, Hylemya antiqua, little house fly, Fannia canicularis, host finding, host-plant attractants, microbial attractants.

~Diptera: Anthomyiidae. 2Diptera: Muscidae. 3paper No. 9470 of the Michigan State University Agricultural Experiment Station. Received for publication May 23, 1980. 419 0098-0331/ 81/ 0300-0419503.00/09 1981PlenumPublishingCorporation

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DINDONIS AND MILLER INTRODUCTION

In field tests conducted in commercial onion fields, both female and male onion flies, Hylemya antiqua (Meigen), were attracted to point sources of damaged onions (Dindonis and Miller, 1980, 1981a). The rotting process seemed to enhance the attractiveness of onions (Dindonis and Miller, 1980, Loosjes, 1976). Moreover, a major volatile of crushed onions, n-dipropyl disulfide (n-Pr2S2), has been identified as a host-finding stimulant for H. antiqua (Matsumoto, 1970). This chemical trapped more onion flies than potted onion plants (Dindonis and Miller, 1980) or onion juice (Loosjes, 1976); however, relatively high release rates of n-Pr2S2 (ca. 1 mg/hr) were required to achieve these catches (Dindonis and Miller, 1981b). The available data suggest that the chemical nature of the optimal hostfinding stimulus for H. antiqua may be: a particular onion volatile, a mixture of onion volatiles, a mixture of onion volatiles in combination with volatiles from associated microorganisms which cause onion rot, or a particular microbial volatile(s). Quantitative changes in each proposed signal could also be expected to affect host finding. This paper reports experiments intended to help characterize the optimal host-finding stimulus for H. antiqua. Attraction to various combinations of microbial by-products and onion volatiles was tested to determine the relative effectiveness of volatile blends. Another experiment assessed onion fly attraction to microorganisms removed from their onion substrate, and concurrently compared the attractiveness of freshly cut and decomposing onion bulbs.

METHODS AND MATERIALS

Experiment 1: Attractancy of Blends of Onion and Microbial Votatiles. The attractancy of six metabolic by-products of soil-inhabiting microorganisms (Pelczar et al., 1972) (n-butanol, 2,3-butanediol, n-butyric acid, 85% aqueous acetyl methyl carbinol, hexanoic acid, propionic acid) and two volatiles common to onions (Whitaker, 1976) and microbes (Pelczar et al., 1972), (ethanal and isopropanol) were tested individually and collectively. In addition, the oviposition stimulant and attractant, n-Pr2S2, was dispensed alone and in combination with the chemicals. A freshly cut onion half (replaced every 3-4 days) and a control trap baited with an empty dispensing vial completed the treatments. n-PrzS2 was dispensed from BEEM TM size 3 polyethylene enclosures charged with 100/~1 of neat material (purity ~>99%). Although this system released optimal rates of n-Pr2S2 (Dindonis and Miller, 1980c), these capsules were not suitable for release of some of the other compounds due to their high

ONION FLY AND LITTLE HOUSE FLY

421

volatility. The objective was to achieve a continuous release of each chemical; however, no attempt was made to maintain equal release rates. Hence, 6 ml of all other compounds were dispensed from 8-ml glass miniscintillation vials capped with white polyethylene stoppers (Brockway Glass, Chicago, Illinois). These dispensers could hold a large reservoir of chemical while the hard polyethylene prolonged release by retarding diffusion through the l-ramthick cap. Vials containing ethanal were embedded cap upward in the soil to reduce further the release rate and pressure of this highly volatile compound. All other vials were placed horizontally within cylindrical brown paper sleeves to reduce chemical degradation by direct sunlight. Onion halves were similarly covered to retard dehydration. During the experiment, the treatments were rerandomized three times, onion halves were replaced every 4th day, and the chemicals (always detectable by the human nose) were replenished whenever necessary to maintain a visible reservoir. The test was conducted September 29 to October 13, 1978, in Hudsonville, Michigan. The treatments were placed beneath acetate cone traps (Dindonis and Miller, 1980) spaced 3 m apart on the border of a muck soil onion field. The four replicates were arranged in a linear randomized complete-block design.

Experiment 2: Attractancy of Microbial Cultures, Freshly Cut and Decomposing Onions. The rate of H. antiqua larval development on onion is accelerated by the presence of bacteria transmitted by the larvae (Friend et al., 1960, Zurlini and Robinson, 1978). Thus, we hypothesized that volatiles specific to these or other microorganisms from decomposing onions might be involved in host finding by H. antiqua females. In an attempt to determine the relative effects of microbial volatiles, inoculum from maggot-infested and decomposing onions was cultured on medium, and the attractancy of such plates and various onion treatments was compared. The eight treatments were: an agar plate with and without n-Pr2S2, an agar plate inoculated with microorganisms from decomposing onions also tested with and without n-Pr2S2, n-Pr2S2 alone, a freshly cut onion half, a decomposing onion half, and an empty plate as a control. The decomposing onion treatment was prepared by placing 15 fieldcollected 2nd-4th instar onion fly larvae on the cut side of an onion half. The infected onion halves were pressed into nonsterilized muck soil and watered daily. Onions conditioned for 4 days were used to inoculate the agar plates and then were placed in the field as the decomposing onion treatment. Potato dextrose agar in 50-mm plastic Petri dishes was inoculated with decomposing onion microorganisms by pressing the cut side of the conditioned onion half into the agar medium for 3 sec. One onion half was used for every five plates. The inoculated plates were incubated at 21~ for 48 hr. Before deploying the plates in the field, the Petri dish covers were replaced with two layers of brown paper toweling tautly secured by a tight-

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fitting plastic ring. This cover allowed volatiles to emanate from the microorganisms and agar while excluding foreign microorganisms as evidenced by the absence of visible colonization of control agar plates during the experiment. n-Pr2S2 was released at ca. 900 #g/hr from size 3 BEEM polyethylene embedding capsules. The capsules were suspended 1.0 cm above the agar and microbial plates by a wire attached to the plate. Treatments were placed within brown paper sleeves and secured beneath acetate cone traps placed on the border of a heavily H. antiqua-infested onion field in Stockbridge, Michigan. The test ran from July 5 to 18, 1979, and consisted of four replicates deployed in a linear randomized complete-block design with a 6-m intertreatment spacing. To maintain relatively constant volatile release rates, the microbial and agar plates were replaced every 24 hr, the decomposing and cut onions every 48 hr, and n-Pr2S2 was replenished as necessary to maintain a visible reservoir within the capsules. Also, treatments were rerandomized within blocks every 4th day, at which time all blocks were moved 3 m in the same direction to guard against contamination due to odors persisting from a previous treatment. RESULTS

Experiment 1: Attractancy of Blends of Onion and Microbial Volatiles. With the exception of n-Pr2S2, no trap baited with a single compound caught more flies than the control trap (Table 1). However, the combination of the eight chemicals (without n-Pr2S2) elicited a significantly greater female response than any of the chemicals tested individually, except n-Pr2S2. Moreover, the combination of eight volatiles plus n-Pr2S2 produced a female catch significantly greater than either the 1-8 combination or n-Pr2S2 alone. The pattern in male response was similar, although fewer significant treatment differences were evident (Table 1). Experiment 2: Attractancy of Microbial Cultures, Freshly Cut and Decomposing Onions. The greatest trap catch of female and male onion flies occurred in traps baited with decomposing onion halves (Table 2). Fewer yet significant numbers of female flies were also caught by all traps containing nPr2S2. Male flies responded similarly to n-Pr2S2. However, with the addition of microbial plates (treatment 7), n-Pr2Sz-baited traps caught no more males than control; furthermore, the microbial cultures alone caught significantly fewer males than the unbaited control trap. Female response to the microbial culture was indistinguishable from that to control. In contrast to the low catch of onion flies, traps baited with the microbial plates caught large numbers of female Fannia canicularis (L,) (Table 2). Moreover, the catch to the microbial plates was further elevated by the

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ONION FLY AND LITTLE HOUSE FLY TABLE 1. ONION FLY RESPONSES TO MICROBIAL BY-PRODUCTS, ONION VOLATILES, AND THEIR COMBINATIONS

Mean number onion flies caught per treatment a Treatments 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. I 1. 12. 13.

Ethanal n-Butanol 2,3-Butanediol n-Butyric acid Acetyl methyl carbinol Hexanoic acid Isopropanol Propionic acid Combination of I-8 n-Dipropyl disulfide 9 + 10 Onion half Control

Female

Male

13.3d 14.3d 9.8d 21.3cd 9.3d 8.0d 15.0d 8.0d 32.8bc 43.3b 73.5a 27.0bc 16.5cd

11.0c 9.8c 8.8c 9.8c 7.5c 6.8c 12.3c 10.3c 16.5abc 25.8ab 30.8a 18.6abe 14.3bc

~Means followed by same letters within columns are not statistically different as determined by 2-way ANOVA followed by a planned F test for mean separation of data transformed to (X + 0.5) ~/2 a d d i t i o n o f n-Pr2S2, which by itself c a u g h t no F. canicularis. V i r t u a l l y no females were t r a p p e d by a n y o f the o t h e r t r e a t m e n t s tested, a n d no male flies were c a u g h t in a n y o f the traps. DISCUSSION

H. antiqua. A l t h o u g h a d d i t i o n a l investigation is necessary, the present e x p e r i m e n t s have a i d e d o u r a t t e m p t to characterize the chemical c o m p o s i t i o n o f a n effective h o s t - f i n d i n g stimulus for 11. antiqua. The hypothesis t h a t a b l e n d o f chemicals, r a t h e r t h a n a single key chemical, is the m o r e effective h o s t - f i n d i n g stimulus is s u p p o r t e d by the significantly larger catch by the c o m b i n a t i o n o f eight chemicals ( E x p e r i m e n t 1) c o m p a r e d to a n y o f the chemicals tested individually. F u r t h e r s u p p o r t is p r o v i d e d by the even greater c a t c h to the c o m b i n a t i o n o f the eight chemicals a n d n-Pr2S2, a n d the significantly g r e a t e r o n i o n fly response to the d e c o m p o s i n g o n i o n t r e a t m e n t s t h a n to o p t i m a l release rates o f n-Pr2S2. P r e v i o u s investigations ( D i n d o n i s a n d Miller, 1981 b) d e m o n s t r a t e d t h a t o n i o n flies were e q u a l l y r e s p o n s i v e to a wide r a n g e o f n-Pr2S: release rates, 1 5 0 / a g / h r to 9 m g / h r ~ f u r t h e r suggesting t h a t once a b o v e t h r e s h o l d , q u a n t i t a t i v e differences o f a stimulus m a y be o f less i m p o r t a n c e t h a n q u a l i t a t i v e differences.

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TABLE 2. FLY RESPONSES TO MICROORGANISMS REMOVED FROM ONION SUBSTRATE, AND TO FRESHLY CUT AND DECOMPOSING ONIONS

Mean number flies caught per treatmenff

Hylemya antiqua Treatments 1. 2. 3. 4. 5. 6. 7. 8.

Decomposing onion half Freshly cut onion half n-Dipropyl disulfide Agar plate 3+ 4 Microorganism culture 3+ 6 Control

Fannia canicularis

Females

Males

Females

Males

32.8a 13.4bc 18.4b 12.4bed 18.0b 8.4cd 14.0bc 7.6c

25.2a 13.4bc 13.8b 8.4cd 10.Sbc 4.4e 7.2de 8.0d

0.4c 0.2c 0.0c 0.2c 0.0c 43.6b 72.0a 0.0c

0 0 0 0 0 0 0 0

aMeans followed by same letters within columns are not statistically different as determined by 2-way ANOVA followed by a planned F test for mean separation of data transformed to

(x + 0.5)~/2 An onion half, renewed every 4 days, did not catch more flies than control (Table 1), whereas an onion half replaced every 2 days did effect a significant catch (Table 2), suggesting a time-dependent decrease of onion volatiles. The decomposing onion, although cut 4 days before exposure, effected the greatest male and female catch, strongly suggesting that the microbial activity on onions produced a blend of volatiles even more stimulating than volatiles from a freshly damaged onion. The import of microbial activity in releasing onion fly behavior has recently been demonstrated by Ellis et al. (1979), who found that reduced oviposition occurred on onion seedlings grown in a relatively sterile medium. The levels of alkyl sulfides released from onions grown in sterilized sand were undetectable (Coley-Smith and King, 1969). Furthermore, soil bacteria are capable of enzymatically cleaving onion volatile precursors which may be exuded by the roots, producing alkyl disulfides, such as n-Pr2S2 (King and Coley-Smith, 1969). Although damaged onion cells emit high levels of sulfurcontaining compounds, the characteristic odor of a healthy onion may be due, in part, to the onion's microbiota. Microorganisms are strongly implicated in production of volatiles stimulatory to the onion fly. The microorganisms removed from their onion substrate and allowed to flourish on agar medium did not enhance onion fly host finding. This may indicate that microbial volatiles alone are not stimulatory. However, the disparate carbon sources and the different physicochemical natures of the media may have altered the expression of certain metabolic pathways with the

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result that differing by-products were produced. Furthermore, the microorganisms emitting the attractive volatiles may have been selected against by the culture medium, or they may have been outcompeted by other microorganisms. That the inoculated plates were actually releasing volatiles was substantiated not only by human perception of an odor (not unlike a rotting vegetable) but also by the F. canicularis trap catch. F. canicularis. The little house fly is reported to breed in a variety of decaying animal and plant matter, including onions (Chillcott 1960). However, in the present study, practically no F. canicularis were caught by traps baited with the decomposing onions which were attractive to H. antiqua. Possibly, a more severely decomposed onion may be a suitable oviposition site. Conversely, traps baited with the microbial plates caught high numbers of 1:. canicularis, but not onion flies. Although of decomposing onion origin, the microbial plates must have produced a volatile profile different from that of decomposing onions and one quite specific for F. canicularis. The behavioral response of onion flies to microbial volatiles appears to be limited to onion-microorganism associations, whereas the muscid fly may respond to microbial volatiles produced from nononion substrates. However, the response of F. canicularis was significantly greater to the microbial plate and n-Pr2S2 combination than to the microbial plates alone, indicating that F. canicularis, like the onion fly, may perceive and respond preferentially to a stimulus consisting of a blend of chemicals. In contrast, the attraction of F. canicularis to fermented sucrose solutions was reportedly due solely to one volatile constituent, ethanol (Hwang et al., 1978). In finding and selecting sites for oviposition, insects capable of integrating the information contained in multicomponent signals, such as those emanating from a decomposing onion, should realize an adaptive advantage if they can select the host or host conditions which will be nutritionally and ecologically optimal for their offspring. Acknowledgments--We thank B. Harrer and M. Kellyfor technicalassistance. This work was funded by a USDA CompetitiveGrant, Agreement No. 5901-0410-9-0229-0.

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1960.A revision of the nearcticspeciesof Fanniinae (Diptera: Muscidae). Can.

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and their effect on germination of sclerotia of Sclerotium eepivorum Berk. Ann. AppL BioL 64:289-301. DINDONIS, L.L., and MILLER,J.R. 1980a. Host-findingresponses of onion and seedcornfliesto healthy and decomposing onions and several synthetic constituents of onion. Environ. Entomol. 9:467-472.

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DINDONIS, L.L., and MILLER,J.R. 1981a. Host-finding behavior of onion flies, Hylemya antiqua. Environ. Entomol. In press. DINDONIS, L.L., and MILLER, J.R. 1981b. Onion fly trap catch as affected by release rates of n-dipropyl disulfide from polyethylene enclosures. J. Chem. Ecol. 7:413-420. ELLIS, P.R., ECI(ENRODE, C.J., and HARMAN, G.E. 1979. Influence of onion cultivars and their microbial colonizers on resistance to onion maggot. J. Econ. Entomol. 72:512-515. FRIEND,W.G., SALKELD,E.H., and STEVENSON,I.L. 1960. Nutrition of onion maggots, larvae of Hylemya antiqua (Meig.), with reference to other members of the genus Hylemya. Ann. N. Y. Acad. Sci. 77:384-393. HWANG, Y., MULLA, M.S., and AXELROD, n. 1978. Attractants for synanthropic flies: Ethanol as attractant for Fannia canicularis and other pest flies in poultry ranches. J. Chem. Ecol. 4:463-470. KING, J.E., and COLEY-SMITH, J.R. 1969. Production of volatile alkyl sulphides by microbial degradation of synthetic alliin and alliin-like compounds, in relation to germination of sclerotia of Sclerotium cepivorum Berk. Ann. Appl. Biol. 64:303-314. LoosJES, M. 1976. Ecology and genetic control of the onion fly, Delia antiqua (Meigen). Agric. Res. Rep. (Versl. landbouwk. Onderz.) 857. Pudoe, Wageningen. 179 pp. MATSUMOTO, Y. 1970. Volatile organic sulfur compounds as insect attractants with special reference to host selection, pp. 133-158, in D. Wood, R. Silverstein, and N. Nakajima (eds.). Control of Insect Behavior by Natural Products. Academic Press, New York. PELCZAR, M.J., Jr., R~ID, R.D., and CHAN, E.C.S. 1972. Microbiology. McGraw-Hill, New York. WHITAKER,J.R. 1976. Development of flavor, odor, and pungency in onion and garlic. Adv. Food Res. 22:73-133. ZURLINI, G., and ROBINSON, A.S. 1978. Onion conditioning pertaining to larval preference, survival rate of development in Delia (-Hylemya)antiqua. Entomol. Exp. Appl. 23:279-286,