Journal of Chemical Ecology, Vol. 20, No. 10, 1994

EVIDENCE FOR VOLATILE CHEMICAL ATTRACTANTS IN THE BEETLE Maladera matrida Argaman (Coleoptera: Scarabaeidae)

GAL

YARDEN

and ARNON

SHANI*

Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel (Received March 30, 1994; accepted June 10, 1994) Abstract--The Maladera matrida beetle (Coleoptera, Scarabaeidae, Melolonthinae), a relatively new species to science, was first identified in Israel in 1983. In the course of field observations it was found that adult M. matrida beetles emerged from the soil at sunset to feed and mate. During the first 20 rain of flight, most of the beetles were males. The females emerged shortly afterwards, and aggregations numbering 20-30 individuals with equal proportions of males and females were eventually formed on peanut plants. Laboratory olfactometer bioassays showed that peanut leaves (food) attracted both males and females. Field-trapping experiments and olfactometer studies showed that M. matrida beetles were highly attracted by live virgin females in the presence of food (cut-up peanut leaves). Another set of field trapping experiments indicated that airborne volatiles produced by live virgin females plus food had the same attracting ability as live virgin females plus food. The attraction exerted by the combination of live virgin females and peanut leave volatiles suggests a synergism effect. Accordingly, we propose a two-stage mechanism of chemical communication in the M. matrida beetles: first, the males cause mechanical damage to the host plant to attract both sexes; later, the females emit attractants (sex pheromone) while eating or shortly thereafter. Key Words--Maladera matrida, Coteoptera, Scarabaeidae, collection of votatiles, field trapping, olfactometer, attractants, host plant volatiles, synergism, aggregation, sex pheromone. INTRODUCTION T h e Maladera rnatrida A r g a m a n b e e t l e ( C o l e o p t e r a , S c a r a b a e i d a e , M e l o l o n t h i n a e ) , a r e l a t i v e n e w c o m e r to I s r a e l , w a s first d e t e c t e d b y K l e i n a n d C h e r t *To whom correspondence should be addressed. 2673 0098-0331/94/10002673507.00/0© t994 PlenumPublishingCorporation

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(1983) and later described as a new species by Argaman (1986, 1990). This species has recently appeared in Saudi Arabia as well (D. Ben-Yakir, personal communication). The M. matrida beetles, like most other Scarabaeidae (Leal et al., 1993), is considered to be a serious polyphagous pest, affecting many agricultural crops. Adult beetles feed on foliage and flowers in orchards, irrigated fields and ornamental gardens. The grubs develop in the soil and cause damage to underground crops such as peanuts and potatoes (Gol'berg et al., 1986, 1989; Harari et al., 1994). Because M. matrida is a new species, basic biological information, mainly about aggregation and mating behavior and possible bioassay, is scanty. By the end of the '80s only three scarab pheromones have been identified (Henzell and Lowe, 1970; Tumlinson et al., 1977; Tamaki et al., 1985), the main problem being the lack of uniform and consistent bioassays (Domek and Johnson, 1987; Bestmann and Vostrowsky, 1988; Leal et al., 1992, 1993). A variety of systems for the collection of airborne volatiles from insects have been reported (e.g., Gaston, 1984, and references therein; Golub and Weatherston, 1984, and references therein; Shani and Lacey, 1984; Jursik et al., 1990; Heath and Manukian, 1992). In these methods attention is paid to a number of factors: protection against contamination, prevention of stress to the insect, avoidance of disturbing the natural environment of the insect, efficiency and speed of the collection from a single insect or from a large number of insects, and simplicity and low cost of the construction and operation of the collection system. The role of host plant volatiles and/or food-type lures with pheromones and their synergistic effect in the attraction of insects has been investigated (Klein et al., 1981; Laddet al., 1981; Domek and Johnson, 1988; Domek et al., 1990; Bartelt et al., 1993; Campbell et al., 1993; Weissling et al., 1993). Semiochemical management of M. matrida populations may require identification of both the insect and host plant volatiles and the synergism between them. The aim of our research was to collect and characterize the volatiles produced and emitted by live virgin beetles as a means of studying the chemical communication in the species and subsequently to apply the findings to pest control. In this paper we present the results of field observations, field-trapping experiments with live beetles and their volatiles, and laboratory olfactometer bioassays. We also describe an all-glass system for the collection of insect volatiles. Finally, we propose a possible mechanism for chemical communication among M. matr/da beetles. METHODS AND MATERIALS

Rearing of Beetles. Third-stage grubs of M. matrida were collected in the field and reared individually in 10-ml plastic boxes containing humid sand. The boxes were kept in a climate-controlled room at 27 _-!-3°C, 60 + 15% relative

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humidity, and a 14L: 10D regime. The grubs were fed with wheat roots. The adult beetles were reared in groups (up to 100 beetles of the same sex in l-liter plastic boxes) and fed with rose flowers. Sexing was performed with the aid of a dissecting microscope by the method described by Gerling and Hefez (1990). Adult beetles were frozen at 0°C for about 3 min and then inspected after the pigydium had been opened gently. Females are distinguished by the presence of two elastic chitinic plates located on each side of the pigydium aperture across the abdomen tip. Males are distinguished by a sex organ, which widens into a funnel-like asymmetric chitinic appendage, reminiscent of the stinger of a bee, for grasping the female (Argaman, 1986, 1990). Collection System for Volatiles. The system (Figure IA) is composed entirely of glass (without any plastic or Tygon piping) in order to prevent contamination. All the tubes are 0.83 cm ID, with spherical S-29 ground glass connections. A strict cleaning routine was followed: new parts of the system were heated at 550°C for 4 hr, and all parts were rinsed with sulfochromic acid, sodium bicarbonate solution, and tap water. The volatiles were collected as follows: By means of an airflow regulator (Mego-Afek, Israel) and air flowmeter (model 10A1190, Fisher and Porter Co., Warrninster, Pennsylvania, capable of discharging 10-117 ml/min), clean dry compressed air (Maxima, Israel) was passed through Poropak Q (Millipore Co., Milford, Massachusetts) and silica blue (Merck Co., Darmstadt, Germany) filters into 2-liter glass flasks containing live beetles. The conditions in the flasks were similar to those in the field, i.e., light intensity of 0.01-0.I lux (darkness), temperature of 24-30°C (ambient temperature), airflow rate of 0.1 liter/min (wind velocity of 0.1 m/sec), and collection of the volatiles over 150 min (flight time in nature). Volatiles were collected separately from male and female virgin, laboratory-reared, and wild beetles from the field whose sexual maturity and status was not known. The volatiles liberated by the beetles were carried over by the airflow and captured in a number of traps connected in series. In most cases three traps were used, the first two being empty tubes cooled with liquid nitrogen and the third conmining an organic solvent, but in some cases the volatiles were trapped in two tubes, both containing organic solvents (cyclohexane or hexane, HPLC grade, Aldrich Chemical Co. Inc., Milwaukee, Wisconsin). After collection of volatiles was completed, the traps were cooled by liquid nitrogen, and the flasks conmining the beetles were rinsed twice with 5 ml of solvent each time. The rinsing solutions were collected for each container separately, dried over calcium chloride if necessary (mainly the traps), and then concentrated with the aid of a slow nitrogen stream up to a final volume of 0.5 ml. The same drying and concentration process was also applied to the solvents in the traps. The concentrates were stored under an inert atmosphere in 1-ml conical tubes stoppered with S-14 ground glass stoppers at 4°C.

I,

¸ L/ FIc. 1. Collection system for volatiles: (A) general view; (B) close-up. The airborne volatiles of the beetles were collected separately from four large groups (up to 150 in each flask) of beetles. The air, cleaned by passing it through Poropak Q and silica blue filters, was passed over live beetles kept in 2-liter glass flasks under conditions imitating those in the field as closely as possible. The volatiles liberated by the beetles were carried over by the airflow and captured by three traps connected in series, two empty tubes cooled with liquid nitrogen, and a tube containing cyclohexane.

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Field Observations of Aggregations. Observations were conducted in peanut fields in the south of Israel at the Kibbutzim Sufa, Holit, and Ze'elim (biological field, where no pesticides were applied) during the summers of 19911993. Flying, eating, and/or mating beetles on the foliage were collected by hand at 5-min intervals during the first 25 min after sunset. Data analysis was performed according to the G-test procedure for replicated goodness of fit, at the 5% level of significance for the deviation from expectation (1 : 1) (Sokal and Rohlf, 1981). Field-Trapping Experiments. The bait comprised three adult M. matrida virgin beetles with or without food. Another set of baits contained a concentrate of volatiles that was collected from hundreds of beetles over a number of 150min periods (each period being defined as a day of collection). We may thus estimate that 10 t~l of cyclohexane solution is about 300 beetle equivalents (BE) per day of collection. The concentrate was applied to Whatman No. 1 filter paper (4.25 cm circle) (the solvent was evaporated for 10 min outside the trap) with or without food (cut-up peanut leaves from a biological field) placed in a perforated 25-ml plastic box connected to the top of a trap. The traps were the type used for trapping Japanese beetles (Ringer Co., Minneapolis, Minnesota); they were washed with detergent and water and then dried in the sun for three days before each field study. The trapping tests were arranged in a randomized complete block design; the distance between traps in a replicate and between replicates was 20 m. The traps were positioned 40 cm above the foliage. Field experiments involving trapping with live virgin beetles as attractants were conducted on May 14, 1992 (29-25°C, 40-50% relative humidity, 0.20.01 lux) and on May 28, 1992 (27-24°C, 50-55% relative humidity, 0.2-0.01 lux) at Kibbutz Sufa. These experiments comprised the following five treatments (six replications): live virgin females with or without food; live virgin males with or without food, and food alone. No empty traps were used (see below). The attracted adults were sexed under a dissecting microscope. Data were analyzed according to the procedure of Sokal and Rohlf (1981), three-way mixed model ANOVA (days, treatments, and blocks) for the two variables (males and females of M. matrida that were trapped), and the Student-Newman-Keuls multiple-range test at the 5 % level of significance. Field experiments involving trapping with volatiles produced by live virgin beetles as the attractant were conducted on May 15, 1993 (32-28°C, 40--45% relative humidity, 0.3-0.01 lux) at Kibbutz Holit and consisted of the following six treatments (seven replications): live virgin females with food, volatiles produced by live virgin females with or without food; volatiles produced by live virgin males with or without food, and cyclohexane with food alone. Data were analyzed by a two-way mixed ANOVA (treatments and blocks) model for the two variables and the Student-Newman-Keuls multiple-range test at the 5 % level of significance.

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Laboratory Olfactometer Bioassays. A Y-shaped glass olfactometer system (2.64 cm ID) was constructed from two 10-cm-long glass tubes and one 15-cm tube joined with a spherical S-35 ground glass connection that allowed an insect to crawl and fly freely through the Y tube. The three ends of the Y tube were closed with plugs that enabled fast insertion and removal of the beetle. A different bait was placed at the end of each of the two arms of the Y tube, and an insect that had been deprived o f food for 24 hr prior to the start of the study was inserted at the tail of the tube. The insect then chose to move into one of the arms against a stream of clean air flowing from the ends of the arm. The air, cleaned by passing it through Poropak Q and silica blue filters, was pumped (pump model 9100, TIF Co., Chicago, Illinois) via flowmeters (9232 Cole Palmer Co., Chicago, Illinois) located at the air inlet and outlet. (By means of smoke, it was verified that the air flow is not turbulent). Each pass lasted 5 min. The olfactometer system was cleaned the same way and maintained under the same conditions as those pertaining to the collection system. The baits were: three virgin females or three males with or without food, food alone, or 10 ~tl of a cyclohexane solution of female volatiles with food. Each type of bait was tested versus the other baits. The results were examined by the G-test procedure at the 5% level of significance (SokaI and Rohlf, 1981). RESULTS AND DISCUSSION

Collection System for Volatiles. The method we report here tbr the collection of volatiles fulfills the criteria required for such a system: protection from contamination; simplicity, rapidity, and flexibility of construction; operation of the system for a large number and wide diversity of insect populations; and low cost (Figure 1A). The airborne volatiles of the beetles were collected separately from four large groups of beetles (up to 150 in each flask--virgin males and females reared in the laboratory, and males and females from the field) (Figure 1B) for comparisons of biological activity and chemical profile. These comparisons are necessary in order to determine the efficacy of the method of collection of the volatiles and the possibility of mass collection o f volatiles from beetles in the field. Collection in empty traps cooled with liquid nitrogen was found to be superior, since it reduced the amount of contaminants (that are present in the solvents) and obviated the need to evaporate large volumes of solvent, thus preventing the loss of components of the volatiles. This technique will thus be used in the future to collect additional quantities of volatiles for structural studies and other analyses using GC-MS, EAG, GC-EAD, preparative separation, NMR, and other spectroscopic techniques. Field Observations of Aggregations. We focused our interest on the sex ratio and time dependence of the formation of aggregations in peanut fields.

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VOLATILE ATTRACTANTS

This aggregation behavior was studied in peanut fields where we conducted our trapping experiment. Such aggregations are not unique to the peanuts and were also found on roses, potatoes, and wild grass surrounding the field. At sunset, mature M. matrida beetles emerged from the ground to search for food and to mate (Figure 2). Most of the beetles captured on the peanut plants during the first 20 min of flight were males (in the first 10 min 85 % of the captured beetles were males, in the next 5 min 67% were males, in the final 5 min 59% were males), but afterwards equal numbers of males and females were captured. Soon thereafter, aggregations of 20-30 beetles were formed on the peanut plants, the aggregations being composed of the two sexes in equal numbers. This 1 : 1 sex ratio was kept in the aggregations throughout the activity period, until feeding and mating was over. Mating was observed in the period from 15 to 25 min after emergence and lasted for at least for 15-20 min while the females continued feeding. These observations indicate that aggregation takes place at the same time as feeding and mating and provides support for the existence of a chemical communication mechanism based on volatiles liberated by the beetles or the

80

20

0 Time

from

Sunset

(rain)

•

Values are total n u m b e r of trapped beetles at the m e n t i o n e d time interval • " Sunset is the starting time, zero, "** Observations of aggregations and mating.

FIG. 2. Field observations of aggregations. The sex ratio and time dependence of the formation of aggregations were examined (P

~7 m Z > Z

>

,.¢

tJ

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attracted by volatiles from virgin females plus food vs. food alone and 60% vs. volatiles from virgin males plus food; 90% of experimental males were attracted by volatiles from virgin females plus food vs. food alone and 75 % vs. volatiles from virgin males plus food. These results indicate that attraction by live females plus food was similar to attraction by volatiles from virgin females plus food, in keeping with the findings of the field trapping experiments (Tables 1 and 2). An interesting point is that females or female volatiles plus food were more attractive to males (78-75%) than to females (59-60%) (Table 3, entries 4 and 5 vs. 1 and 2). This may indicate the presence of a sex pheromone released by the females to attract males, in addition to the attraction by the food volatiles. In general, our findings that in the field and in the olfactometer female volatiles have the same effect as live female beetles on the behavior of the tested beetles are in keeping with the olfactometer study of Harari et al. (1994). The only exception lies in the preference of virgin females toward females and food vs. males and food (entry 1): while we found a small preference of females towards females and food, Harari et al. (1994) reported no preference in such an experiment. The findings of Harari et al. (1994) are based solely on olfactometer studies, whereas our conclusions are based on field trapping, which is a completely different environment in terms of ambient conditions and other factors distinguishing a natural from an artificial state. Although Harari et al. (1994) did not find evidence for the existence of an aggregation pheromone released either by males or females, we cannot, at this stage, ignore or exclude such a possibility under field conditions. Biological Behavior. The findings of our field and laboratory experiments indicate the operation of a possible two-step mechanism of chemical communication in M. matrida. The first step is the liberation of plant volatiles as a result of the mechanical injury caused by the males, which emerge first. These volatiles attract both sexes until a 1 : 1 sex ratio is reached. The second step is the excretion of an attracting mixture (perhaps a sex or aggregation pheromone) by the females while they eat the leaves of the plant or just after eating. The attraction may be due to a combination of insect pheromone(s) and plant volatiles, which are liberated during the process of eating, and the two agents may act synergistically. Acknowledgments--The authors wish to thank the Office of the Chief Scientist. Ministry of Agriculture, for supporting the study.

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tion ofAnomala cupera Hope (Coleoptera: Scarabaeidae) sex pheromone. Appl. EntornoL ZooL 27:289-291. LEAL, W.S., SAWADA, M., and HASEGAWA,M. 1993. The scarab beetle Anomala cupera utilizes the sex pheromone of Popillia japonica as a minor component. J. Chem. Ecol. 19:1303-1313. SHANI, A., and LACEY, M.J. 1984. Convenient method applicable to single insects for collection and measurements of blend ratios of airborne pheromones from artificial sources. J. Chem. Ecol. 10:1677-1692. SOKAL, R.R., and ROHL~, F.J. 1981. Biometry. W.H. Freeman. San Francisco. TAMAKI, Y., SUGIE, H., and NOGUCHI, H. 1985. Methyl (Z)-5-tetradecenoate: Sex attractant pheromone of the soybean beetle, Anomala rufocupera Motschulsky (Coleoptera: Scarabaeidae). Appl. Entomol. Zool. 20:359-361. TUMLINSON,J.H., KLEIN, M.G., DOOLITTLE,R.E., LADD, T.L., and PROVEAUX,A.T. 1977. Identification of the female Japanese beetle sex pheromone: Inhibition of male response by an enantiomer. Science 197:789-792. WElSSLING,T.J., GIBLIN-DAVlS,R.M., and SCHEFERAHN,R.H. 1993. Laboratory and field evidence for male-produced aggregation pheromone in Rhynchophorus cruentatus (F) (Coleoptera: Scarabaeidae). J. Chem. Ecol. 19:1195-1203.