Journal of Chemical Ecology, Vol. 1 7, No. 6, 1991

EVIDENCE FOR MALE-PRODUCED AGGREGATION PHEROMONE IN AMERICAN PALM WEEVIL, Rhynchophorus palmarum (L.) (COLEOPTERA:

CURCULIONIDAE):

D I D I E R R O C H A T , 1'* A R I E L G O N Z A L E Z V . , 2 D O M I N I Q U E

M A R I A U , 3 A L E X A N D E R V I L L A N U E V A G . , 2 a n d P I E R R E Z A G A T T I ~

I Laboratoire des Mddiateurs Chimiques, INRA Domaine de Brouessy, 78114 Magny-les-Hameaux, France

2palmeras de la Costa S.A. Subgerencia tdcnica y Departamiento de sanidad vegetal

Calle 54 No. 10-81, AA 050364, Bogotd, Colombia

31nstitut de Recherche pour les Huiles et les Oldagineux, CIRAD DOpartement Entomologie

Avenue du Val de Montferrand, 34032 Montpellier Cedex, France

(Received November 21, 1990; accepted February 14, 1991)

Abstract--Field trapping of the American palm weevil (APW), Rhyncho- phorus palmarum, showed that the combination of caged male APWs and palm stem was much more attractive to APWs of both sexes than palm stem alone. Caged female APWs did not enhance the attractiveness of the palm. Caged APWs without palm stem were not attractive. Virgin laboratory-bred males were highly attractive to APWs of both sexes in a two-choice pitfall olfactometer, whereas virgin laboratory-bred females were not. Adsorbent- trapped volatiles from virgin laboratory-bred males reproduced the effect of living males, giving evidence for a male-produced aggregation pheromone in this species. Wild-mated APWs of both sexes were as responsive to the aggregation pheromone as virgin laboratory-bred APWs. This is the first record of chemical communication in this species. These results have prompted investigations into the chemical identification of the aggregation pheromone.

Key Words--Aggregation pheromone, field trapping, laboratory bioassay, Coleoptera, Curculionidae, Rhynchophorus palmarum.

*To whom correspondence may be addressed.

1221

0098-0331/91/0600-1221 $06.50/0 �9 1991 Plenum PuNishing Corporation

1222 ROCHAT ET AL.

INTRODUCTION

The American palm weevil (APW), RhynchophoruspaImarum (L.), is a serious pest on coconut, Cocos nucifera L., and oil palm, Elaeis guineensis Jacq., trees in the neotropical region (Lepesme et al., 1947; Genty et al., 1978). It is occa- sionally a pest on sugarcane, Saccharum officinarum L. (Restrepo et al., 1982). The APW adult is the major known vector of red ring disease caused by the nematode Rhadinaphelenchus cocophilus Goodey (Griffith, 1968, 1987). APW larvae develop in the stems, buds, and rachis of leaves and inflorescences within a large variety of Palmae species (Lepesme et al., 1947; Hagley, 1965). The only way to prevent red ring disease is to eliminate the adults and larvae of the APW, since the difficulty and cost of nematicide treatments are extreme.

Extensive studies have been conducted on the development and larval rear- ing of palm weevils from the genus Rhynchophorus (Wilson, 1963; Giblin- Davis et al., 1989). In contrast, not much is known about the chemical ecology of the adults. They have been caught in traps baited with palm pieces (Morin et al., 1988; Rochat, 1990). It has been observed for a long time that wounds on palms are attractive to the APW (Mariau, 1968), but nothing is known about the role of the APW itself in the aggregation of the weevils on trees.

This study reports investigations carried out in the field and in the labo- ratory and presents evidence to document the existence of a male-produced aggregation pheromone in the APW.

METHODS AND MATERIALS

Field Experiments

The attractiveness of seven combinations of living APWs and oil palm stem was studied (Table 1). The experiment was conducted in an oil palm plan- tation of northern Colombia. The bait weevils were caught on oil palm trees felled before the experiment. These weevils were mated as shown by the hatch- ing of the eggs laid by females trapped in the field. Four insects (four males or four females or two males + two females) were placed in wire mesh cages (8 x 5 x 3 cm). The traps consisted of plastic jerricans (38 x 30 x 17 cm) from which one side had been removed. Palm-baited jerricans were filled with stem pieces from the same freshly felled oil palm tree. Other jerricans were filled with wood shavings as shelters for the insects. Cages containing weevils were placed at the bottom of the jerricans onto a half mango as food for the weevils. The bait weevils could also feed on palm stem pieces in palm-baited traps. Wire cages were hidden by the stem pieces or the shavings. No killing agent was added to the trap because of the use of living weevils as bait, and

AGGREGATION PHEROMONE OF AMERICAN PALM WEEVIL 1223

TABLE 1. A P W CAPTURES OBTAINED WITH DIFFERENT COMBINATIONS OF 4 LIVING

APW AND PALM STEM

Mean captures (~nl/4) Sex ratio Treatment per 4-day period (range)" (Male:Female)

4 males without stem 0.5 (0-2)a 2:0 4 females without stem 0.5 (0-1)a 1 : 1 2 males + 2 females without stem 0.8 (0-1)a 1:2 Stem without insects 3.0 (1-4)b 1:0.6 Stem with 4 females 3.3 (3-4)b 1:0.9 Stem with 2 males + 2 females 3.0 (2-9)b 1:2.6 Stem with 4 males 12.3 (5-18)c 1 : 1.0 F(6, 21) 13.66 (P < 0.0001) --

aData were subjected to a log (ni + 1) transformation for homoscedasticity purposes. Statistical reference is to a one-way analysis of variance on the transformed variate. Treatments followed by the same letter do not differ significantly at 5 % level according to Newman-Keuls test on the means of transformed values.

lured weevils could freely leave the jerr icans. Seven traps were baited with each o f the different combinat ions of palm and APW. The 49 traps were placed 80 m apart on the ground according to a Latin-square design, each one at the base o f a palm tree. The weevils caught in the traps were collected twice a day at ca. 2 hr after dusk and ca. 3 -4 hr after sunrise. This regime took into account the daily rhythm of the A P W flights (Hagley, 1965; Lucchini and Morin, 1985; Rochat, 1990). Weevi ls were collected for only four consecutive days since we observed that older pieces of pa lm stem were not attractive to APWs (Rochat, 1990).

The experiment consisted of four four-day trapping periods with, each time, random permutation of the Latin-square design, random redistribution of bait weevils into the wire cages, and replacement o f the decaying palm material by fresh material. Two consecutive trapping periods were separated by three days.

The data were subjected to a one-way analysis of variance on the trans- formed variate log (n i q- 1), ni being the total number of insects caught in the seven traps of the ith p a l m - A P W combinat ion during one four-day period. Dif- ferences between means were tested using the Newman-Keuls test at the 5 % level (Dagnelie, 1975).

Laboratory Bioassays

Insects. Weevi ls of two distinct histories were used: wild-mated weevils from Equador or Colombia and virgin weevils reared on sugarcane in the lab- oratory from a strain that originated in Equador. Sexes were separated on arrival

1224 ROCHAT ET AL.

of the wild insects or when the reared adults eclosed. These large weevils (35- 45 mm long) were kept individually in cylindrical plastic boxes (105 x 75 mm) with a wire mesh cover and fed with sugarcane. They were maintained at 27 __+ 2~ and 85 + 10% relative humidity on a 12 : 12 light-dark regime.

Volatile Collections. Five virgin APW males were placed in a cylindrical glass jar (6 x 22 cm) with three sugarcane pieces. Three pieces of the same size and from the same stem were placed in a similar jar as a control. Volatiles emitted by APWs and/or sugarcane were simultaneously trapped onto two glass cartridges connected downstream to the jars during a two-day period. Each car- tridge was filled with 0.5 g of Supelpak-2 adsorbent (16-50 mesh; purified Amberlite XAD-2 resin, Supelco). The volatiles were collected in a room main- tained at 27 + 2~ with a 12 : 12 light-dark regime. A regulated airflow (500 ml/min) passed through the glass systems. The cartridges were eluted with 2.5 ml of methylene chloride (HPLC grade, Waters). The eluates were then con- centrated at 100 /zl under a nitrogen flow (150 ml/mn) and stored at - 3 0 ~ until use.

Insect Bioassays. Insects were bioassayed using a two-choice pitfall olfac- tometer adapted from Pierce et al. (1981). The olfactometer consists of a cir- cular arena (40 x 14 cm) connected to two glass jars. The floor of the arena is made of Plexiglas and covered with white paper to allow the weevils to walk. A filter paper disk closes each jar at 4 cm from below the floor of the arena to prevent any visual stimulus when weevils are introduced into the jar as bait. Filter papers also close the two jars when volatiles are used as bait. The pitfall olfactometer is placed at the center of a 70 • 70-cm arena surrounded by white walls and homogeneously lighted with four red painted fluorescent tubes (18 W, daylight type D) giving a 40-1ux lighting at the olfactometer level.

Bioassays were run during the first half of the scotophase at 27 ___ 2~ The weevils were transferred to the testing room in their individual boxes half an hour prior to an experiment. A single weevil was released at the center of the olfactometer arena and its position was recorded after 30 min. Weevils were classified as responding only if they fell into a jar (stimulus or control) or clung to the underside of the arena floor in a jar.

Male and female individual responses to three different stimuli versus a control were recorded: (1) three virgin laboratory-bred males versus no insects; (2) three virgin laboratory-bred females versus no insects; (3) virgin laboratory- bred male effluvia + sugarcane volatiles versus sugarcane volatiles alone. One microliter of methylene chloride extract containing the volatiles to be tested (0.1 male-day-equivalent) was applied to a filter paper (17 x 17 mm) placed into a glass jar just prior to the test.

The responses of APWs also were recorded in a control treatment with two empty jars. Twenty to 35 of both wild-mated and virgin laboratory-bred weevils were bioassayed in each of the different cases.

AGGREGATION P H E R O M O N E OF AMERICAN PALM WEEVIL 1225

Data Analysis

Two criteria were used to quantify the APW responses. First, the propor- tion of tested weevils that responded after 30 min; the proportions of weevils that responded in presence of a stimulus (living males, living females, male + sugarcane volatiles) were compared with that obtained in the absence of a stim- ulus (control treatment) using the X 2 test for two independant samples (Siegel, 1956). Secondly, the number of APW choices for the stimulus jar was com- pared with the number of choices for the associated control jar using the non- parametric binomial test (Siegel, 1956) with the null hypothesis of an equal probability of choosing the control or the stimulus.

Comparisons between sexes for the same situation and between wild-mated and virgin laboratory-bred insects were analyzed by X 2 tests.

R E S U L T S

Field Experiments (Table 1)

The bait of palm stem + four males was very attractive to APWs, signif- icantly more so than all other baits. The baits of palm stem + four females and palm stem without caged APWs were equally attractive to APWs and signifi- cantly more attractive than the three baits that did not contain palm stem but only caged APWs.

Laboratory Bioassays

Few APWs responded in the absence of an experimental stimulus (control treatment with two empty jars), and the responses occurred randomly between the two empty jars (Table 2).

The introduction of three virgin laboratory-bred males as bait induced a significant response by both virgin laboratory-bred and wild-mated APWs after 30 min. Weevil choice for the male-baited jar was highly significant (Table 2).

There was very little response to three virgin laboratory-bred females as bait (Table 2). Only virgin laboratory-bred females had a slightly heightened activity level. No significant choices occurred to the female-baited jar by any category of test weevils.

The volatiles from males and sugarcane induced high numbers of weevils to respond (Table 2). The effect was slightly stronger in virgin females than males. Both laboratory-bred and virgin APWs preferred the male + sugarcane volatiles to the sugarcane volatiles alone.

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AGGREGATION PHEROMONE OF AMERICAN PALM WEEVIL 1227

The percentages of wild-mated males responding to any stimulus were not statistically greater than the responses obtained in the control treatment (Table 2), probably because many males entered unbaited jars in the control treatment.

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DISCUSSION

The requirement for palm stem to be a component of an attractive stimulus for APWs agreed with previous observations that palm tissues were attractive to the palm weevils (Nadarajan, 1984; Morin et al., 1988). The male effect in combination with palm stem volatiles was aggregative since male as well as female APWs were caught in a nearly 1 : 1 sex ratio. Synergy for attraction between beetle-produced and host-plant chemicals has been found frequently in Coleoptera, especially in bark beetles and stored-product insects (Borden, 1985). A similar synergistic relationship was shown for the pea and bean weevil, Sitona lineatus L., (Blight and Wadhams, 1987). Meksongsee and Sakulpanich (1975) showed that coconut + male Rhynchophorus vulneratus Panzer baits were much more attractive than coconut + female R. vulneratus baits, but synergy between males of this species and the host plant has not been proved.

The presence of APW females with males seemed to modify the male- produced aggregating effect, but the lower number of males (two instead of four) used for this bait could explain in part the weak catches. However, recent field trapping data (Lacerda Moura, 1 personal communication) support the hypothesis that females modify the release by males of an aggregation phero- mone or that females release a antiaggregative stimulus.

The few weevils caught in traps baited only with caged APWs might indi- cate either that males do not release the aggregation stimulus in the absence of host-plant volatiles or that flying weevils might be lured only by the simulta- neous perception of host and male APW stimuli. The hypothesis of sonic com- munication (Dumortier, 1963; Haskell, 1974; Rudinsky and Ryker, 1977) in APWs was refuted by the laboratory observations in which attraction was induced solely by captured host-plant plus male volatiles.

Among the numerous olfactometers described in the literature to investi- gate aggregation pheromones in adults of coleopteran species (Hardee et al., 1967; Payne et al., 1976; Borden et al., 1979; Phillips and Burkholder, 1981; Pierce et al., 1981; Faustini et al., 1982b), the two-choice pitfall olfaetometer

l CEPLAC/CEPEC, Km 22, Rod. Ilheus/ltabuna, CX. Postal 7, 45.600-Itabuna-Ba., Brazil.

1228 ROCHAT ET AL.

proved to be highly discriminative. This type of olfactometer gave accurate responses by the APW too. Our single-insect laboratory bioassay eliminated the need to work with large olfactometers adapted for multiple-insect tests with these very large beetles. Furthermore it avoided any possibility that bioassayed weevils could have emitted pheromones that biased the results. It avoided also any tactile and visual interaction between individuals, which are extremely strong in APW group formation, especially under artificial conditions (Rochat, 1987).

The attractiveness of the living males without palm in pitfall bioassays appears to contradict the results in the field. However, the time and space scales were totally different in the two experiments, as was the physiological stage of the bait weevils.

Mating seemed not to modify the response by APWs to the aggregation pheromone since the responses of mated and virgin APWs did not differ. This result is consistent with the fact that APWs have considerable longevity and are able to copulate several times during their life (Wilson, 1963). The retention of a high response capability would ensure that weevils are able to aggregate on favorable sites throughout their life. Phillips and Burkholder (1981) reported that mated and virgin Sitophilus oryzae L. were equally responsive to aggre- gation pheromone. O'Ceallachain and Ryan (1977) also demonstrated that mat- ing did not affect the response of Tribolium confusum L. to its aggregation pheromone.

The APW (Rhynchophorus palmarum) belongs the subfamily Rhyncho- phorinae as do the Sitophilus spp. Male-produced aggregation pheromones have been reported in three species from this latter genus (Phillips and Burkholder, 1981; Faustini et al., 1982a; Walgenbach et al., 1983). The existence of a male- produced aggregation pheromone in the APW is a new report of such a phero- mone system in this Curculionidae subfamily.

This first record of chemical communication in this pest has opened new fields of investigation for a potential improvement of APW and red ring disease control. These results have prompted chemical investigations to identify the APW aggregation pheromone. Synthetic pheromone could be used as a weevil lure in combination with either palm stems or synthetic host-plant kairomones. Studies on the chemical identification and synthesis of the APW aggregation pheromone are underway.

Acknowledgments--We thank Palmeras del Ecuador S.A. (Equador) for providing wild wee- vils. We thank the Institut de Recherche pour les Huiles et les Ol6agineux (France), Fedepalma (Colombia), and P. Genty (INDUPALMA, Colombia) for financial and technical assistance. We also thank R. ValMe for insect rearing; J.I. Lacerda Moura (CEPLAC, Brazil) for communicating unpublished results; J.H. Borden, B. Fr6rot, B. Lalanne-Cassou, G. Lempefi~re, and M. Renou for critical readings of the manuscript; and A. Bishop for English revision.

AGGREGATION PHEROMONE OF AMERICAN PALM WEEVIL 1229

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