to host fruit odor - Springer Link

Journal of Chemical Ecology, VoL 21, No. 8, 1995

BIOASSAY APPROACHES TO ASSESSING BEHAVIORAL RESPONSES OF PLUM CURCULIO ADULTS (COLEOPTERA: CURCULIONIDAE) TO HOST FRUIT ODOR

RONALD

J. P R O K O P Y , I'* S Y L V I A a n d P. L A R R Y

S. C O O L E Y , I

PHELAN z

M Department of Entomology University of Massachusetts Amherst, Massachusetts 01003 2Department of Entomology Ohio Agricultural Research and Development Center Wooster, Ohio 44691 (Received October 7, 1994; accepted March 14, 1994) Abstract--We evaluated several approaches to developing a simple, sensitive, and reliable laboratory bioassay of responses of overwintered adult plum curculios (PCs), Conotrachelus nenuphar (Herbst), to host fruit odor or its attractive components. A high proportion of assayed PCs responded positively to odor of wild plums under no-choice, moving-air conditions in a wind tunnel and under dual-choice, still-air conditions in enclosed Petri dishes. Positive response to controls lacking host odor, however, was much greater in the wind tunnel, arguing in favor of bioassays under dual-choice conditions in still air to provide greater PC discrimination. Response to host odor (from wild plums or hexane extract of wild plums or Liberty apples) in Petri dish bioassay chambers proved greatest: (1) during the scotophase of PCs under total dark or dim red light conditions, (2) when Petri dishes were completely enclosed, (3) when PCs were starved for 24 or 48 hr, and (4) when PCs were tested within seven weeks after apple tree petal fall. Neither the sex of a PC nor the direction in which a PC was obliged to move to find the source of host odor (upward through a port in the Petri dish lid or downward through a port in the base) had a substantial effect on level of response to host odor or discrimination of host odor from a nonodorous control. We conclude that an enclosed Petri dish bioassay chamber of the type described here should be a valuable asset in the process of chemically identifying components of host fruit odor attractive to PCs. Key Words--Fruit odor, bioassay methods, Conotrachelus nenuphar. *To whom correspondence should be addressed.

1073 0098-0331/95/0800-1073507.50/0© 1995PtenumPublishingCorporation

1074

PROKOPY ET AL.

INTRODUCTION

The plum curculio (PC), Conotrachelus nenuphar (Herbst), is among the most damaging of all insect pests of stone and pome fruit in eastern and central North America (Racette et al., 1991). Despite this economic importance, currently there exists no effective trap for monitoring the appearance, abundance, or disappearance of PC adults in commercial orchards (Racette et al., 1992; Prokopy et al., 1993). In contrast, effective monitoring traps are available for essentially all other key frugivorous insect pests of North American stone and pome fruit orchards (Prokopy and Croft, 1994). They also are available for several other economically important Curculionidae. For example, traps baited with pheromone in combination with host plant volatiles are used to monitor the cotton boll weevil, Anthonomus grandis Boheman (Dickens, 1989), the palm weevil, Rhynchophorus palmarum (L.) (Jaff6 et al., 1993; Oehlschlager et al., 1993), the palmetto weevil, Rhynchophorus cruentatus (Fabricius) (Giblin-Davis et al., 1994), and the rice weevil, Sitophilus oryzae (L.) (Phillips et al., 1993). Traps baited with pheromone alone are employed in monitoring the sweetpotato weevil, Cylas formicarius (Fabricius) (Jansson et al., 1992), legume-infesting Sitona weevils (Neilsen and Jensen, 1993) and the pepper weevil, Anthonomus eugenii Cano (Eller et al., 1994), while traps baited with host plant volatiles alone have been used to monitor Hylobius and Pachylobius weevils in pine forests (Hunt and Raffa, 1989). The principal native host of PC is suspected to be wild plum, Prunus americana (Maier, 1990). Within the past 150 years, however, PC has expanded its host range onto introduced commercial apple, pear, plum, peach, and cherry trees (Maier, 1990). Following eclosion in mid-summer, adults feed on host fruit somewhat briefly and then migrate to hibernation sites in nearby woods or hedgerows (Lafleur et al., 1987). In spring, overwintered adults move toward host trees, beginning when apple buds are in the tight cluster stage of development and peaking several weeks later at fruit set (Lafleur and Hill, 1987). Until petal fall, activity within host trees is largely nocturnal, with adults spending a majority of daylight hours in the ground cover beneath trees (Racette et al., 1991). After petal fall, activities of feeding and egg-laying on host fruit occur both diurnally and nocturnally but tend to be concentrated from mid- or late afternoon until dawn the next day (Racette et al., 1991). Our ultimate goal is to develop an effective monitoring trap for PC adults that incorporates both attractive odor and attractive visual stimuli. Toward this end, we have found that overwintered PC adults respond positively to the odor of intact developing host fruit under both field (Butkewich and Prokopy, unpublished data) and laboratory (Butkewich and Prokopy, 1993) conditions and that adults respond positively to odor of the same or opposite sex (Prokopy and

ASSESSING RESPONSES OF PLUM CURCULIO

1075

Cooley, unpublished data). Identification of host fruit or pheromonal chemicals eliciting attraction cannot proceed efficiently, however, without a simple, sensitive, and reliable laboratory bioassay procedure. The laboratory assay approach we had employed until now involved hanging candidate fruit or cotton wicks containing fruit extracts from ends of wooden crosspieces enclosed in clear plastic cylinders, allowing an introduced adult to climb the central stem supporting the crosspiece, and continuously observing the response pattern of the adult thereafter (Butkewich and Prokopy, 1993). Although reasonably sensitive and reliable, this bioassay procedure proved too cumbersome for use on a large scale. Our objective here was to develop an efficient approach to bioassaying response of PC adults to attractive host fruit odor. To this end, we assessed PC responses to fruit odor under wind-tunnel versus still-air conditions, under photophase versus scotophase conditions, and under various modified still-air conditions. We also assayed responses of adults of varying physiological states with respect to hunger and age and responses of females versus males. The result has been development of a still-air, choice-test bioassay approach that is simple, sensitive, and reliable.

METHODS AND MATERIALS

PCs used in the assays were obtained as adults from unmanaged apple trees from mid-May (petal fall) to early June by tapping tree limbs and collecting fallen adults on sheets of cloth placed beneath the trees. Collected and unsexed PCs were placed in groups of 20-30 in waxed, lid-covered paper cups (500 cc), maintained in the laboratory at 26°C and 80% relative humidity, and provided with several fresh-picked wild plum or apple fruit that were renewed every other day. Unless stated otherwise, the scotophase in the maintenance room extended from 10:00 to 18:00 hr, and fruit were removed from the cups 24 hr before testing. Owing to the limited number of available PCs, the same PCs were used in more than one experiment, but never in more than one experiment per week. Individuals used in experimental treatments were chosen at random from the available pool of PCs. Intact fruit used in assays were wild plums, picked fresh (when 11-13 mm diam.) about two weeks after petal fall. For several assays, we used a hexane extract of unsprayed domestic plums cv. Fellenberg or domestic apples cv. Liberty. For extracts, fruit were picked fresh about one week after petal fall (when 0.7 g in weight) and were soaked in hexane (1 g fruit/1 ml hexane) for 24 hr at 25°C. In tests, 50/~1 of raw extract was applied using a micropipet to a 1-cm 3 piece of cotton dental wick (Absorbal, Wheat Ridge, Colorado) and allowed to evaporate for 4 min before use. As a control treatment, we used

1076

PROKOPY ET AL.

either 12-mm-diam wooden beads painted with green artist pigment to approximate the color of wild plums or 1-cm 3 pieces of cotton dental wick, each treated with 50 t~l of hexane and allowed to dry for 4 min before use. The wind tunnel (Phelan, unpublished) consisted of a 78-cm-long × 34cm-wide × 10-cm-high Plexiglas box that was open at both ends and divided into four 7-cm lanes by Plexigtas dividers. Air, purified by a charcoal filter, was blown through the lanes at 0.4 m/sec by a fan attached to the filter by a plastic bag. The tunnel was illuminated by fluorescent white light (130 lux) or by an incandescent red lamp (1 lux). Assays were conducted under no-choice conditions. Five minutes before a trial, a treatment or control substance was placed 3 cm downwind of the filter at the upwind end of a lane, separated from the lane by wire mesh. Within 30 sec of the start of a trial, a single PC was introduced into the midway point of each lane, immediately after which the downwind end was closed with wire screen. Each PC was observed continuously until it reached the upwind screen (recorded as a positive response) or until 5 min elapsed without reaching the upwind screen (trial terminated). Still-air test chambers consisted of plastic Petri dishes (90 mm diam × 15 mm high) modified in the following way (Figure 1). Two holes, 10 mm diam. and 40 mm apart, were bored through a Petri dish lid using a heated cork borer. A tapered disposable polyethylene micropipet tip, I0 mm diam. at the base, 30 mm tall, and 7 mm diam at the tip (after cutting) was fitted snugly into each

.../ FIG. 1. An enclosed dual-choice Petri dish bioassay apparatus in standard orientation. The dark spheres represent a wild plum (left) and a wooden model of a plum (right),

ASSESSING RESPONSES OF PLUM CIARCUL|O

1077

hole so that the base was flush with the lid of the dish and the tip protruded above the lid. A 35-ml transparent polystyrene cup was centered over each tube, enclosing it. One of the cups contained a treatment inserted within 5 min of the start of a trial. The other cup contained an appropriate control. Within 2 rain of the start of a trial, a single PC was placed in the bottom of the dish. It could choose to remain in the dish or to crawl up the inner surface of one of the two tubes and enter a cup containing a treatment or control. In initial tests, the location of each PC was observed every 15 min up to 4 hr. Thereafter, it was observed only at 3 hr. Data were analyzed according to a G test (P < 0.05) (Sokal and Rohlf, 1981) in which the number of PCs that entered a treatment cup was compared with the number that entered a control cup. In addition, to provide a common criterion for judging the power of a treatment to attract PCs under various Petri dish assay conditions, we adopted a response index (RI) used by Phillips et al. (1993) for assessing laboratory attraction of rice weevils to odors of grain. RI = [(T - C)/totall × 100, where T is the number of PCs responding to a treatment, C is the number of PCs responding to the control, and total is the number of PCs evaluated in the test.

RESULTS

Wind-Tunnel versus Still-Air Conditions. In wind-tunnel assays conducted under white light 8-12 hr after the onset of the photophase of PCs (4-8 hr before the scotophase), 3% of PCs responded positively to a wild plum and 3% to a wooden plum model within 1 min (Table I. experiment 1). Response levels at 5 min (the end of a trial) were 27 and 20%, respectively. Even though substantial numbers of PCs in nature have been observed to feed and oviposit during this part of the photophase (Owens et al., 1982; Racette et al., 1991), we speculated that response to host fruit might be greater during the scotophase. Indeed, this proved to be so. In assays carried out under dim red light 0-4 hr after the onset of the scotophase, 40% of PCs responded positively to a wild plum and 13% to a wooden plum model within 1 min (Table 1, experiment 2). Response levels at 4 rain were identical to those at 5 min: 67% to a wild plum and 30% to a wooden plum model. Response to hexane extract of plums compared with hexane alone under the same conditions as experiment 2 yielded results very similar to those of experiment 2 (Table 1, experiment 3L Despite the significantly greater proportion of PCs responding to intact plums or plum extract compared with controls during scotophase under windtunnel conditions, we believed that this bioassay lacked sufficient discrimination and was too time-consuming to be useful when conducting large numbers of assays associated with identification of chemical components of fruit or sex odor.

1078

PROKOPY ET AL,

TABLE 1. P C RESPONSE TO WILD PLUM (WOODEN MODEL AS CONTROL) OR HEXANE EXTRACT OF PLUMS (HEXANE AS CONTROL) UNDER WIND-TUNNEL OR STILL-AIR (PETRI DISH) TEST CONDITIONS DURING PHOTOPHASE OF P C (WHITE LIGHT) OR SCOTOPHASE OF P C (RED LIGHT) u

Assay condition

PC responding (%)

Experiment

Air

PC phase

Light

Treatment stimulus

1

Wind

Photo

White

Plum

2

Wind

Scoto

Red

Plum

3

Wind

Scoto

Red

Extract

4 5

Still Still

Photo Scoto

White Red

Plum Plum

6

Still

Scoto

Red

Extract

Response at (rain)

Treatment

Control

1 5 1 5 1 5

3 27 40 ~' 67 ~ 37 h 63 h

3 20 13 30 10 37

240 30 60 120 180 240 30 60 120 180 240

47 t' 37 ~' 60 h 70 h 73 h 73 t' 30 b 43 h 57 t' 63 t' 63 h

17 7 10 t0 10 10 3 3 3 3 3

"Thirty PCs were tested per experiment. bTreatments are significantly different from control at P < 0.05.

In particular, PCs were prone to move upwind even in clean air. We reasoned that a choice test under still-air conditions might prove more rewarding. Using still-air Petri dish test chambers, we repeated the treatments of the three wind-tunnel experiments. In each case, PC discrimination between treatment and control exceeded that in the comparable wind-tunnel experiment. Thus, when tested during the photophase, 47 vs. 17 % of PCs had responded positively to a wild plum compared with a wooden plum model by 4 hr (Table 1, experiment 4). In a similar test during the scotophase, response levels at 4 hr were 73 vs. t0%, respectively (Table 1, experiment 5). When hexane extract of plums was compared with hexane alone during the scotophase, response levels at 4 hr were 63 vs. 3 %, respectively (Table 1, experiment 6). For experiments 5 and 6, we provide response levels at 30, 60, 120, 180, and 240 min. In each experiment, response to treatment as well as control reached a maximum at 3


1079

hr. In each experiment, 3 % of the PCs assayed changed location from a treatment cup to a control cup or vice versa between observation periods. We concluded from this set of six experiments that assays conducted in still air in Petri dishes under dim red light early in the scotophase of PC permitted a substantially greater level of PC discrimination than assays carried out in a wind tunnel or during the photophase of PC. Unless stated otherwise, all remaining assays were conducted in still air in Petri dishes. Photophase versus Scotophase Conditions. To confirm and expand our conclusions with respect to effects of photophase versus scotophase on PC response levels, we conducted two additional experiments in which PCs were evaluated under photophase or scotophase conditions using plum extract as the treatment substance. When assayed early in the scotophase, PCs responded identically under conditions of dim red light or total darkness: 61% to plum extract versus 3% to control, respectively (Table 2, experiment 1). For PCs entrained for one week to a photophase from 18:00 to 10:00 hr the next day, response levels to plum extract versus control were 60 vs 4% when the assay was initiated at 10:00 hr under red light but 25 vs. 2% when initiated at 10:00 hr under white light (Table 2, experiment 2). For PCs entrained for one week to a photophase from 05:00 to 21:00 hr, response levels to plum extract versus control were 23 vs. 6% when the assay was initiated at 10:00 hr under red light and 27 vs. 2% when initiated at 10:00 hr under white light (Table 2, experiment 2). These results show that PC response to host odor under dim red light does not differ from that in total darkness and confirm that response to host odor is substantially greater when assays are initiated at the beginning of scotophase under dim red light (or total darkness) than otherwise. All remaining assays were conducted under this condition. Closed versus Open-to-Air Petri Dishes. We hypothesized that the level of PC response to host odor in Petri dish test chambers might be even greater if air were allowed to pass into the chambers rather than maintaining closed air conditions. We reasoned that PCs might be less likely to adapt or habituate to host odor in dishes that permitted exchange of interior with exterior air. Results showed, however, that discrimination between plum extract and control was slightly greater in Petri dishes that were closed (58 vs. 8%) than in Petri dishes having an entirely screened bottom that permitted entry of air from below when dishes were raised 2 cm above floor level (56 vs. 19%) (Table 2, experiment 3). Orientation of Petri Dishes. We asked whether PC response to host odor differed according to whether PCs in closed Petri dishes were obliged to crawl upward through tubes to reach sources of host odor in cups resting upon lids of dishes (our standard orientation) or were obliged to crawl downward through tubes to reach sources of host odor in cups when dishes, tubes, and cups were flipped 180 degrees so that dishes rested on cups beneath. Results revealed little

1080

PROKOPY ET AL, TABLE 2. P C RESPONSE TO WILD PLUM (WOODEN MODEL AS CONTROL) IN

EXPERIMENTS 6 AND 7 TO HEXANE EXTRACT OF PLUMS (HEXANE AS CONTROL) IN EXPERIMENTS 1, 2, 3, AND 4 OR TO HEXANE EXTRACT OF APPLES (HEXANE AS CONTROL) IN EXPERIMENTS 5 AND 8 UNDER TREATMENT MODIFICATIONS OF STANDARD STILL-AtR PETRI DISH ( P D ) ASSAY CONDITION"

PC responding (%) Experiment

Assay condition

PC assayed (N)

1

Scotophase, red light Scotophase, darkness Scotophase, red light Scotophase. white light Photophase, red light Photophase, white light PD enclosed PD screened bottom O d o r above PD Odor below PD PD tubes without Fluon PD tubes with Fluon PC deprived 0 h PC deprived 24 h PC deprived 48 h PC deprived 72 h PC tested May 31 PC tested July 5 PC tested July 19 Female PC Male PC

36 36 48 48 48 48 36 36 50 50 56 56 40 40 40 40 36 36 36 69 31

2

3 4 5 6

7

8

Treatment

Control

RI'

61 ~' 611' 60/' 25 I' 23 j' 27 j' 58 ~' 56 ~' 62 ~' 64/' 68 t' 38 I' 38 73/' 75/' 35/' 69 t' 64t, 47 ~' 70 ~' 68 I'

3 3 4 2 6 2 8 19 8 14 9 13 40 13 8 0 I1 6 14 I0 10

58 58 56 23 17 25 50 36 54 50 59 25 -3 60 68 35 58 58 33 59 58

"Standard assays employed mixed sexes of PCs starved lbr 24 hr and were conducted early in the scotophase of PC under dim red light conditions in enclosed Petri dishes in which fruit-odorcontaining and control cups were placed on lids o f dishes and no Fluon was used to coat the exterior of micropippet tubes. All assays except experiment 7 were conducted between June I and June 30, 1'Treatments are significantly different from control at P < 0.05. ' Response index; see text for definition.

response difference between these two assay conditions: 62% to plum extract versus 8% to control under the standard orientation compared with 64 and 14%, respectively, under the reverse orientation (Table 2, experiment 4). Because it was substantially less effort to set up Petri dishes for assays under the standard dish orientation, we adopted this position rather than the reverse orientation for

all tests. Clean versus Fluon-Coated Micropipet Tubes in Petri Dishes. Even though


108 |

only 3% of assayed PCs were observed to change cups during the course of experiments 5 and 6 of Table 1, we speculated that coating the outside of each micropippet tube with Fluon (polytetrafluoroethylene), a substance to which insect tarsi cannot adhere, might prevent any PCs whatsoever from changing location. Unexpectedly, results showed that PC response was only 38 % to apple extract (versus 13% to control) under such assay conditions compared with 68% to apple extract (versus 9% to control) under standard conditions with clean micropipette tubes (Table 2, experiment 5). Observations indicated that oftentimes PCs that had climbed up inside surfaces of tubes to reach open tips were reluctant to crawl down Fluon-coated outside surfaces to reach sources of odor. Physiological State of PCs. Under standard Petri dish assay conditions, we compared response patterns of PCs in varying physiological states with respect to hunger or age. For PCs deprived of all food for 0, 24, 48, or 72 hr before testing, proportions responding to an intact plum versus a wooden plum model were 38 vs. 40%, 73 vs. 13%, 75 vs. 8% and 35 vs. 0%, respectively (Table 2, experiment 6). For PCs collected in late May (within two weeks of apple tree petal fall), proportions responding to apple extract versus control were 69 vs. 11% on May 31, 64 vs. 6% on July 5 and 47 vs. 14% on July 19 (Table 2, experiment 7). We conclude from these tests that nonstarved PCs do not discriminate between fruit odor and control, that PCs starved for 72 hr are perhaps too weak to respond strongly to fruit odor, and that response to fruit odor begins to fade by two months after apple petal fall. when collected overwintering PCs held under laboratory conitions begin to die in substantial numbers. We further conclude that deprivation of food for 24 hr rather than 48 hr is likely to pose less threat to the long-term survival of collected PCs and is the deprivation period of choice where the intent is maximizing PC response to host odor while preserving the health of PCs for future testing. Sex of PCs, Using Thomson's (1932) criterion for determining the sex of PCs, we attempted to segregate females from males before testing but were unable to do so effectively without injuring some PCs. We did kill and sex 100 assayed PCs after termination of a test of response to apple extract and Ibund 69 to be females, Among the 69 females, response to apple extract versus control was 70 vs. 10%. Among the 31 males, response was 68 vs. 10%, respectively (Table 2, experiment 8). Thus, females and males appear to respond similarly to host odor.

DISCUSSION

Our findings indicated that about 65% of PCs assayed in a wind tunnel early in the scotophase moved upwind in an airstream flowing at 0.4 m/sec in response to odor of wild plums or hexane extract of wild plums before the

1082

PROKOPY ET AL.

proportion responding reached a plateau. Similarly, about 68% of PCs assayed in still-air Petri dishes early in the scotophase responded positively to such host odor before reaching a plateau. The principal difference between the results of these two bioassay approaches was the degree of PC response to controls, which averaged 34% in the wind tunnel but only 7% in Petri dishes. It should not be surprising that PCs responded to a similar extent to host fruit odor under windtunnel and still-air conditions given that PCs appear to use host odor as an informational cue both when locating host trees under windy field conditions (Butkewich and Prokopy, unpublished data) and when locating individual host fruit within a tree under calm conditions (Butkewich and Prokopy, 1993). The lower level of response of PCs to controls in closed Petri dish bioassays than in wind-tunnel bioassays argues in favor of conducting bioassays in still air to maximize PC discrimination. Petri dishes proved to be convenient still-air test chambers. We estimate it requires about 70 min for one person to set up 100 Petri dishes for assay (each dish containing a test substance, a control substance, and a single PC) and about 40 min to record the data 3 hr later and disassemble the dishes. We do not recommend introducing more than one PC per Petri dish owing to the army of sounds emitted by PCs that could attract, repel, or otherwise affect PCs (Webb et al., 1980). Use of response indices (Table 2) facilitates comparison among experiments. Response indices were greater for PCs assayed at the beginning of scotophase under dim red light than during photophase or under white light, were no different for assays under dim red light or total darkness, were greater for Petri dishes that were enclosed than for Petri dishes whose bottom was screened and open to incoming air, were no different for Petri dishes in which PCs crawled upward to reach cups than for Petri dishes in which PCs crawled downward to reach cups, were greater for bioassays without Fluon compared to bioassays where Fluon was used, were greater for PCs starved for 24 or 48 hr before testing than for unstarved PCs or PCs starved for 72 hr, were greater for overwintered PCs tested two or seven weeks after petal fall than for PCs tested nine weeks after petal fall, and were no different for females and males. Laboratory procedures reported recently for bioassaying responses of curculionid adults to host odor or pheromonal stimuli include use of an olfactometer under moving air for assaying palm weevils during photophase (Jaff6 et al., 1993) and a variety of olfactometers under still-air, two-choice conditions for assaying palm weevils (Rochat et al., 1991), rice weevils (Phillips et al., 1993), and banana weevils, Cosmopolites sordidus (Germar), (Budenberg et al., 1993) during scotophase. Our results support the latter approach as being the most effective for PCs. Currently, we are attempting to identify the compound(s) in host fruit odor attractive to PCs. Such a compound(s) alone, or in combination with pheromonal


1083

components, might serve as an effective odor stimulus for use in traps for monitoring or possibly even directly controlling PCs. Acknowledgments--We thank Gabriella Gonzales and Luis Galarza for assistance and Julia Connelly for typing the manuscript. This research was supported by a USDA Northeast Regional IPM Competitive Grant and by Massachusetts Argicultural Experiment Station Project 604.

REFERENCES BUDENBERG, W.J., NDIEGE, I.O., KARAGO,F.W., and HANSSON, B,S. 1993. Behavioral and electrophysiological responses of the banana weevil Cosmopolites sordidus to host plant volatiles. J. Chem. EcoL 19:267-277. BUTKEWlCH, S.L., and PROKOPY, R.J. 1993. The effect of short-range host odor stimuli on host fruit finding and feeding behavior of plum curculio adults. J. Chem. Ecol. 19:825-835. DICKENS, J.C. 1989. Green leaf volatiles enhance aggregation pheromone of boll weevil. Anthonomus grandis. Entomol. E~rp. Appl. 52:191-203, ELLER, F.J., BARTELT.R.J., SHASHA,B.S., SCHUSTER,D.J., RILEY, D.G., STANSLY, P.A., MUELLER, T.F., SHULER, K.D., JOHNSON, B., DAVIS, J.H., and SUTHERLAND,C.A. 1994. Aggregation pheromone for the pepper weevil, Anthonomus eugenii Cano: Identification and field activity. J. Chem. Ecol. 20:1537-1555. GIBLIN-DAVIS, R.M., WEISSUNG, T.J,, OEHLSCHLAGER,A.C., and GONZALEZ, L.M. 1994. Field response of Rhynchophorus cruentatus to its aggregation pheromone and fermenting plant volatiles. Fla. Entomol. 77:164-177. HUNT. D.W.A,, and RAEFA, K.F. 1989. Attraction of Hylobius radicis and Pachylobius picivorus to ethanol and turpentine in pitfall traps. Environ, EntomoL 18:35t-355. JAFFE, K., SANCHEZ~P., CERDA, H. HERNANDEZ,J.V., JAFFI~. R., URDANETA,N., GUERRA, G., MARTINEZ, R.. and MIRAS, B, 1993. Chemical ecology of the palm weevil, Rhynchophorus palmarum (L): Attraction to host plants and to a male-produced aggregation pheromone. J. Chem. Ecol. t9:1703-1720. JANSSON, R.K., MASON, L.J., HEATH, R.R., SORENSEN, K,A., HAMMOND,A.M., and ROBINSON, J.V. 1992. Pheromone trap monitoring system for sweetpotato weevil in the southern United States: Effects of trap type and pheromone dose. J. Econ. Entomol. 85:416-423. LAELEUR, G., and HILL, S.B, 1987. Spring migration, within-orchard dispersal and apple tree preference of plum curculio in southern Quebec. J. Econ. Entomol. 80:1173-1187. LAFLEUR~ G., HILL, S.B., and VINCENT, C. 1987. Fall migration, hibernation site selection and associated winter mortality of plum curculio in a Quebec apple orchard, J. Econ. Entomol. 80:1152-1172. MAma, C.T. 1990. Native and exotic rosaceous hosts of apple, plum and quince curculio larvae in northeastern United States. J. Econ. EntomoL 83:1326-1332. NIELSEN, B.S., and JENSEN, T,S. 1993. Spring dispersal of Sitona lineatus: The use of aggregation pheromone traps for monitoring, Entomol. Exp. Appl. 66:21-30. OEHLSCHLAGER,A.C., CHINCHILLA,C.M., GONZALEZ,L.M., JIRON, L.F., MEXZON, R., and MORGAN, B. 1993. Development of a pheromone-based trapping system for Rhychophorus palmature. J. Econ. Emomol. 86:1381-1392. OWENS, E.D., HAUSCHILD,K.I., HUBBELL,G.L., and PROKOPY,R.J. 1982. Diurnal behavior of plum curcutio adults within host trees in nature. Ann. Entomol. Soc. Am, 75:357-362. PHILLIPS, T.W., JIANG, X.L., BURKHOLDER,W.E., PHILLIPS, J.K., and TRAN, H.Q, 1993. Behavioral responses to food volatiles by two species of stored-product Coleoptera, Sitophilus oryzae and Tribolium castaneum, J. Chem. Ecol. 19:723-734~

1084

PROKOPY ET AL.

PROKOPY. R.J., and B.A. CROFT. 1994. Apple insect pest management, pp. 543-585, in R.L. Metcalf and W H . Luckmann (eds,). Introduction to Insect Pest Management, 3rd ed. John Wiley & Sons, New York. PROKOPY, R.J., GALARZA,G., and PHELAN, P.L. 1993. Monitoring plum curculio in orchards: New hope for a more effective method. Proc. N. EngL Fruit Meet. 99:70-76. RACETTE, G., CHOUINARD,G., HILL, S.B,, and VtNCENT, C. 1991. Activity of adult plum curculio on apple trees in spring. J. Econ. Entomol. 84: 1827-1832. RACETTE, G,, CHOUmARO,G., VINCENT, C., and HILL, S.B. 1992, Ecology and management of plum curculio, Conotrachelus nenuphar, in apple orchards, Phytoprotection 73:85-100. ROCHAT, D.. GONZALEZ, A. MARIAU, D., VILLANUEVA,A., and ZAGATTI, P. 199I. Evidence for a male-produced aggregation pheromone in American palm weevil, Rhynchophorus pahnarum. J. Chem. Ecol. 17:1221-1230. SOKAL, R,R., and F.J. ROHLF. 1981. Biometry. W.H, Freeman, New York, THOMSON, R.R. 1932. Sex differentiation of adults of Conotrachelus nenuphar. J. Econ, EntontoL 25:807-810. WEBB, J.C., CALKINS, C.O., CARLYSLE, T., and BENNER, J. 1980. Analysis of stress sounds produced by the pIum curculio. J, Ga. Entomol. Soc. 15:419-427.