Journal of Chemical Ecology, Vol. 17, No. 2, 1991

FLIGHT ACTIVITY OF Heliothis virescens (F.) FEMALES (LEPIDOPTERA: NOCTUIDAE) WITH REFERENCE TO HOST-PLANT VOLATILES 1

E.R. MITCHELL,*

F.C. TINGLE,

and R . R . H E A T H

Insect Attractants, Behavior, and Basic Biology Research Laboratory Agricultural Research Service, U.S. Department of Agriculture Gainesville, Florida 32604

(Received June 11, 1990; accepted September 18, 1990) Abstract--Heliothis virescens (F.) females responded positively via upwind flight in laboratory assays to volatiles emitted from methylene chloride washes of fresh whole leaves of host plants including cotton, tobacco, and a weed species, Desmodium tortuosum (Swartz) de Candolle. Except for D. tortuosum, the response increased positively with dose; the steepest slope occurred with an extract of cotton squares (flower buds). Almost all of the moths that landed on the extract dispenser also oviposited. Moths simulated by extracts from cotton squares exhibited a full array of behaviors (upwind flight, contact with the dispenser, examination of the cloth snbstrate with antennae, and oviposition) expected of gravid individuals seeking sites to propagate the species.

Key Words--Plant attractant, host phenology, oviposition stimulant, Heliothis subflexa, Heliothis armigera, Lepidoptera, Noctuidae.

INTRODUCTION M a n y p h y t o p h a g o u s insects use airborne v o l a t i l e s e m i t t e d f r o m plants to locate t h e i r hosts. T h e recent d e v e l o p m e n t o f b i o a s s a y systems for studying host-plant finding and o v i p o s i t i o n a l b e h a v i o r u n d e r c o n t r o l l e d e n v i r o n m e n t a l conditions in the l a b o r a t o r y has intensified interest in characterization o f the specific b e h a v i o r s r e g u l a t e d by v o l a t i l e e m i s s i o n s f r o m plants and identification o f the *To whom correspondence should be addressed. 1This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or the recommendation of its use by USDA. 259

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active compounds. Information on volatile chemicals that attract phytophagous insects to their host to stimulate oviposition upon arrival could be of benefit to plant-breeding programs. For example, selective elimination or reduction in the level of these chemicals possibly could impart a degree of "resistance" to otherwise acceptable cultivars. Heliothis and Helicoverpa spp. rank among the most destructive pests of crops in the United States and around the world. The evidence is growing that many of these species use plant constituents for host location and accep- tance. Allelochemicals that stimulate females to oviposit have been isolated for Helicoverpa zea (Boddie) (corn) (Wiseman et al., 1988), Heliothis subflexa (Guen6e) (Physalis angulata L.) (Mitchell and Heath, 1987), and Heliothis virescens (F.) (tobacco) (Jackson et al., 1984; Mitchell et al., 1990). Mitchell et al. (1990) also isolated oviposition stimulant compounds for H. virescens from a lateseason wild host plant, Desmodium tortuosum (Swartz) de Candolle, and leaves and squares (i.e., flower buds) of cotton. In each of the cases cited above, the techniques used to demonstrate oviposition activity were unable to differentiate between tactile and olfactory stimuli. Other workers, however, have demonstrated a relationship between plant volatiles and attraction/oviposition stimulation in Heliothis. Rembold and Tober (1985) showed that Heliothis armigera (HiJbner) females (an oligophagous species) responded differentially in oviposition trials to odors obtained by pulling air over seedlings of two cultivars of pigeonpea, Cajanus cajan L. Millsp. Tingle et al. (1989) showed that mated Heliothis subflexa females (a monophageous species) flew upwind in wind-tunnel bioassays to a methanol wash of fresh whole leaves from its host plant, groundcherry (P. angulata), culminating in ca. 50% of the responding moths depositing one or more eggs. The present study reports on the flight and ovipositional responses of females of a sibling species of H. subflexa, H. virescens, in a wind-tunnel system to extracts of selected cultivated host plants (cotton and tobacco) and D. tortuosum, a lateseason weed host.

METHODS AND MATERIALS

A Plexiglas flight tunnel (60 cm wide x 60 cm high x 195 cm long) was used to observe flight and ovipositional responses of females to volatiles from crude extracts of three different plant hosts. The extracts were obtained by dipping ca. 400 g fresh plant material for 30 sec in 1 liter of solvent (methylene chloride), which was filtered and stored in l-liter glass containers at 0~ until needed (Mitchell and Heath, 1987). Extracts were prepared from cotton (variety McNair 220) leaves and squares (flower buds) and from leaves of D. tor-

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tuosum and tobacco [ susceptible: NC 2326, and resistant: TI 1112 (Jackson et

al., 1983)]. All plants were grown in small field plots using conventional cultural practices. The extracts were concentrated in a rotary evaporator before testing. All plants were in the flowering stage except tobacco, which was in the prebloom stage. All test insects were reared in our laboratory on the modified pinto bean diet using methods described by Guy et al. (1985) and Mitchell et al. (1988). The insects were sexed in the pupal stage and held separately in 3.8-liter paper cans with screened tops until emergence. Upon eclosion, the sexes were confined together for two days for mating (21 females and 14 males/cage) in 5.5liter plastic cages with screened tops. The moths were fed a 10% honey-water solution and held under a reversed 14 : 10-hr light-dark cycle in a holding room that was maintained at 25-26~ and 60-70% relative humidity. Preliminary studies indicated that > 95 % of the females mated under these conditions, as evidenced by the presence of a spermatophore in the bursa copulatrix. On the day of testing, the moths were sexed by gently squeezing the abdomen to extrude the genitalia, and the females were confined in a 25 x 25 x 25-cm Plexiglas holding cage and placed in the flight tunnel room (3 x 2.6 x 2.1 m) ca. 1 hr before scotophase. An intake vent in the wall allowed a continuous flow of fresh unfiltered air from the outside into the room, which was maintained at the same environmental conditions as the holding room. An electric timer was used to control overhead fluorescent lights (two banks of two 40-W bulbs). Three incandescent 25-W red light bulbs that were equally spaced above the tunnel remained on continuously. The light level during scotophase was 1.4 lux. Air was pulled through the tunnel at ca. 0.4 m/sec, when measured at the center of the tunnel, and exhausted opposite the intake duct via a 30-cmdiam. flexible pipe equipped with a fan. The flight tunnel was the same as described by Tingle et al. (1989). Crude extract (gram equivalent dosages) was deposited on white muslin cloth dispensers secured over the end of a glass cylinder (3.5-cm opening) with a rubber band. After a 5-min evaporation period, the dispenser was placed at the upwind end of the tunnel. Air was blown through the cylinder with an aquarium pump at the rate of 1 liter/min to provide a continuous flow (plume) of the extract odor through the tunnel. The location and form of the plume was verified by introducing smoke into the dispenser system and observing the smoke trail. A moth was removed from the holding cage and placed into a cylindrical 4 x 6.5-cm plastic release cage with screened ends. After placement of the release cage into the downwind end of the flight tunnel, the moth was released immediately and observed for 2 min. Behavioral responses, including random and oriented flight, and contacts, landings, and oviposition on extract-treated substrates were recorded. Ten to 16 H. virescens females (mean = 14) were

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tested individually in each replicate; each treatment was replicated 3-13 times. The data were converted to arcsin ~/percentage and analyzed using ANOVA or regression analysis (Steel and Torrie, 1960).

RESULTS AND DISCUSSION

Heliothis virescens (Hv) females demonstrated upwind flight to volatiles emitted from cloth dispensers treated with each of the host-plant extracts (Figure 1). The flight sequence was similar to that previously described for a sibling species, H. sublfexa, to extracts of whole leaves from its host, groundcherry (Tingle et al., 1989). Typically, most of the females began nondirectional (random) flight after emerging from the release cage at the downwind end of the tunnel. Random flight usually began in an upwind direction. Shortly after exiting the release cage, moths detecting the odor plume flew upwind in a zigzag pattern towards the extract dispenser. A few moths flew directly to the dispenser and landed, probed the cloth substrate with their ovipositor, and deposited an egg. Others flew to within 5 cm of the dispenser and hovered for a short time

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dosages (gram equivalents) to methylene chloride washes of whole leaves of susceptible (ST) and resistance (RT) tobacco, cotton (CL), and Desmodium tortuosum (DES) in wind-tunnel assays. Differences between means within each response category were not significant (P < 0.05; ANOVA) (Steel and Torrie, 1960). The response totals do show a progressive increase with dosage for whole-leaf washes of tobacco and cotton.

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HELIOTHIS RESPONSE TO PLANT VOLATILES

before veering away or landing on the cloth substrate. The mean oriented flight response to control dispensers treated with methylene chloride was only 6.7 +_ 2.6 % (eight replicates). The few females orienting to the control dispenser flew upwind < 100 cm before veering away and continued flying about in random fashion or landed on the tunnel wall. None of these females contacted the control dispenser. With the exception of D. tortuosum, the total response (oriented upwind flight, contact, and landing) recorded for Hv to each leaf extract showed a progressive increase with dose (Figure 1). The steepest slope occurred with an extract from cotton squares (Figure 2). It is unknown whether the apparent difference in response to extracts of cotton squares and cotton leaves was quantitative or qualitative. A comparable situation also might exist with tobacco and D. tortuosum; however, floral parts of these plants were not examined. The flowering stage of corn, cotton, soybean, and tobacco is the most preferred phenological state for oviposition by H. zea (Johnson et al., 1975), although females will oviposit on certain host plants in the absence of flowering parts. Roome (1975) reached similar conclusions concerning the oviposition behavior of H. armigera in corn and sorghum. He also suggested that "flowering" corn and sorghum crops attract adults that, once in the crop, are "trapp e d " by suitable physiological cues from the plants. Thus, the increased response of females to extracts of cotton squares versus cotton leaves suggests

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that, like the aforementioned species, Hv also are more strongly attracted to plants in the reproductive phase of their life cycle. A test was conducted to establish whether attraction of Hv to cotton and tobacco was due to specific chemicals emitted by these plants or if the attraction response was due to the ubiquitous "green odor" factor. Using the flight-tunnel assay described earlier, female Hv were exposed to a 1-g equivalent dose of a methylene chloride wash of fresh whole leaves of cotton, susceptible tobacco, or elderberry (Sambucus simpsonii Rehd.), a nonhost weed species. Tingle and Mitchell (1986) showed that a methylene chloride wash (among others) of whole elderberry leaves was effective in deterring oviposition by Hv in laboratory assays. The mean percent flight responses for the three extracts were: cotton, 55.6 ___3.5a; tobacco, 50.0 +__5.4a; and elderberry, 23.6 + 7.4 b ]means +SE, with different letters indicating significant differences, P < 0.05, Duncan's multiple-range test (Duncan, 1955)]. These results suggest that the flight responses recorded for Hv to cotton and tobacco extract were due to chemical qualities different from what is typically referred to as the green factor. It is unknown, however, whether the attractants from cotton and tobacco are the same compounds. The total flight response (upwind flight, contact, and landing) by Hv to D. tortuosum was ca. 50% for each of the three doses tested. However, ca. twothirds of the moths flying upwind and contacting the dispensers treated with D. tortuosum extract did not land. Mitchell et al. (1990) reported that D. tortuosum possessed compounds that stimulated oviposition by Hv females in laboratory assays. In Florida, D. tortuosum typically becomes abundant in late August and September in tobacco fields following harvest. Jackson and Mitchell (1984) showed that although Hv can survive to pupation on D. tortuosum, this species is less suitable than tobacco as a host. Thus, due to the proximity of D. tortuosum to moth emergence sites in abandoned tobacco fields, a strong longrange attractant is not needed for Hv females to locate and oviposit on D. tortuosum. The propensity for Hv to oviposit on D. tortuosum located outside of tobacco fields relative to D. tortuosum in tobacco fields is unknown. There was no significant difference among treatments in the mean percentage of Hv moths (13.9 +__ 1.5) depositing an egg upon contact with the cloth substrates. In a recent study using a system of individual chambers specifically designed to evaluate oviposition responses, Mitchell et al. (1990) showed that Hv females laid significantly more eggs on cloth substrates treated with whole-leaf wash extracts of leaves from susceptible or resistant tobacco, D. tortuosum, and cotton leaves or squares compared to the number of eggs laid on control cloths. Moreover, in competitive trials in which Hv females were given a choice between susceptible tobacco-leaf extract and extract from leaves of resistant tobacco, cotton, or D. tortuosum, significantly more eggs

HELIOTHIS RESPONSE TO PLANT VOLATILES

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were deposited on cloths treated with susceptible tobacco extract (Mitchell et al.., 1990). In this study, the response period was limited to 2 m i n / m o t h . This necessarily restricted the total number o f eggs that a female could deposit within the observation period. Nevertheless, females were stimulated to exhibit an array o f behaviors (upwind flight, contact, surface examination with antennae, and oviposition) characteristic o f gravid individuals seeking sites to propagate the species. W o r k by Jackson et al. (1984) suggests that the chemicals eliciting egg laying in Hv most likely are contact stimuli. Thus, the chemicals that elicit long-range orientation ( > 1 m ) and landing probably are different from those eliciting the oviposition response. The role o f plant volatiles in the selection and colonization process o f oligophagous species.such as Hv, and indeed most insect species regardless o f their degree o f host specificity, is poorly understood. Elucidation o f the phytochemicals governing these processes will enhance greatly an understanding o f the factors driving the establishment and development o f insect pest populations on crops. Knowledge o f the behavioral effects o f such phytochemicals offers opportunities for creative management o f crop insect pests via genetic modification o f the host's chemical profile. Alternately, chemically defined plant attractants may be used alone or combined with insect sex attractant pheromones to monitor pest populations or for direct control as toxic baits by combining plant attractants, pheromones, and insecticides. Acknowledgments--We gratefully acknowledge the technical assistance of P. Hall, W. Copeland, F. Adams, and B. Dueben of this laboratory; and D.M. Jackson, ARS USDA, Oxford, North Carolina, and G. Herzog, Tifton, Georgia, for providing the tobacco and cotton seed, respectively.

REFERENCES DUNCAN, D.B. 1955. Multiple range and multiple F tests. Biometrics 11:1-2. GuY, R.H., LEPPLA,N.C., RYE, J.R., GREEN,C.W., BARRETTE,S.L., and HOLLIEN,K.A. 1985. Trichoplusia Hi, pp. 487-494, in P. Singh and R.F. Moore, (eds.). Handbook of Insect Rearing, Vol. 2. Elsevier Science Publishers B.V., Amsterdam. JACKSON, D.M., and MITCHELL, E.R. 1984. Growth and survival of tobacco budworm (Lepidoptera: Noctuidae) larvae fed Florida beggarweed (Fabaceae) and tobacco (Solanaceae). J. Econ. Entomol. 77:960-965. JACKSON, D.M., CHEATHAM, J.S., PITTS, J.M., and BAUMHOVER, A.H. 1983. Ovipositional response of tobacco budworm moths (Lepidoptera: Noctuidae) to tobacco Introduction 1112 and NC 2326 in cage tests. J. Econ. Entomol. 76:1303-1308. JACKSON, D.M., SEVERSON, R.F., JOrINSON,A.W., CHAPLIN,J.F., and STEPHENSON, M.G. 1984. Ovipositional response of tobacco budworm moths (Lepidoptera: Noctuidae) to cuticular chemical isolates from green tobacco leaves. Environ. Entomol. 13:1023-1030. JOHNSON, M.W., STINNER, R.E., and RABB, R.L. t975. Ovipositional response of Heliothis zea (Boddie) to its major hosts in North Carolina. Environ. Entomol. 4:291-297.

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MITCHELL, E.R., and HEATH, R.R. 1987. Heliothis subflexa (Gn.) (Lepidoptera: Noctuidae): Demonstration of oviposition stimulant from groundcherry using novel bioassay. J. Chem. Ecol. 13:1849-1858. MITCHELL, E.R., HINES, R.W., and COPELAND, W.W. 1988. Heliothis subflexa (Lepidoptera: Noctuidae): Establishment and maintenance of a laboratory colony. Fla. Entomol. 71:212214. MITCHELL, E.R., TINGLE, F.C., and HEATH, R.R. 1990. Oviposition response of three Heliothis species (Lepidoptera: Noctuidae) to allelochemicals from cultivated and wild host plants. J. Chem. Ecol. 16:1817-1827. REMBOLD, H., and TOBER, H. 1985. Kairomones as pigeonpea resistant factors against Heliothis armigera. Insect Sci Appl. 6:249-252. ROOME, R.E. 1975. Activity of adult Heliothis armigera (Hb.) (Lepidoptera: Noctuidae) with reference to the flowering of sorghum and maize in Botswanna. Bull. Entomol. Res. 65:523530. STEEL, R.G.D., and TORRIE,J.H. 1960. Principles and Procedures of Statistics. McGraw-Hill, New York. TINGLE, F.C., and MITCHELL, E.R. 1986. Behavior of Heliothis virescens (F.) in presence of oviposition deterrents from elderberry. J. Chem. Ecol. 12:1523-1531. TINGLE, F.C., HEATH, R.R., and MITCHELL, E.R. 1989. Flight response of Heliothis subflexa (Gn.) females to an attractant from groundcherry, Physalis angulata L. J. Chem. Ecol. 15:221231. WISEMAN,B.R., GROSS, H.R., WIDSTROM, N.M., WAISS, A.C., and JONES, R.L. 1988. Resistance of corn to Heliothis zea. South. Coop. Ser. Bull. 337:21-30.