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Semiochemicals from froth of egg pods attract ovipositing female Schistocerca gregaria
J. Insect Physiol. Vol. 41, No. 8, pp. 711-716, 1995
Copyright 0 1995 Elsevier Science Ltd
Pergamon
0022-1910(95)00016-X
Printed in Great Britain. All rights reserved 0022-1910195 $9.50 + 0.00
Semiochemicals from Froth of Egg Pods Attract Ovipositing Female Schistocerca . gregarla R. K. SAINI,*t
M. M. RAI,*
A. HASSANALI,*
J. WAWIYE,*
H. ODONGO*
Received 7 July 1994; revised 16 December 1994
A chemical signal, originating from the froth of egg pods attracts gravid female S’chistocerca gregariu (Orthoptera: Acrididae) to common egg laying sites. Behavioural experiments indicated that females preferred to oviposit in moist sand contaminated with froth (60% egg laying vs 34% in sterilized sand). Extracts and volatiles collected from froth were also attractive to gravid females. In fact, froth volatiles elicited the strongest egg laying response (80% egg laying). Results with froth extracts obtained by sequential extraction with solvents of increasing polarity suggest that both non-polar and polar compounds are involved in the attraction of gravid females. Electroantennogram recordings with extracts and volatiles collected from froth confirmed the presence of olfactory receptors on the antennae that are responsive to compounds in the extracts and the volatile collections. Schistocerca gregaria
Locust
Oviposition
Aggregation
Female Schistocerca gregaria (Forskal) have been observed to deposit their egg pods in sites where other females are ovipositing even though other areas have environmental factors that appear to be suitable (Popov, 1958; Stower et al., 1958; Uvarov, 1977). Norris (1963, 1970) found that both visual and chemical cues were important in aggregating females to common egg laying sites. She found that living decoys of either sex, immature or mature males, and last instar hoppers could be used as decoys and were functional even in total darkness. The fact that chemical stimuli may also be involved was reconfirmed when Norris (1970) showed that papers contaminated by mature adults and by hoppers were attractive to gravid females. Responses to locust extracts in several solvents were, however, quite variable and active extracts were more readily prepared from mature males than from newly ecdysed adults. Norris (1970) concluded that the active substances, believed to be a pheromone, were present on the locust’s entire body. She also showed that antennatectomized females were able to find live decoys but that they were much less responsive to dead ones. However, when decoys were enclosed and could no longer be touched, the response was greatly diminished. Accordingly, she suggested that (ICIPE),
Egg laying
Electroantennograms
chemotactile receptors on other parts of the body in addition to the antennae and possibly the palpi also promoted oviposition. Norris found no attraction to sand contaminated with exposed froth plugs and faeces such as might be present at previously used oviposition sites. Norris (1970) also reported that exuviae from Locusta migratoria (Reiche & Fairmaire) were attractive to ovipositing females of S. gregaria but to a lesser extent than those from conspecific locusts. This suggested that the oviposition aggregation pheromone was at least partially species specific. Lauga and Hatte (1977, 1978) showed that female L. migratoria were strongly attracted over a distance of 0.5 m to sand into which gregarious females had laid egg pods. No work relating to the identification of these attractants has been reported in the literature. The present studies were designed to determine if a chemical signal from egg pods mediated oviposition, and if so, to establish the nature and to trace the source of the signal.
INTRODUCTION
*International Centre of Insect Physiology and Ecology Box 30772, Nairobi, Kenya. tTo whom all correspondence should be addressed.
Pheromone
MATERIALS AND METHODS Insects
Crowded desert locust, S. gregaria were obtained from the International Centre of Insect Physiology and Ecology (ICIPE) locust colony which originated from the locust culture of the Desert Locust Control Organization for Eastern Africa (DLCO-EA) in Addis Ababa,
P.O.
711
R. K. SAINI
712
Ethiopia. Insects were reared under crowded conditions using aluminium cages (50 x 50 x 50 cm). The room temperature was maintained between 30-35°C with a 12: 12 h 1ight:dark cycle. In all the experiments 13-15 day-old locusts were used.
et al.
of the solvents. The extracts were stored at -20°C until use. Amounts of extracts corresponding to 1 or 2 pod equivalents were tested individually or in combination in two- or four-choice bioassays. Collection
Experimental
In an initial experiment, two-choice behavioural bioassays were undertaken in standard aluminium locust rearing cages (50 x 50 x 50 cm). All sides of the cage were made from wire mesh except for the front which had a sliding glass door. The ceiling had an electric bulb at the rear end. Locusts were provided with four aluminium oviposition tubes (each 10.0 cm 1 x 4.0 cm dia.) at the front of the false floor and close to the sliding door. Two of these tubes were filled with sterilized sand (control) with moisture content of 15% (15 ml water to 100 g sand) while the other two were filled with moist sand containing two egg pods. The two sets of tubes were placed 18 cm apart at an intertubal distance of 3.5 cm. The initial standard rearing cages however, proved to be inappropriate for bioassays as it was observed that the egg laying tubes were too narrow and if one was occupied by an egg laying adult, any other gravid female was forced to oviposit in a control tube. In addition, the tubes were placed in a row and their placement could not be altered. Heating was also found to be asymmetrical. new larger of the above reasons, Because (60 x 60 x 60 cm) four-choice behavioural cages made of aluminium were constructed. The sides and roof of these cages were made of wire mesh; the back was of metal and the front had a removable glass door. The ceiling had a 60 W electric bulb in the middle. The false floor in the middle had four egg laying cups (8.5 cm dia., 9 cm depth) placed at distances of 13 cm from each other and 22 cm diagonally. For two-choice experiments, two of the diagonally placed cups were blocked by turning them upside down. Cleaning and conditioning
of sand
Sand for oviposition cups was sieved using wire mesh (2 mm’) and washed successively with hexane, ethyl acetate, methanol and finally with distilled water. It was then dried and heat-sterilized by baking in an oven at 150°C for 24 h. Sterilized sand was moistened by adding 15 ml water per 100 g of sand. Extraction
of volatiles
cages
of eggs and froth
Froth and egg extracts were obtained either by sequential or by single solvent extractions. For sequential extraction, eggs and froth derived from pods were allowed to dry at ambient temperature (25-27°C) for 6 h. Each part was then placed in a dropping funnel (50 ml) and 6 ml hexane added. After 10 min, the hexane extract was removed and the extraction repeated with ethyl acetate (6 ml) followed by methanol (6 ml). Single-solvent extractions of froth were carried out in the same way with hexane, ethyl acetate and methanol, each time using 25 ml
Volatiles were collected on traps using activated charcoal (S&l00 mesh, Chromopack). Before use, the charcoal was cleaned by Soxlet extraction with dichloromethane (Merck) for 72 h, followed by thermal treatment at 250°C under nitrogen (20 ml/min). Volatiles were collected from 1-day-old froth plugs placed in a short glass tube connected to charcoal traps prepared by packing charcoal (ca I .2 g) between two glass wool plugs in 6 cm long x 8 mm id. glass tubes. Nitrogen, cleaned by passing it through a short plug of activated charcoal, carried volatiles from froth to the charcoal trap at the rate of 3.6 ml/min at room temperature. Trapping was carried out for 12 h and the adsorbed material eluted with 8 ml HPLC grade dichloromethane (Aldrich Ltd). Behavioural
bioassays
Egg laying responses of ovipositing S. gregaria females were determined in three types of experiments: (1) Gravid females were offered a choice between sterilized sand and sand containing two egg pods in the standard rearing cages (Expt. 1). Two pairs of insects were used for each of the 30 replicates. Experiment 2 was a repeat of Expt. 1 in the new large behaviour chambers using 6 pairs of locusts for each of the 17 replicates. In all subsequent bioassays, only the larger behaviour chambers were used. (2) Gravid females were given a choice between sterilized sand (control), sand mixed with only eggs from two egg pods, sand mixed with froth from two egg pods or sand mixed with two complete egg pods (Expt. 3). In Expt. 4 the treatments were similar except that the sand with two egg pods was substituted with sand from which egg pods had been removed. (3) Gravid females were offered a choice between various sands contaminated with either sequentially extracted or single solvent extracts of froth or eggs (Expts 5-7) mixtures of froth extracts (Expt. 8) or froth volatiles (Expt. 9). In these experiments, the extracts or volatiles from froth (two froth equivalents) were delivered on strips of filter paper (10 x 2.5 cm). These impregnated filter paper strips were then placed into the egg laying containers about 1 cm below the surface of the moistened, sterilized sand. Filter paper strips treated with similar amounts of respective solvents were used as control stimuli. In all the experiments, except where indicated, each replicate consisted of eight pairs of insects tested for 4 days. The egg pods laid were counted daily by removing the egg laying cups. These were then replaced by freshly prepared egg laying cups with their respective treatments. In all four-choice experiments (in order to avoid any
EGG
POD ATTRACTION
713
TABLE 1. Responses of ovipositing Schistocerca greguriu females to sand mixed with egg pods in standard locust rearing (A) and in new enlarged oviposition cages (B) Egg pods deposited
Treatments
Expt. No.
No. of replicates
(A) Standard
(B)
cages Sterilized sand (SS) SS + 2 egg pods
30
New cages
17
Sterilized sand SS + 2 egg pods
No.
%
23 32
4l.P 58.2
35 68
34.0b 66.0
Means within an experiment followed by the same letter are not significantly different (P i 0.05). N = 2 pairs of insects per test in (A) and N = 6 in (B).
position effect) the position of the egg laying cups was rotated in 4 x 4 Latin square design. In two choice experiments, the cups were rotated diagonally. All the bioassays were conducted in the laboratory at 30 f 2”C, relative humidity of 4@45% and 12:12, L:D photoperiod. Test insects were fed daily with seedlings and wheat bran (Triticum sp.). The results were analysed using Wilcoxon’s T test (Wilcoxon, 1945) and Friedman’s test (Friedman, 1940). Electroantennograms
60 s before and after each stimulation with the test compounds. The amplitude of the response to the test compound was expressed as a percentage of the mean response to solvent control stimuli presented 60 s before and after stimulation with the test compound. EAGs were recorded from at least 10 gravid female antennae for each stimulus.
(EAGs)
RESULTS
EAGs were recorded utilizing glass pipettes filled with locust saline (Hoyle, 1951) into which Ag-AgCI wires were inserted. The indifferent electrode was inserted into the pedicel of an amputated antenna while the recording electrode was placed over a few segments of the flagellum. The signal was amplified by a Grass P16 amplifier (Universal a.c./d.c. amplifier, Grass Instruments Co., MA, U.S.A.), displayed on an oscilloscope and recorded using a chart recorder. The odour delivery system and stimulation technique was as described by Saini and Hassanali (1992). EAGs were recorded to hexane, ethyl acetate and methanol extracts of froth and from volatiles (in dichloromethane) collected from froth at the following dosages: 0.1, 0.2, 0.3, 0.4 and 0.5 froth equivalents (one froth equivalent is the volatiles contained in the froth from one egg pod). To correct for the gradual diminishing of the EAG response during an experiment and to allow comparison among the different preparations, an equivalent amount of the respective solvent was applied
Initial two-choice experiments (Expt. 1) undertaken in standard rearing cages in which gravid females were given a choice between sterilized sand or sand in which previously laid egg pods were mixed, indicated a small preference for the latter (Table 1). However, when these experiments were repeated in larger, new oviposition cages (Expt. 2), females laid twice as many eggs in sand containing egg pods than in sterilized sand (control) (Table 1). Results of Expts 3 and 4 in which females were given four choices indicated that ovipositing females preferred to oviposit in sand contaminated with either entire egg pods or froth alone as compared to sand mixed with eggs alone or the sterilized sand control (Table 2). Even sand in which egg pods had been laid previously but subsequently removed was shown to be attractive to gravid females (Table 2, Expt. 4). These results clearly indicated that chemicals emanating from the froth of egg pods caused ovipositing females to aggregate at a
TABLE 2. Responses of ovipositing Schistocerca gregariu females to sand mixed with eggs, or froth
or Sand contaminated
with egg pods (in each case the source
was from 2 egg pods) Egg pods deposited
Expt. No.
Treatments Sterilized sand (SS) SS + eggs SS + froth SS + egg pods Sterilized sand (SS) SS + eggs SS + froth Contaminated sand (CS)*
No. of replicates
No.
%
8
12 10 21 19 39 37 68 98
19.4b 16.1b 33.9” 30.6” 16.1b 15.36 28.1” 40.5”
25
Means within an experiment followed by the same letter are not significantly *Sand from which egg pods had been removed.
different
(P < 0.05).
714
R. K. SAINI
et al.
TABLE 3. Responses of ovipositing Schistocerca gregaria females to extracts of: eggs or froth, extracted with hexane (Hex), ethyl acetate (EtAc) and methanol (MeOH) respectively and froth extracted seperately in the above solvents Egg pods deposited Expt. No.
Treatments
No. of replicates
5 Eggs (sequential
6 Froth
7 Froth
extraction) Sterilized sand (SS) SS + Hex extract SS + EtAc extract SS + MeOH extract (sequential extraction) Sterilized sand (SS) SS + Hex extract SS + EtAc extract SS + MeOH extract (separate extraction) Sterilized sand (SS) SS + Hex extract SS + EtAc extract SS + MeOH extract
Means within
an experiment
followed
%
15 16 13 12
26.8= 28.6” 23.P 21.4”
29 76 33 56
14.9b 39.2 17.0b 28.9”
10 25 21 15
14.1’ 35.2” 29.6ab 2l.lk
25
10
by the same letter are not significantly
common egg laying site. Neither eggs alone (Table 2) nor their extracts (Table 3) were attractive. Froth extracts which were obtained either by sequential extractions or separately by single solvent extraction elicited significant attraction of gravid females (Table 3). When froth was extracted sequentially the hexane and methanol extracts evoked significantly more egg laying than the ethyl acetate extract and the control (Table 3). However, when separate solvent extracts of froth were tested, sand impregnated with the hexane extract was found to be the most preferred for egg laying, followed by the ethyl acetate extract. The methanol extract was intermediate in activity between the control and the ethyl acetate extract (Table 3). When ovipositing females were given a choice between different combinations of froth extracts (sequentially extracted with hexane, ethyl acetate and methanol) in 1: 1 or 1: 1: 1 ratio, sand containing both hexane and methanol extracts elicited the highest egg laying response (Table 4). Volatiles collected from froth were also very attractive and evoked significantly more egg laying as compared to sterilized sand (Table 5). The magnitude of electroantennograms (EAGs) recorded in response to the hexane, methanol and ethyl acetate extracts of froth and also from froth volatiles increased with increasing doses (Fig. 1). The captured
No.
8
different
(P -C 0.05).
volatiles however, evoked the highest EAG responses. These electrophysiological investigations confirm the presence of antenna1 olfactory receptors for the attractive compounds in the froth.
DISCUSSION
Gregarious locusts must mate and oviposit in ways that would ensure temporal and spatial cohesiveness of succeeding hopper generations. In this way, the gregarious integrity of the locust population is ensured. In a number of locust species, including the desert locust, this has been achieved through synchronization of maturation of the adults and group oviposition at common egg-laying fields (Popov, 1958; Stower et al., 1958; Loher, 1960; Norris, 1962, 1963, 1964; Amerasinghe, 1978). Mediation by pheromones has been implicated in both these processes (Loher, 1960; Norris, 1963, 1964, 1970; Amerasinghe, 1978), and part of the pheromone system modulating synchrony in S. gregaria has been characterized (Mahamat et al., 1993). In the desert locust, females tend to lay their eggs in areas where other females have oviposited, even though environmentally suitable alternatives may be available (Popov, 1958; Stower et al., 1958; Uvarov, 1977). Our results clearly indicate that froth from egg pods of
TABLE 4. Responses of ovipositing Schistocercagregaria females to binary and ternary mixtures of froth extracts (extracted sequentially with hexane (Hex), ethyl acetate (EtAc) and methanol (MeOH) respectively) Egg pods deposited Expt. No.
8
SS = sterilized
No. of replicates
Treatments SS SS SS SS
+ + + +
MeOH MeOH MeOH EtAc +
sand. Means
+ Hex + EtAc + EtAc + Hex Hex
followed
12
by the same letter are not significantly
No.
%
21 14 28 17
26.3”b 17.5’ 35.W 21.2k
different
(P < 0.05).
EGG TABLE
5.
POD ATTRACTION
715
Responses of ovipositing Schistocerca gregariu females to dichloromethane) collected from froth plugs (G-1 day old)
volatiles
(in
Egg pods deposited Expt. No.
No. of replicates
Treatments
9
Sterilized sand (SS) SS + volatiles
Means
followed
by the same letter are not significantly
S. gregariu is a source of chemical signals that attract conspecific ovipositing females and suggest that these signals may play an important role in group oviposition once a suitable site has been selected. Because of its foamy constitution, it is very likely that the egg froth constitutes an effective controlled-release source of the attractive chemicals, ensuring their attraction over a duration advantageous to the insect. Our results differ from those of Norris (1970) who, surprisingly, failed to observe any attraction to contaminated sand with exposed froth plugs and faeces and concluded that the oviposition stimulus for S. gregaria was nonvolatile and perceived by chemotactile means. On the other hand, Lauga and Hatte (1977) found that sand used for previous egg-laying by females of L. migratoriu was attractive to conspecific gravid females. These authors postulated that egg-laying females produced a pheromone that attracts gravid females of both phases. The pheromone was shown to be volatile, inducing attraction over a distance of 0.5 m to sand in which eggs have been laid. Our behavioural and electrophysiological results with S. gregaria are in agreement with those obtained for L. migratoria. However, females could still utilize a chemotactile stimulus in their oviposition behaviour. In fact, ovipositing females were often seen touching contaminated sand with their antennae and palpi, suggesting the perception of short-range or contact signals. Moreover, our results with froth extracts obtained by sequential extraction with solvents of increasing polarity (hexane, ethyl acetate and methanol) suggest that both non-polar
600 r
n Methanol extract * Hexane extract
+ Ethyl acetate 0 Volatiles
extract
I’;+ 0.2
0.3 Egg
0.4
pod equivalents
FIGURE 1.Electroantennogram (EAG) responses of gravid S. gregaria females to froth extracts and volatiles. Responses are expressed as a percentage of the reference signal (respective solvent), N = 10 antennae (egg pod equivalent refers to volatiles contained in the froth of one egg pod).
22
different
No.
%
33 142
18.9b 81.1”
(P < 0.001)
and polar compounds mediate the oviposition process. It is conceivable that one or more polar compounds extracted in methanol may operate in whole or in part through contact. Norris (1963, 1970) observed that ovipositing female S. gregaria are attracted by other individuals at different developmental stages, alive or dead, as well as to locust extracts and shed skin. While we have not specifically repeated Norris’ experiments, her observations can be explained by recent results on the identity of the components of the aggregation pheromone complex of the desert locust (Obeng-Ofori et al., 1993, 1994a, b; Torto et al., 1994). These studies have demonstrated the existence of three sets of aggregationinducing volatiles in S. gregaria: a blend emitted by live nymphs of both sexes that is specific to nymphs (Obeng-Ofori et al., 1993, 1994a); a blend emitted by older males that elicits aggregation in all stages and sexes of the adults (Obeng-Ofori et al., 1993, 1994a; Torto et al., 1994); and a blend derived from nymphal faeces and those of young adults that elicits aggregation in all stages and sexes of the insect (Obeng-Ofori et al., 1994b). Norris’ experiments did not effectively delineate behavioural elements involved in oviposition from those involved in aggregation, and it is likely that in some of her experiments she observed responses of the females to the aggregation pheromone or residual components of the pheromone rather than to oviposition attractants. Significantly, Norris (1970) found extracts derived from older males more potent in eliciting attraction from gravid females than those from younger adults, which is consistent with the recent finding that the adult aggregation pheromone is produced by older males (Tort0 et al., 1994). It is possible that elements of oviposition behaviour in S. gregaria are modulated by both the froth-derived oviposition pheromone system and the adult aggregation pheromone. Complete characterization of the former would allow the relative role of the two systems in the oviposition process of S. gregaria to be clearly elucidated. Compounds involved in aggregating ovipositing females may be useful in dispersing the oviposition sites to the detriment of the gregarious phase populations or could be used in concentrating locusts in areas where pathogens or other biocontrol agents could cause high mortality. The pheromone might also be used in traps for monitoring populations.
R. K. SAINI
716 REFERENCES
Amerasinghe F. P. (1978) Pheromonal effects on sexual maturation, yellowing, and the vibration reaction on immature male desert locusts, Schistocerca gregaria. J. Insect Physiol. 24, 309-3 14. Friedman M. (1940) A comparison of alternate tests of significance for the problem of m ranking. Ann. Math. Statist. 11, 8692. Hoyle G. (1951) Potassium ions and insect nerve muscle. J. Exp. Biol. 30, 121-135. Lauga J. and Hatte M. (1977) Proprietes gregarisantes acquises par le sable dans lequel ont pondu a de nombreuses resprises des femelles de Locusta migraloria migratorioides (Orthoptera: gregaires Acrididae) R. & F. Acrida 6, 307-3 11. Lauga J. and Hatte M. (1978) L’active gregarisante du sable de ponte chez Locusta migratoria L.: action sur le comportement et la reproduction des individus. Ann. Sci. Naturelles. 2001. Biol. Anim. Paris 20, 37752. Loher W. (1960) The chemical acceleration of the maturation process and its hormonal control in the male of the desert locust. Proc. R. Sot. Land. Ser. B 153, 38c397. Mahamat H., Hassanali A., Odongo H., Torto B. and Bashir S. El. (1993) Studies on the maturation-accelerating pheromone of the desert locust, Schistocerca gregaria (Forskal). Chemoecology 4, 159-164. Norris M. J. (1962) Group effects on the activity and behaviour of adult males of the desert locust, (Schistocerca gregaria Forsk.) in relation to sexual maturation. Anim. Behao. 10, 275-291. Norris M. J. (1963) Laboratory experiments on gregarious behaviour in ovipositing females of the desert locust, (Schistocerca gregaria Forskal). Ent. Exp. Appl. 6, 2799303. Norris M. J. (1964) Accelerating and inhibiting effects of crowding on sexual maturation in two species of locusts. Nature 203, 784785. Norris M. J. (1970) Aggregation response in ovipositing females of the desert locust, with special reference to the chemical factor. J. Insect Physiol. 16, 149331515. Obeng-Ofori D., Torto B. and Hassanali A. (1993) Evidence for mediation of two releaser pheromones in the aggregation
et al.
behaviour of the gregarious desert locust, Schistocerca gregaria (Forskal) (Orthoptera: Acrididae). J. Chem. Ecof. 19, 1665-1676. Obeng-Ofori D., Njagi P. G. N., Torto B., Hassanali A. and Amiani H. (1994a) Sex differentiation studies relating to releaser aggregation pheromones of the desert locust, Schistocerca gregaria. Em. Exp. Appl. 73, 85-91. Obeng-Ofori D., Torto B., Njagi P. G. N., Hassanali A. and Amiani H. (1994b) Fecal volatiles as part of the aggregation pheromone complex of the desert locust, Schistocerca gregaria (Forskal) (Orthoptera: Acrididae). J. Chem. Ecol. 20, 2078-2087. Popov G. B. (1958) Ecological studies on oviposition by swarms of the desert locust, (Schistocerca gregaria Forskal) in Eastern Africa. Anti-Locust Bull. 31, l-70. Saini R. K. and Hassanali A. (1992) Olfactory sensitivity of tsetse to phenolic kairomones. Insect Sci. Applic. 13, 95-104. Stower W. J., Popov G. B. and Greathead D. J. (1958) Oviposition behaviour and egg mortality of the desert locust, (Schistocerca gregariu Forskal) on the coast of Eritrea. Anti-Locust Bull. 30, l-33. Torto B., Obeng-Ofori D., Njagi P. G. N., Hassanali A. and Amiani H. (1994) Aggregation pheromone system of adult gregarious desert locust (Schistocerca gregaria Forskal). J. Chem. Ecol. 20, 1749-1762. Uvarov B. (1977) Grasshoppers and Locusts. A Handbook of General Acridology, Vol. 2. Cambridge University Press, London. Wilcoxon F. (1945) Individual comparisons by ranking methods. Biomet. Bull. 1, 8&83.
Acknowledgements-We are grateful to the staff of the locust rearing unit especially Messrs Ndugo, Ongudha and Nganga for regular supply of insects and to J. Andoke and P. Ahuya for technical assistance. This work was supported by funds from a consortium of donors coordinated by International Fund for Agricultural Development (IFAD) through the Consultative Group on Locust Research (CGLR), United Nations Development Programme (UNDP), Swedish Agency for Research Cooperation with Developing Countries (SAREC) and Arab Fund for Economic and Social Development (AFESD) to whom we are grateful.
Copyright 0 1995 Elsevier Science Ltd
Pergamon
0022-1910(95)00016-X
Printed in Great Britain. All rights reserved 0022-1910195 $9.50 + 0.00
Semiochemicals from Froth of Egg Pods Attract Ovipositing Female Schistocerca . gregarla R. K. SAINI,*t
M. M. RAI,*
A. HASSANALI,*
J. WAWIYE,*
H. ODONGO*
Received 7 July 1994; revised 16 December 1994
A chemical signal, originating from the froth of egg pods attracts gravid female S’chistocerca gregariu (Orthoptera: Acrididae) to common egg laying sites. Behavioural experiments indicated that females preferred to oviposit in moist sand contaminated with froth (60% egg laying vs 34% in sterilized sand). Extracts and volatiles collected from froth were also attractive to gravid females. In fact, froth volatiles elicited the strongest egg laying response (80% egg laying). Results with froth extracts obtained by sequential extraction with solvents of increasing polarity suggest that both non-polar and polar compounds are involved in the attraction of gravid females. Electroantennogram recordings with extracts and volatiles collected from froth confirmed the presence of olfactory receptors on the antennae that are responsive to compounds in the extracts and the volatile collections. Schistocerca gregaria
Locust
Oviposition
Aggregation
Female Schistocerca gregaria (Forskal) have been observed to deposit their egg pods in sites where other females are ovipositing even though other areas have environmental factors that appear to be suitable (Popov, 1958; Stower et al., 1958; Uvarov, 1977). Norris (1963, 1970) found that both visual and chemical cues were important in aggregating females to common egg laying sites. She found that living decoys of either sex, immature or mature males, and last instar hoppers could be used as decoys and were functional even in total darkness. The fact that chemical stimuli may also be involved was reconfirmed when Norris (1970) showed that papers contaminated by mature adults and by hoppers were attractive to gravid females. Responses to locust extracts in several solvents were, however, quite variable and active extracts were more readily prepared from mature males than from newly ecdysed adults. Norris (1970) concluded that the active substances, believed to be a pheromone, were present on the locust’s entire body. She also showed that antennatectomized females were able to find live decoys but that they were much less responsive to dead ones. However, when decoys were enclosed and could no longer be touched, the response was greatly diminished. Accordingly, she suggested that (ICIPE),
Egg laying
Electroantennograms
chemotactile receptors on other parts of the body in addition to the antennae and possibly the palpi also promoted oviposition. Norris found no attraction to sand contaminated with exposed froth plugs and faeces such as might be present at previously used oviposition sites. Norris (1970) also reported that exuviae from Locusta migratoria (Reiche & Fairmaire) were attractive to ovipositing females of S. gregaria but to a lesser extent than those from conspecific locusts. This suggested that the oviposition aggregation pheromone was at least partially species specific. Lauga and Hatte (1977, 1978) showed that female L. migratoria were strongly attracted over a distance of 0.5 m to sand into which gregarious females had laid egg pods. No work relating to the identification of these attractants has been reported in the literature. The present studies were designed to determine if a chemical signal from egg pods mediated oviposition, and if so, to establish the nature and to trace the source of the signal.
INTRODUCTION
*International Centre of Insect Physiology and Ecology Box 30772, Nairobi, Kenya. tTo whom all correspondence should be addressed.
Pheromone
MATERIALS AND METHODS Insects
Crowded desert locust, S. gregaria were obtained from the International Centre of Insect Physiology and Ecology (ICIPE) locust colony which originated from the locust culture of the Desert Locust Control Organization for Eastern Africa (DLCO-EA) in Addis Ababa,
P.O.
711
R. K. SAINI
712
Ethiopia. Insects were reared under crowded conditions using aluminium cages (50 x 50 x 50 cm). The room temperature was maintained between 30-35°C with a 12: 12 h 1ight:dark cycle. In all the experiments 13-15 day-old locusts were used.
et al.
of the solvents. The extracts were stored at -20°C until use. Amounts of extracts corresponding to 1 or 2 pod equivalents were tested individually or in combination in two- or four-choice bioassays. Collection
Experimental
In an initial experiment, two-choice behavioural bioassays were undertaken in standard aluminium locust rearing cages (50 x 50 x 50 cm). All sides of the cage were made from wire mesh except for the front which had a sliding glass door. The ceiling had an electric bulb at the rear end. Locusts were provided with four aluminium oviposition tubes (each 10.0 cm 1 x 4.0 cm dia.) at the front of the false floor and close to the sliding door. Two of these tubes were filled with sterilized sand (control) with moisture content of 15% (15 ml water to 100 g sand) while the other two were filled with moist sand containing two egg pods. The two sets of tubes were placed 18 cm apart at an intertubal distance of 3.5 cm. The initial standard rearing cages however, proved to be inappropriate for bioassays as it was observed that the egg laying tubes were too narrow and if one was occupied by an egg laying adult, any other gravid female was forced to oviposit in a control tube. In addition, the tubes were placed in a row and their placement could not be altered. Heating was also found to be asymmetrical. new larger of the above reasons, Because (60 x 60 x 60 cm) four-choice behavioural cages made of aluminium were constructed. The sides and roof of these cages were made of wire mesh; the back was of metal and the front had a removable glass door. The ceiling had a 60 W electric bulb in the middle. The false floor in the middle had four egg laying cups (8.5 cm dia., 9 cm depth) placed at distances of 13 cm from each other and 22 cm diagonally. For two-choice experiments, two of the diagonally placed cups were blocked by turning them upside down. Cleaning and conditioning
of sand
Sand for oviposition cups was sieved using wire mesh (2 mm’) and washed successively with hexane, ethyl acetate, methanol and finally with distilled water. It was then dried and heat-sterilized by baking in an oven at 150°C for 24 h. Sterilized sand was moistened by adding 15 ml water per 100 g of sand. Extraction
of volatiles
cages
of eggs and froth
Froth and egg extracts were obtained either by sequential or by single solvent extractions. For sequential extraction, eggs and froth derived from pods were allowed to dry at ambient temperature (25-27°C) for 6 h. Each part was then placed in a dropping funnel (50 ml) and 6 ml hexane added. After 10 min, the hexane extract was removed and the extraction repeated with ethyl acetate (6 ml) followed by methanol (6 ml). Single-solvent extractions of froth were carried out in the same way with hexane, ethyl acetate and methanol, each time using 25 ml
Volatiles were collected on traps using activated charcoal (S&l00 mesh, Chromopack). Before use, the charcoal was cleaned by Soxlet extraction with dichloromethane (Merck) for 72 h, followed by thermal treatment at 250°C under nitrogen (20 ml/min). Volatiles were collected from 1-day-old froth plugs placed in a short glass tube connected to charcoal traps prepared by packing charcoal (ca I .2 g) between two glass wool plugs in 6 cm long x 8 mm id. glass tubes. Nitrogen, cleaned by passing it through a short plug of activated charcoal, carried volatiles from froth to the charcoal trap at the rate of 3.6 ml/min at room temperature. Trapping was carried out for 12 h and the adsorbed material eluted with 8 ml HPLC grade dichloromethane (Aldrich Ltd). Behavioural
bioassays
Egg laying responses of ovipositing S. gregaria females were determined in three types of experiments: (1) Gravid females were offered a choice between sterilized sand and sand containing two egg pods in the standard rearing cages (Expt. 1). Two pairs of insects were used for each of the 30 replicates. Experiment 2 was a repeat of Expt. 1 in the new large behaviour chambers using 6 pairs of locusts for each of the 17 replicates. In all subsequent bioassays, only the larger behaviour chambers were used. (2) Gravid females were given a choice between sterilized sand (control), sand mixed with only eggs from two egg pods, sand mixed with froth from two egg pods or sand mixed with two complete egg pods (Expt. 3). In Expt. 4 the treatments were similar except that the sand with two egg pods was substituted with sand from which egg pods had been removed. (3) Gravid females were offered a choice between various sands contaminated with either sequentially extracted or single solvent extracts of froth or eggs (Expts 5-7) mixtures of froth extracts (Expt. 8) or froth volatiles (Expt. 9). In these experiments, the extracts or volatiles from froth (two froth equivalents) were delivered on strips of filter paper (10 x 2.5 cm). These impregnated filter paper strips were then placed into the egg laying containers about 1 cm below the surface of the moistened, sterilized sand. Filter paper strips treated with similar amounts of respective solvents were used as control stimuli. In all the experiments, except where indicated, each replicate consisted of eight pairs of insects tested for 4 days. The egg pods laid were counted daily by removing the egg laying cups. These were then replaced by freshly prepared egg laying cups with their respective treatments. In all four-choice experiments (in order to avoid any
EGG
POD ATTRACTION
713
TABLE 1. Responses of ovipositing Schistocerca greguriu females to sand mixed with egg pods in standard locust rearing (A) and in new enlarged oviposition cages (B) Egg pods deposited
Treatments
Expt. No.
No. of replicates
(A) Standard
(B)
cages Sterilized sand (SS) SS + 2 egg pods
30
New cages
17
Sterilized sand SS + 2 egg pods
No.
%
23 32
4l.P 58.2
35 68
34.0b 66.0
Means within an experiment followed by the same letter are not significantly different (P i 0.05). N = 2 pairs of insects per test in (A) and N = 6 in (B).
position effect) the position of the egg laying cups was rotated in 4 x 4 Latin square design. In two choice experiments, the cups were rotated diagonally. All the bioassays were conducted in the laboratory at 30 f 2”C, relative humidity of 4@45% and 12:12, L:D photoperiod. Test insects were fed daily with seedlings and wheat bran (Triticum sp.). The results were analysed using Wilcoxon’s T test (Wilcoxon, 1945) and Friedman’s test (Friedman, 1940). Electroantennograms
60 s before and after each stimulation with the test compounds. The amplitude of the response to the test compound was expressed as a percentage of the mean response to solvent control stimuli presented 60 s before and after stimulation with the test compound. EAGs were recorded from at least 10 gravid female antennae for each stimulus.
(EAGs)
RESULTS
EAGs were recorded utilizing glass pipettes filled with locust saline (Hoyle, 1951) into which Ag-AgCI wires were inserted. The indifferent electrode was inserted into the pedicel of an amputated antenna while the recording electrode was placed over a few segments of the flagellum. The signal was amplified by a Grass P16 amplifier (Universal a.c./d.c. amplifier, Grass Instruments Co., MA, U.S.A.), displayed on an oscilloscope and recorded using a chart recorder. The odour delivery system and stimulation technique was as described by Saini and Hassanali (1992). EAGs were recorded to hexane, ethyl acetate and methanol extracts of froth and from volatiles (in dichloromethane) collected from froth at the following dosages: 0.1, 0.2, 0.3, 0.4 and 0.5 froth equivalents (one froth equivalent is the volatiles contained in the froth from one egg pod). To correct for the gradual diminishing of the EAG response during an experiment and to allow comparison among the different preparations, an equivalent amount of the respective solvent was applied
Initial two-choice experiments (Expt. 1) undertaken in standard rearing cages in which gravid females were given a choice between sterilized sand or sand in which previously laid egg pods were mixed, indicated a small preference for the latter (Table 1). However, when these experiments were repeated in larger, new oviposition cages (Expt. 2), females laid twice as many eggs in sand containing egg pods than in sterilized sand (control) (Table 1). Results of Expts 3 and 4 in which females were given four choices indicated that ovipositing females preferred to oviposit in sand contaminated with either entire egg pods or froth alone as compared to sand mixed with eggs alone or the sterilized sand control (Table 2). Even sand in which egg pods had been laid previously but subsequently removed was shown to be attractive to gravid females (Table 2, Expt. 4). These results clearly indicated that chemicals emanating from the froth of egg pods caused ovipositing females to aggregate at a
TABLE 2. Responses of ovipositing Schistocerca gregariu females to sand mixed with eggs, or froth
or Sand contaminated
with egg pods (in each case the source
was from 2 egg pods) Egg pods deposited
Expt. No.
Treatments Sterilized sand (SS) SS + eggs SS + froth SS + egg pods Sterilized sand (SS) SS + eggs SS + froth Contaminated sand (CS)*
No. of replicates
No.
%
8
12 10 21 19 39 37 68 98
19.4b 16.1b 33.9” 30.6” 16.1b 15.36 28.1” 40.5”
25
Means within an experiment followed by the same letter are not significantly *Sand from which egg pods had been removed.
different
(P < 0.05).
714
R. K. SAINI
et al.
TABLE 3. Responses of ovipositing Schistocerca gregaria females to extracts of: eggs or froth, extracted with hexane (Hex), ethyl acetate (EtAc) and methanol (MeOH) respectively and froth extracted seperately in the above solvents Egg pods deposited Expt. No.
Treatments
No. of replicates
5 Eggs (sequential
6 Froth
7 Froth
extraction) Sterilized sand (SS) SS + Hex extract SS + EtAc extract SS + MeOH extract (sequential extraction) Sterilized sand (SS) SS + Hex extract SS + EtAc extract SS + MeOH extract (separate extraction) Sterilized sand (SS) SS + Hex extract SS + EtAc extract SS + MeOH extract
Means within
an experiment
followed
%
15 16 13 12
26.8= 28.6” 23.P 21.4”
29 76 33 56
14.9b 39.2 17.0b 28.9”
10 25 21 15
14.1’ 35.2” 29.6ab 2l.lk
25
10
by the same letter are not significantly
common egg laying site. Neither eggs alone (Table 2) nor their extracts (Table 3) were attractive. Froth extracts which were obtained either by sequential extractions or separately by single solvent extraction elicited significant attraction of gravid females (Table 3). When froth was extracted sequentially the hexane and methanol extracts evoked significantly more egg laying than the ethyl acetate extract and the control (Table 3). However, when separate solvent extracts of froth were tested, sand impregnated with the hexane extract was found to be the most preferred for egg laying, followed by the ethyl acetate extract. The methanol extract was intermediate in activity between the control and the ethyl acetate extract (Table 3). When ovipositing females were given a choice between different combinations of froth extracts (sequentially extracted with hexane, ethyl acetate and methanol) in 1: 1 or 1: 1: 1 ratio, sand containing both hexane and methanol extracts elicited the highest egg laying response (Table 4). Volatiles collected from froth were also very attractive and evoked significantly more egg laying as compared to sterilized sand (Table 5). The magnitude of electroantennograms (EAGs) recorded in response to the hexane, methanol and ethyl acetate extracts of froth and also from froth volatiles increased with increasing doses (Fig. 1). The captured
No.
8
different
(P -C 0.05).
volatiles however, evoked the highest EAG responses. These electrophysiological investigations confirm the presence of antenna1 olfactory receptors for the attractive compounds in the froth.
DISCUSSION
Gregarious locusts must mate and oviposit in ways that would ensure temporal and spatial cohesiveness of succeeding hopper generations. In this way, the gregarious integrity of the locust population is ensured. In a number of locust species, including the desert locust, this has been achieved through synchronization of maturation of the adults and group oviposition at common egg-laying fields (Popov, 1958; Stower et al., 1958; Loher, 1960; Norris, 1962, 1963, 1964; Amerasinghe, 1978). Mediation by pheromones has been implicated in both these processes (Loher, 1960; Norris, 1963, 1964, 1970; Amerasinghe, 1978), and part of the pheromone system modulating synchrony in S. gregaria has been characterized (Mahamat et al., 1993). In the desert locust, females tend to lay their eggs in areas where other females have oviposited, even though environmentally suitable alternatives may be available (Popov, 1958; Stower et al., 1958; Uvarov, 1977). Our results clearly indicate that froth from egg pods of
TABLE 4. Responses of ovipositing Schistocercagregaria females to binary and ternary mixtures of froth extracts (extracted sequentially with hexane (Hex), ethyl acetate (EtAc) and methanol (MeOH) respectively) Egg pods deposited Expt. No.
8
SS = sterilized
No. of replicates
Treatments SS SS SS SS
+ + + +
MeOH MeOH MeOH EtAc +
sand. Means
+ Hex + EtAc + EtAc + Hex Hex
followed
12
by the same letter are not significantly
No.
%
21 14 28 17
26.3”b 17.5’ 35.W 21.2k
different
(P < 0.05).
EGG TABLE
5.
POD ATTRACTION
715
Responses of ovipositing Schistocerca gregariu females to dichloromethane) collected from froth plugs (G-1 day old)
volatiles
(in
Egg pods deposited Expt. No.
No. of replicates
Treatments
9
Sterilized sand (SS) SS + volatiles
Means
followed
by the same letter are not significantly
S. gregariu is a source of chemical signals that attract conspecific ovipositing females and suggest that these signals may play an important role in group oviposition once a suitable site has been selected. Because of its foamy constitution, it is very likely that the egg froth constitutes an effective controlled-release source of the attractive chemicals, ensuring their attraction over a duration advantageous to the insect. Our results differ from those of Norris (1970) who, surprisingly, failed to observe any attraction to contaminated sand with exposed froth plugs and faeces and concluded that the oviposition stimulus for S. gregaria was nonvolatile and perceived by chemotactile means. On the other hand, Lauga and Hatte (1977) found that sand used for previous egg-laying by females of L. migratoriu was attractive to conspecific gravid females. These authors postulated that egg-laying females produced a pheromone that attracts gravid females of both phases. The pheromone was shown to be volatile, inducing attraction over a distance of 0.5 m to sand in which eggs have been laid. Our behavioural and electrophysiological results with S. gregaria are in agreement with those obtained for L. migratoria. However, females could still utilize a chemotactile stimulus in their oviposition behaviour. In fact, ovipositing females were often seen touching contaminated sand with their antennae and palpi, suggesting the perception of short-range or contact signals. Moreover, our results with froth extracts obtained by sequential extraction with solvents of increasing polarity (hexane, ethyl acetate and methanol) suggest that both non-polar
600 r
n Methanol extract * Hexane extract
+ Ethyl acetate 0 Volatiles
extract
I’;+ 0.2
0.3 Egg
0.4
pod equivalents
FIGURE 1.Electroantennogram (EAG) responses of gravid S. gregaria females to froth extracts and volatiles. Responses are expressed as a percentage of the reference signal (respective solvent), N = 10 antennae (egg pod equivalent refers to volatiles contained in the froth of one egg pod).
22
different
No.
%
33 142
18.9b 81.1”
(P < 0.001)
and polar compounds mediate the oviposition process. It is conceivable that one or more polar compounds extracted in methanol may operate in whole or in part through contact. Norris (1963, 1970) observed that ovipositing female S. gregaria are attracted by other individuals at different developmental stages, alive or dead, as well as to locust extracts and shed skin. While we have not specifically repeated Norris’ experiments, her observations can be explained by recent results on the identity of the components of the aggregation pheromone complex of the desert locust (Obeng-Ofori et al., 1993, 1994a, b; Torto et al., 1994). These studies have demonstrated the existence of three sets of aggregationinducing volatiles in S. gregaria: a blend emitted by live nymphs of both sexes that is specific to nymphs (Obeng-Ofori et al., 1993, 1994a); a blend emitted by older males that elicits aggregation in all stages and sexes of the adults (Obeng-Ofori et al., 1993, 1994a; Torto et al., 1994); and a blend derived from nymphal faeces and those of young adults that elicits aggregation in all stages and sexes of the insect (Obeng-Ofori et al., 1994b). Norris’ experiments did not effectively delineate behavioural elements involved in oviposition from those involved in aggregation, and it is likely that in some of her experiments she observed responses of the females to the aggregation pheromone or residual components of the pheromone rather than to oviposition attractants. Significantly, Norris (1970) found extracts derived from older males more potent in eliciting attraction from gravid females than those from younger adults, which is consistent with the recent finding that the adult aggregation pheromone is produced by older males (Tort0 et al., 1994). It is possible that elements of oviposition behaviour in S. gregaria are modulated by both the froth-derived oviposition pheromone system and the adult aggregation pheromone. Complete characterization of the former would allow the relative role of the two systems in the oviposition process of S. gregaria to be clearly elucidated. Compounds involved in aggregating ovipositing females may be useful in dispersing the oviposition sites to the detriment of the gregarious phase populations or could be used in concentrating locusts in areas where pathogens or other biocontrol agents could cause high mortality. The pheromone might also be used in traps for monitoring populations.
R. K. SAINI
716 REFERENCES
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Acknowledgements-We are grateful to the staff of the locust rearing unit especially Messrs Ndugo, Ongudha and Nganga for regular supply of insects and to J. Andoke and P. Ahuya for technical assistance. This work was supported by funds from a consortium of donors coordinated by International Fund for Agricultural Development (IFAD) through the Consultative Group on Locust Research (CGLR), United Nations Development Programme (UNDP), Swedish Agency for Research Cooperation with Developing Countries (SAREC) and Arab Fund for Economic and Social Development (AFESD) to whom we are grateful.