Insect Sci. Applic. Vol. 20, No. 1, pp. 1-5, 2000

The Role of Different Components of the Pheromone Emission of Mature Males of the Desert Locust, Schistocerca gregaria (Forskål) (Orthoptera: Acrididae) in Accelerating Maturation of Immature Adults

Hassane Mahamat, Ahmed Hassanali and Hezekiel Odongo
International Centre of Insect Physiology and Ecology (ICIPE), P.O. Box 30772, Nairobi, Kenya

E-mail: ahassanali@icipe.org

Corresponding author: AH.

(Accepted 29 March 2000)

Code Number: ti00001

Abstract– Different blends of five chromatographically prominent components of the pheromone emission of gregarious-phase mature desert locust Schistocerca gregaria (Forskål) (Orthoptera: Acrididae) males were bioassayed for their maturation-accelerating effects on immature counterparts. The blend of all five components, namely anisole, veratrole, benzaldehyde, phenylacetonitrile and 4-vinylveratrole was as effective as the emission from live mature males in accelerating the onset of mating in immature males. Subtraction of anisole had no significant effect but that of any one of the other four components significantly reduced the acceleration potency of the resulting blends. The maximum reduction occurred in the absence of phenylacetonitrile which appears to be critical to the activity of the blend. The magnitude of reduction in maturation acceleration that resulted from the subtraction of the other three components suggests that, although present in lesser relative amounts, these compounds contribute significantly to the activity of the full blend. Some differences were found between the onset of integumental yellowing and mating which indicate that blends of slightly different compositions are involved in promoting these two physiological processes.

Key Words: desert locust, Schistocerca gregaria, maturation acceleration, phenylacetonitrile, benzaldehyde, veratrole, 4-vinylveratrole, anisole

Résumé–On a essayé différents mélanges de 5 substances les plus importantes obtenues lors de la chromatographie des sécrétions phéromonales des mâles adultes en phase grégaire du criquet du désert (Schistocerca gregaria) Forskal (Orthoptera: Acrididae), afin de déceler leurs effets biologiques de hâter le début d’accouplement chez les sujets immatures de la même espèce. Le mélange de cinq substances dont l’anisole, le vératrole, le bénzaldéhyde, le phénylacétonitrile et le 4-vinylvératrole accélérait efficacement le début d’accouplement au sein des sujets mâles immatures au même titre que le phéromone dégagé par les mâles adultes vivants. L’exclusion de l’anisole du mélange n’avait pas d’effet significatif mais par contre, la suppression de l’une ou l’autre des quatre substances réduisait significativement la capacité du mélange d’accélérer le début d’accouplement. La réduction maximale avait lieu à l’absence du phénylacétonitrile qui semble être indispensable pour l’activation du mélange. L’importance de la réduction de l’accélération du début d’accouplement résultant de l’exclusion des autres substances suggère que celles-ci contribuent significativement à l’activation du mélange des cinq éléments, quand bien même à des concentrations relativement faibles. Quelques différences ont été notées entre le début du jaunissement du tégument et le début d’accouplement, ce qui indique que les mélanges avec des compositions légèrement différentes participent dans l’activation de ces deux processus physiologiques.

Mots Clés: criquet du désert, Schistocerca gregaria, accélération de la maturité, phénylacétonitrile, anisole, vératrole, bénzaldéhyde, 4-vinylvératrole

Introduction

Observations made by Norris (1952, 1954, 1957), Loher (1960) and Amerasinghe (1978) suggested that the accelerating effect of mature males of the desert locust, Schistocerca gregaria, on the sexual maturation of their immature male and female counterparts was mediated by a volatile pheromone. This observation was confirmed by demonstrating that trapped volatiles of the mature male desert locust also accelerated the maturation of young males and females, as evidenced by yellowing and copulation (Mahamat et al., 1993) and by earlier increase in the level of vitellogenin in females (Mahamat et al., 1997). Gas chromatography-linked mass spectrometric (GC-MS) analysis of the volatiles showed the presence of anisole, veratrole, benzaldehyde, phenylacetonitrile and 4-vinylveratrole (Fig.1) in the approximate ratio of 6:3:7:79:5. In addition, a series of other benzene derivatives, including guaiacol, phenol, benzylnitrile, benzyl alcohol and 2-benzoyloxyphenylacetonitrile were present in smaller amounts (Mahamat et al., 1993). In a parallel study, emissions of older and mature males were also found to elicit aggregation of the adult stages of the desert locust (Obeng-Ofori et al., 1994; Torto et al., 1994). Thus, the adult male volatiles have a dual function: that of an adult aggregant and a maturation accelerant. The main behaviourally active components were shown to be phenylacetonitrile, guaiacol, phenol and benzaldehyde in aggregation assays (Torto et al., 1994). The ethers, such as anisole and veratrole, were electrophysiologically active but did not elicit significant aggregation. On the other hand, veratrole has been shown to be part of the oviposition-inducing signal associated with egg-froth volatiles (Saini et al., 1995; Rai et al., 1997). In the present study, a subtraction procedure (Owaga et al., 1988) was used to identify compositionally prominent components of the mature male volatiles that contribute to maturation acceleration in the desert locust.

Materials and Methods

Insects

Gregarious desert locust, S. gregaria, from the ICIPE colony originating from a stock obtained from the Desert Locust Control Organisation for Eastern Africa (DLCO-EA) in Addis Ababa, Ethiopia and another from the stock collected from the Tokar Delta in the Sudan, were used in the study. Locusts (300-400) of both sexes were bred under crowded conditions in aluminium cages (50 x 50 x 50 cm). These insects were reared in a special room (4.5 x 4.5 m) which was well aerated by a duct system (10-15 air changes/h) that maintained a negative pressure, at a temperature of 30-35°C and 12:12 (L:D)h cycle. Fresh wheat shoots and bran were provided daily for the insects. Immature male adult desert locusts used in this study were 1-2 days old (after moult) and sexually mature male adults were 4-5 weeks old (after moult). Males were selected for the present study because their integumental yellowing associated with maturation is relatively more prominent than in females (Norris, 1954; Loher, 1960).

Preparation and dispensing of synthetic blends

A 2% stock solution of a blend of the five major components, anisole (I), veratrole (II), benzaldehyde (III), phenylacetonitrile (IV) and 4-vinylveratrole (V) (Aldrich Ltd) was prepared by dissolving appropriate amounts of the compounds in the ratio 6:3:7:79:5 in paraffin oil. Five other similar stock solutions were prepared each containing only four of the five compounds, i.e. with one of the five compounds missing. The differences in weights between solutions and that containing the full blend were compensated by adding appropriate amounts of paraffin oil.

Solutions containing different blends (2.0 g each) were dispensed from 4-ml vials (Aldrich Ltd) with plastic screw caps with 5-mm-diameter vents to control the release of volatiles. Pilot experiments with vents of different sizes had shown that under the conditions of the experiment (33 ± 2°C, relative humidity 60%) the 5-mm-diameter vent gave a relatively stable release rate of 6.8 µg/day of the major component (phenylacetonitrile), comparable, on the average to that of three 24 to 28-day-old mature males from our crowd-reared colony.

Fig. 1. Major components of volatile emission of mature Schistocerca gregaria males

Bioassays

Bioassays were carried out in two-chamber cages (each 30 x 15 x 15 cm) as previously described (Mahamat et al., 1993; Assad et al., 1997). The chamber consisted of a lower part, where the test insects were placed, separated from the upper part, where the source of the pheromone was located. The two chambers were separated by a wire gauze covered with a black cloth. No visual or tactile contact between insects from the two chambers was possible.

The experiments were set up in a well aerated room (5.4 x 1.6 m) maintained at 32 ± 2°C, 60% relative humidity and 12:12 (L:D) h cycle similar to the rearing rooms described above. Only 12 experimental cages (2 per treatment) were placed in the room at a time, well spaced out to avoid proximity effects between different treatments and undue concentration of the volatiles in the room.

A group of three newly moulted immature (1 to 2 day-old) males (referred to as recipient or test insects) were combined with sexually mature males, immature males, or one of six dispensers containing different blends of the test compounds described above (referred to as signal sources). The signal sources were placed in the upper chamber and the recipients in the lower chamber. Treatments are shown in Table 1. Each was replicated six times.

Recipient insects were checked daily for signs of maturation as assessed by: (1) changes in colour (yellowing) in accordance with stage III classification developed by Norris (1954) and recently used in our laboratory (Mahamat et al., 1993; Assad et al., 1997); (2) copulation time carried out with young males that had begun to develop coloration on the abdomen (one test male was withdrawn from each cage and its mating behaviour in the presence of a mature (4-week old) female observed for 6 h and copulatory activities, if any, were recorded).

Data analysis

Data on yellowing and mating were subjected to analysis of variance (ANOVA) using SAS Initiative Inc. Cary, North Carolina 27512-8001,1987. Means for different treatments were compared using the least significance difference (LSD) test.

Results

Table 2 summarises the results of exposure of immature males of S. gregaria to different blends of the five major compounds released by their mature counterparts. In the majority of treatments, integumental yellowing more or less coincided with the onset of copulation activity; however, there were small differences in two treatments, one associated with volatiles from mature males (11.00 ± 0.21 for yellowing and 13.89 ± 0.32 for copulation), and the other with synthetic blend minus 4-vinylveratrole (V) (20.39 ± 0.44 for yellowing and 23.39 ± 0.16 for copulation).

Of the synthetic blends, the blend of the five major components was as effective as the natural emission from mature males in inducing the onset of copulation. However, its yellowing effect was significantly slower than that of the natural mature males emission. Omission of anisole (I) from the blend did not affect the maturation time (14.17 ± 0.23 for yellowing; 14.89 ± 0.21 for copulation) compared to the treatment with the full blend, showing that this compound does not play any significant role in the process. On the other hand, subtraction of any of the other components led to significant delay in both integumental yellowing and onset of copulatory activity. The maximum delay occurred with the blend in which phenylacetonitrile (IV) was missing. The number of days to copulation in the treatment involving this blend (26.89 ± 0.37 days) was not significantly different from the control with only immature males (27.50 ± 0.12 days), although yellowing occurred somewhat earlier (day 26.05 ± 39 in the former compared to 27.50 ± 0.12 in the control). The next large delay occurred in the blend in which benzaldehyde (III) was omitted (maturation time ~24 days) followed by those in which the two ethers, veratrole (II) and 4-vinylveratrole (V), were missing (maturation time about 19-20 days).

Discussion

We previously showed that the volatile emission associated with gregarious-phase mature males of S. gregaria contained pheromone constituents responsible for accelerating the sexual maturation of immature males and females (Mahamat et al., 1993). In the present study, the roles of five chromatographically prominent components of mature male emission (anisole, benzaldehyde, veratrole, phenylacetonitrile and 4-vinylveratrole, I-V) on the onset of integumental yellowing and copulation were studied. In the treatment involving exposure of immature males to a synthetic blend of the five compounds, released in approximately the same ratio and rate as emissions from control mature males, the insects mated at approximately the same time as those exposed to the volatiles from live mature males (Table 2). This shows that the synthetic blend contained the components largely responsible for maturation-accelerating effect of male pheromonal emission. On the other hand, the blend was slightly but significantly less effective in accelerating integumental yellowing, suggesting that other minor components in male volatiles (Mahamat et al., 1993) may also contribute to this process.

In the treatments involving subtraction of one of the five components, only the omission of anisole (I) had an insignificant effect on the maturation-accelerating effect of the remaining blend. The absence of each of the other four components caused varying degrees of reduction in maturation acceleration, with the omission of phenylacetonitrile (IV) having the most pronounced effect. Indeed in the absence of phenylacetonitrile, the timing of copulation was not significantly different from that of immature control, although that of integumental yellowing was marginally faster (Table 2). Interestingly, this compositionally dominant component of the gregarious-phase pheromonal emission of adult males, is also the most potent in eliciting behavioural aggregation in adults (Torto et al., 1994). Despite the dominant role of phenylacetonitrile in accelerating maturation, the other components (benzaldehyde, veratrole and 4-vinylveratrole, III, II and V respectively,) also contribute significantly to this effect. In fact, the magnitude of delay in maturation resulting from the omission of one of these compounds, particularly benzaldehyde, relative to that associated with the full blend, appears to be quite disproportionate and suggests that the pheromone components do not exert their effects separately and additively, but as a blend in which the constituents may interact in a rather intricate way. Studies on the physiological basis of action of the compounds may help throw light on the phenomenon.

In summary, maturation assays conducted in the present study with synthetic blends in which one of the five test constituents was missing provide an interesting insight into the way these constituents interact as a blend. Although the presence of phenylacetonitrile is clearly critical for maturation acceleration, the assays reveal that the relatively minor components contribute disproportionately to the activity of the blend. Secondly, although integumental yellowing coincided with sexual maturation in most treatments, this was not always so, and suggests that a somewhat different blend of male volatiles is responsible for inducing this process.

Acknowledgements–The authors are grateful to Drs Baldwyn Torto and Peter Njagi for reviewing the manuscript. The study was undertaken as part of desert locust semiochemicals project funded by IFAD, UNDP, SAREC and AFESD, whose support is gratefully acknowledged.

References

Printed in Kenya. All rights reserved © 2000 ICIPE

The following images related to this document are available:

Photo images