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Courtship inhibition pheromone in desert locusts, Schistocerca gregaria
Journal of Insect Physiology 48 (2002) 991–996 www.elsevier.com/locate/jinsphys
Courtship inhibition pheromone in desert locusts, Schistocerca gregaria Karsten Seidelmann ∗, Hans-Jo¨rg Ferenz Martin-Luther University, Institute of Zoology, Animal Physiology, 06099 Halle, Germany Received 18 April 2002; received in revised form 30 July 2002; accepted 30 July 2002
Abstract Male desert locusts in the gregarious phase release phenylacetonitrile (PAN) on becoming sexually mature. It has been assumed that this chemical is responsible for aggregation of adult desert locusts. However, PAN has repellent characteristics and is involved in sexual behavior. Mature males release PAN as a volatile to serve as a kind of olfactory concealment during mating and to prevent competing males from homosexual encounters. We conclude that PAN is a courtship-inhibiting pheromone exclusively used under crowded conditions in dense populations when high sperm competition occurs among desert locust males. By chemically enhancing their mate guarding, gregarious males improve the protection of their mate from rivals and ensure their reproductive success. 2002 Published by Elsevier Science Ltd. Keywords: Behavior; Chemical communication; Locust; Reproduction; Phenylacetonitrile
1. Introduction Under exceptionally favorable conditions when desert locusts, Schistocerca gregaria, breed successfully, they aggregate into huge swarms which, in biblical proportions, invade and devastate agricultural areas. It is believed that semiochemicals play an important role in the establishment of such plagues (see, e.g., Ferenz, 1990; Pener, 1991; Pener and Yerushalmi, 1998). A variety of aromatic compounds released by desert locusts has been identified and is proposed to be the aggregation pheromone system of adults (Torto et al., 1994). Phenylacetonitrile (PAN) is a predominant component of this mixture. However, we have recently shown that PAN appears rather to be a repellent (Seidelmann et al., 2000; Seidelmann et al., 2002). PAN is a volatile phenol derivative released exclusively by gregarious mature adult male desert locusts. Immature males, females, hoppers, and solitary mature adults do not produce this substance (Luber et al., 1993; ∗ Corresponding author. Tel.: +49-345-55-26476; fax: +49-34555-27152. E-mail address: [email protected] (K. Seidelmann).
0022-1910/02/$ - see front matter 2002 Published by Elsevier Science Ltd. PII: S 0 0 2 2 - 1 9 1 0 ( 0 2 ) 0 0 1 7 8 - 6
Obeng-Ofori et al., 1994). The site and control of its production are unknown. The restriction of PAN to the reproductive phase of gregarious male locusts led us to the assumption that it might play a role in sexual behavior and mate recognition in locust swarms. The mating behavior of gregarious desert locusts has been described in some detail (Popov, 1958; Loher, 1959; Uvarov, 1977). It includes a slow approach of the male to the female, interrupted by stops in a semi-stilted posture. But on the whole no complicated sequence of courtship behavior can be observed. Finally the interested male jumps on the back of the female from a short distance, though he may be rejected by the female. Females are receptive shortly before oviposition. Pairing, if successful, is quickly followed by copulation. After the sperm transfer the male remains on the female and only leaves at the termination of egg laying. If the pair is separated prior to egg laying, the female will mate immediately with other males. However, although in locusts swarms many mature males are around, the guarding male is usually not attacked by rivals (Popov, 1958). As mature males produce PAN only in the presence of other mature males (Seidelmann et al., 2000), we speculated that the repellent PAN might act like a courtship-inhibiting pheromone (Seidelmann et al.,
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K. Seidelmann, H.-J. Ferenz / Journal of Insect Physiology 48 (2002) 991–996
2002). Thus we investigated courtship and mating in the presence and absence of PAN.
2. Materials and methods 2.1. Insects Our desert locust culture, Schistocerca gregaria (Orthoptera, Acrididae), originated from specimens collected in 1991 in Niger. The insects were reared in the laboratory under crowded (‘gregarious’) conditions as described previously (Seidelmann et al., 2000). 2.2. Behavioural assay Experiments were done with truly gregarious locusts taken directly from the culture. We used only locusts which were engaged in pairing to ensure the right stage of development and intensive sexual behavior. Each couple was placed individually into numbered small cages and the males were marked by a small dot of white water-soluble non-toxic paint on the pronotum. For experiments with an additional competing male the female of each separated couple was placed in an open field (660 cm2) together with the new test male. After pair formation the original male was introduced to monitor mating attempts. Arranging the pairs usually lasted about 30 min; the assay was terminated after 120 min. At least five independent attacks or pairing attempts had to be observed. Manipulations of the pheromone bouquet of the test males was done by keeping mature males isolated in small cages (10 x 10 x 20 cm) for 7 days. Gregarious mature male desert locusts cease PAN emission within a few days after isolation. After 1 week of isolation, male PAN release cannot be detected any more (Seidelmann et al., 2000). PAN (Sigma, Germany) was dissolved in dichloromethane (Merck, Germany). 1.0 µl of the 10% solution (v/v) was applied topically on the prothorax of the test male with a microliter syringe. In control experiments we applied dichloromethane only. For experiments without an additional competing male, separated pairs were introduced into small observation cages (10 x 10 x 20 cm). Re-pairing was monitored at 30 min intervals for 2 hrs. In these mate-choice experiments, we marked one of the females by clipping off the very tip of their forewings. This marking technique is commonly used and does not influence male mate choice.
described above and kept in a large cage. After 1, 30, 60, or 120 min each locust was put into a 40 ml airtight glass vial for 180 min at 30 °C. After 150 min a SPME fiber (PDMS 100 µm, SUPELCO) was introduced and evaporated PAN absorbed for 30 min. Then the PANloaded fiber was placed into the split/splitless injector (splittless mode, 220 °C) of a Hewlett Packard 5890A gas chromatograph equipped with a 30 m x 0.32 mm ID polar capillary column (EC-wax, 0.25 µm, Alltech). Helium was used as carrier gas, set at 1 ml min–1. A gradient of 10 °C min–1 from 40–240 °C was used. The PAN FID peak was identified by comparison with authentic reference material. The amount of PAN was calculated from a standard curve obtained by applying various amounts of PAN on a metal mesh under the same conditions. From the measured PAN doses a curve for PAN evaporation over time can be calculated. The evaporation curve was modeled as an exponential function (Fig. 1). The measured PAN amounts represent the area under this curve. After antidifferentiation the unknown parameters of the function were calculated from the data by the non-linear regression module of STATISTICA (StatSoft Inc. 2000). It can be estimated that during the bioassay time (30 to 120 min) the amount of PAN released from the treated locusts is in the range from 104 to 42 ng hr–1. These doses are in agreement with the PAN amounts normally released by mature gregarious males (Torto et al., 1994; Seidelmann et al., 2000).
3. Results A first experiment was designed to confirm that mating males are able to guard a gravid female. We took mating pairs and forcibly separated them. The female was placed in the small observation arena and offered a
2.3. Determination of PAN concentration The rate of PAN evaporation from the locust body surface was determined in the following way: Test locusts were treated with 1 µl of the PAN solution as
Fig. 1. Standard curve for the time-dependent release of topically applied PAN. Values are the means ± SD of 4–5 replicates for each dose.
K. Seidelmann, H.-J. Ferenz / Journal of Insect Physiology 48 (2002) 991–996
gregarious mature male (but not her original mate). As soon as pairing had taken place, the original male mate was added. In all experiments this first mate approached the couple but did not attack (Fig. 2 A). The male showed some interest in the pair but did not jump onto the couple or fight for possession of the female. No takeover attempts could be observed (Table 1). Obviously the copulating male was able to repel the competing male. Since PAN is a repellent (Seidelmann et al., 2002), the volatiles including PAN naturally emitted by a mature gregarious male might be responsible for this effect. We confirmed our interpretation by an experiment
Fig. 2. Mating experiments to demonstrate the role of PAN in mate guarding of S. gregaria. A, control experiment with 2 gregarious males; B, test of the guarding ability of an isolated male (PAN production ceased); C, as D but isolated male treated with PAN; D, mating after treatment of the female with PAN; E, mate choice test with one PAN-treated and one control female (females, gray; males, white; male from original couple with gray thorax; female from original couple with white thorax). For further explanations see text.
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Table 1 Effect of PAN on pairing attempts of male desert locusts
Experiment
A B C D E
Treatment
gregarious male isolated male isolated male + PAN female + PAN female + PAN (choice)
n
11 12 12 20 12
% attacks or pairing attempts Treated
Control
0 100 0 0 0
– – 100 80 92
with mature males which were kept isolated for 7 days and ceased PAN emission. Gregarious pairs were separated as in the first experiment and each female locust was placed in the arena (Fig. 2 B). An isolated mature male was added and, after successful pairing, the original gregarious male mate was introduced into the arena. In all experiments the added male quickly jumped onto the couple and made attempts to copulate as though not recognizing the presence of the copulating male competitor (Table 1). To demonstrate that PAN indeed keeps the rival male off the couple, we repeated this experiment but treated the isolated males prior to pairing with 1 µl of the PAN solution on the pronotum (Fig. 2 C). Under the experimental conditions the evaporation rate of the applied PAN is in the range of the natural emission of gregarious males. Now the added gregarious male tolerated the copulating couple and did not attack it or try to jump on top of it (Table 1). In the control experiment with application of dichloromethane only all males attacked the couple as in untreated isolated males (Fig. 2 B). These results demonstrate that indeed the PAN odor is necessary to repel rivals. In a further experiment we tested whether PAN can be effective if not presented in combination with other male characteristics or male defensive behaviors. Thus, again we separated copulating pairs. Both partners were placed in a small observation cage and re-pairing was monitored at 30 min intervals for 2 hrs (Fig. 2 D). In the control group we observed re-pairing in 80% of the tests within 30 min, and pairing then lasted for the 2 hr observation period. In the experimental group, 1 µl PAN solution was placed on the prothorax of each female prior to the re-pairing experiment. Following this treatment no pairing could be observed within the 2 hr observation period (Table 1, Fig. 2 D). Obviously, the PAN odor overrides all other attracting signals specific to mature females and prevents males from courting and pairing with the PAN-treated females. PAN alone and not male characteristics or male behavior repels rivals. A last experiment was designed to assure that PAN does not change the motivation of the test male to mate. Thus, we provided a male with two females, one of
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which was treated with PAN and the other served as control (Fig. 2 E). For this experiment copulating pairs were treated as described in experiment D. Then an extra female was added to each pair in the observation arena. This female was taken from another separated couple. All males avoided the PAN-treated female: within 30 min, 9 males (75%) were engaged in pairing with the control female, and within 60 min a total of 11 males (92%). One male made no mating attempts at all (Table 1). This choice experiment confirms the repellent properties of PAN overriding the attractiveness of a mature female.
4. Discussion The presence of the main component of the mature desert locust male pheromone bouquet, PAN, is strongly associated with both sexual maturity and gregarious phase (Luber et al., 1993; Seidelmann et al., 2000; Torto et al., 1994). These findings suggested that PAN has an important function in desert locust reproductive behavior. Our results of mating and take-over experiments provide evidence that PAN emitted by mating males prevents approach and copulatory behavior of rivals. Male desert locusts use PAN for a chemically enhanced mate guarding; it serves an important function to avoid sperm competition. We observed a significant last-male precedence in desert locusts when two males mate successively with a female before oviposition (unpublished results). This finding is in agreement with the earlier results of HunterJones (1960). If a female mates with two males prior to oviposition, the last male will father the majority of the offspring. Therefore, to ensure his reproductive success, a male desert locust must prevent his mate from accepting another male until she has laid eggs. We did not find a spermatophore or a mating plug deposited by the male that might physically block the female reproductive tract. Thus, desert locust males have to guard their inseminated mate to keep off rivals and to avoid sperm competition. However, postcopulatory mate guarding may be costly in terms of lost opportunities to mate with other females. In S. gregaria, pairing and copulation occur when the female carries well-developed eggs and is close to oviposition. Only at this stage does a female accept a courting male. It appears to signal readiness to accept a male by a certain behavior (Popov, 1958) or, perhaps, by a short-range sex pheromone (Torto et al., 1995). Therefore, the operational sex ratio is strongly male biased and the chance of finding an unguarded receptive female is rather low, while the probability of the current female mate being captured and copulated by a male rival is high. Costs of precopulatory mate guarding in terms of lost mating opportunities should be negligible in the light
of the fitness benefits due to ensured fatherhood of the actual mate’s offspring. Thus, mating desert locust males remain in physical contact with the female after the separation of the genitalia until oviposition has taken place (Popov, 1958; Stower, 1958). Under conditions of high local competition for mates, as in S. gregaria swarms, the most important precopulatory trait to avoid take-overs and sperm competition is mate concealment (Simmons and Siva-Jothy, 1998). When the female releases a mate-attracting pheromone, males should respond by releasing quickly a pheromone to mask or diminish the female’s attractant (Happ, 1969; Happ and Wheeler, 1969; Happ et al., 1970). PAN is such a concealing or courtship-inhibiting pheromone, a kind of olfactory camouflage that appears to hide the receptivity-signaling female and thus protects the couple from competing males. It acts as an antiaphrodisiac to conspecific males and prevents them from attempts to court and to mount a guarded female. Male-to-male repellents reducing the attractiveness of a receptive female are also known from some moth species (e.g. Jacobs and Adler, 1984; Hirai et al., 1978; Pliske, 1975) and beetles (e.g. Happ, 1969; Happ et al., 1970; Nijholt, 1973). PAN can also be called an abstinon (Schlein et al., 1981), because it marks mature gregarious males and prevents wasteful homosexual activity. In this respect PAN is a synergistic signal to the yellow coloration of gregarious males. However, PAN does not fully meet the common aspects of both a courtship-inhibition pheromone and an abstinon: a courtship-inhibition pheromone is usually placed on a female after copulation; abstinons are normally not used for mate guarding. PAN appears to be a new type of a male-specific courtshipinhibiting pheromone which combines both functions resulting in an efficient mechanism for mate guarding ensuring reproductive success. Desert locusts may occur in both extremes of the abundance scale: In the solitary phase individuals are present in very low population densities in their recession areas. However, during gradations and swarm formation, very high population densities can be reached. For adult male locusts these extreme situations require different tactics and adaptations in mating strategies and sperm competition. While under low population densities the ability to locate or attract a mate will be the key for reproductive success of a male locust, the avoidance of sperm competition becomes the main problem under crowded conditions. To ensure mating success, males must be able to guard a gravid female and to protect her from mating with other males until she has used his sperm to fertilize the egg batch. The production of a courtship-inhibiting pheromone only in the presence of male competitors (Seidelmann et al., 2000) appears to be a specific and useful adaptation to crowded conditions in a phase-polymorphic species. In another locust species, the African migratory locust
K. Seidelmann, H.-J. Ferenz / Journal of Insect Physiology 48 (2002) 991–996
Locusta migratoria, with a very similar phase polymorphism, a precopulatory mate guarding has been described (Parker and Smith, 1975). Males of L. migratoria deposit a spermatophore blocking the female’s spermathecal duct until oviposition. In this species there is no need to guard the female after successful copulation until oviposition in order to avoid sperm competition. However, males guard a mature female in order to be the first male mating after the spermatophore has been expelled during oviposition. The operational sex ratio is rather balanced as females accept guarding males, and all mature females are worth being guarded. Take-overs are therefore not so costly for the loser. Hoppers of L. migratoria release PAN (Niassy et al., 1999), but its function needs further clarification. Males of this species do not use this pheromone (personal observations). To date, courtship-inhibiting pheromones have not been detected for L. migratoria although in this locust phase polymorphism and aggregation pheromones have been studied intensively. Recently, the existence of an aggregation pheromone system in desert locusts has been questioned. First, because the attractance of PAN previously established by olfactometer experiments (Torto et al., 1994) could not be confirmed (Seidelmann et al., 2000; Seidelmann et al., 2002) and, secondly, because aggregation can be fully induced by various non-pheromonal factors (Ha¨ gele and Simpson, 2000; Simpson et al., 2001). Furthermore, the major component guaiacol has been shown to be unspecifically derived from gut bacteria which metabolize digestive waste material (Dillon et al., 2000). Our results show that PAN, the key component of the suggested desert locust aggregation pheromone bouquet, has a completely different and previously unsuspected function. To our knowledge it is the first identified courtship-inhibiting pheromone in grasshoppers.
Acknowledgements We thank S. Hertel for excellent technical assistance. Support from the Deutsche Forschungsgemeinschaft (Se 1043) is gratefully acknowledged.
References Dillon, R.J., Vennard, C.T., Charnley, A.K., 2000. Exploitation of gut bacteria in the locust. Nature 403, 851. Ferenz, H.J., 1990. Locust pheromones — Basic and applied aspects. Boletin de Sanidad Vegetal, Fuere de Serie 20, 29–37 Also as Proceeding of the 5th International Meeting of the Orthopterists’ Society 1989. Ha¨ gele, B.F., Simpson, S.J., 2000. The influence of mechanical, visual and contact chemical stimulation on the behavioural phase state of solitarious desert locusts (Schistocerca gregaria). Journal of Insect Physiology 46, 1295–1301.
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Happ, G.H., 1969. Multiple sex pheromones of the mealworm beetle Tenebrio molitor. Nature 222, 180–181. Happ, G.M., Wheeler, J., 1969. Bioassay, preliminary purification and effect of age, crowding and mating on the release of sex pheromone by female Tenebrio molitor. Annals of the Entomological Society of America 62, 846–851. Happ, G.M., Schroeder, M.E., Wang, J.C.H., 1970. Effects of male and female scent on reproductive maturation in young female Tenebrio molitor. Journal of Insect Physiology 16, 1543–1548. Hirai, K., Shorey, H.H., Gaston, L.K., 1978. Competition among courting male moths: male-to-male inhibitory pheromone. Science 202, 644–645. Hunter-Jones, P., 1960. Fertilization of eggs of the desert locust by spermatozoa from successive copulations. Nature 185, 336. Jacobs, M., Adler, V.E., 1984. A male tobacco budworm pheromone inhibitory to courtship. Journal of Environmental Sciences and Health A 19 (4), 469–476. Loher, W., 1959. Contributions to the study of the sexual behaviour of Schistocerca gregaria (Forskal). Proceedings of the Royal Entomological Society London (A) 34, 49–56. Luber, K., Wieting, J., Zeeck, E., Ferenz, H.J., 1993. Isolation and characterization of a volatile aromatic infochemical released by sexual maturing gregarious male desert locusts, Schistocerca gregaria. Verhandlungen der Deutschen Zoologischen Gesellschaft 86, 261. Niassy, A., Torto, B., Njagi, P.G.N., Hassanali, A., Obeng-Ofori, D., Ayertey, J.N., 1999. Intra- and interspecific aggregation responses of Locusta migratoria migratoriodes and Schistocerca gregaria and a comparison of their pheromone emissions. Journal of Chemical Ecology 25, 1029–1042. Nijholt, W.W., 1973. The effect of male Trypodendron lineatum (Coleoptera: Scolytidae) on the response of field populations to secondary attraction. Canadian Entomologist 105, 583–590. Obeng-Ofori, D., Torto, B., Njagi, P.G.N., Hassanali, A., Amiani, H., 1994. Fecal volatiles as part of the aggregation pheromone complex of the desert locust, Schistocerca gregaria (Forskal) (Orthoptera: Acrididae). Journal of Chemical Ecology 20, 2077–2087. Parker, G.A., Smith, J.L., 1975. Sperm competition and the evolution of the precopulatory passive phase behaviour in Locusta migratoria migratorioides. Journal of Entomology 49, 155–171. Pener, M.P., 1991. Locust phase polymorphism and its endocrine relations. Advances of Insect Physiology 23, 1–79. Pener, M.P., Yerushalmi, Y., 1998. The physiology of locust phase polymorphism: an update. Journal of Insect Physiology 44, 365– 377. Pliske, T.E., 1975. Courtship behavior and use of chemical communication by males of certain species of ithomiine butterflies (Nymphalidae, Lepidoptera). Annals of the Entomological Society of America 68, 935–942. Popov, G.B., 1958. Ecological studies on oviposition by swarms of the desert locust in Eastern Africa. Anti-Locust Bulletin 31, 70. Schlein, Y., Galun, R., Ben-Eliahu, M.N., 1981. Abstinons. Male-produced deterrents of mating in flies. Journal of Chemical Ecology 7, 285–290. Seidelmann, K., Luber, K., Ferenz, H.J., 2000. Analysis of release and role of benzyl cyanide in male desert locusts, Schistocerca gregaria. Journal of Chemical Ecology 26, 897–1910. Seidelmann, K., Warnstorff, K., Ferenz, H.J., 2002. Phenylacetonitrile is a male specific repellent in desert locusts, Schistocerca gregaria. Journal of Chemical Ecology 28 (in press) Simmons, L.W., Siva-Jothy, M.T., 1998. Sperm competition in insects: mechanisms and the potential for selection. In: Birkhead, T.R., Møller, A.P. (Eds.), Sperm competition and sexual selection. Academic Press, San Diego. Simpson, S.J., Despland, E., Ha¨ gele, B.F., Dodgson, T., 2001. Gregarious behavior in desert locusts is evoked by touching their back
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legs. Proceedings of the National Academy of Sciences USA 98, 3895–3897. Stower, W.J., Popov, G.B., Greathead, D.J., 1958. Oviposition behaviour and egg mortality of the desert locust (Schistocerca gregaria) on the coast of Eritrea. Ant-Locust Bulletin 30, 33. Torto, B., Obeng-Ofori, D., Njagi, P.G.N., Hassanali, A., Amiani, H., 1994. Aggregation pheromone system of adult gregarious desert locust Schistocerca gregaria (Forskal). Journal of Chemical Ecology 20, 1749–1762.
Torto, B., Njagi, P.G.N., Hassanali, A., 1995. Sex pheromone studies in the desert locust Schistocerca gregaria (Forskal) (Orthoptera: Acrididae). Proceedings of the 12th Annual Meeting International Society of Ch Ecol., Santiago. Uvarov, B.P., 1977. Grasshoppers and Locusts, Vol 2. Centre for Overseas Pest Research, Cambridge.
Courtship inhibition pheromone in desert locusts, Schistocerca gregaria Karsten Seidelmann ∗, Hans-Jo¨rg Ferenz Martin-Luther University, Institute of Zoology, Animal Physiology, 06099 Halle, Germany Received 18 April 2002; received in revised form 30 July 2002; accepted 30 July 2002
Abstract Male desert locusts in the gregarious phase release phenylacetonitrile (PAN) on becoming sexually mature. It has been assumed that this chemical is responsible for aggregation of adult desert locusts. However, PAN has repellent characteristics and is involved in sexual behavior. Mature males release PAN as a volatile to serve as a kind of olfactory concealment during mating and to prevent competing males from homosexual encounters. We conclude that PAN is a courtship-inhibiting pheromone exclusively used under crowded conditions in dense populations when high sperm competition occurs among desert locust males. By chemically enhancing their mate guarding, gregarious males improve the protection of their mate from rivals and ensure their reproductive success. 2002 Published by Elsevier Science Ltd. Keywords: Behavior; Chemical communication; Locust; Reproduction; Phenylacetonitrile
1. Introduction Under exceptionally favorable conditions when desert locusts, Schistocerca gregaria, breed successfully, they aggregate into huge swarms which, in biblical proportions, invade and devastate agricultural areas. It is believed that semiochemicals play an important role in the establishment of such plagues (see, e.g., Ferenz, 1990; Pener, 1991; Pener and Yerushalmi, 1998). A variety of aromatic compounds released by desert locusts has been identified and is proposed to be the aggregation pheromone system of adults (Torto et al., 1994). Phenylacetonitrile (PAN) is a predominant component of this mixture. However, we have recently shown that PAN appears rather to be a repellent (Seidelmann et al., 2000; Seidelmann et al., 2002). PAN is a volatile phenol derivative released exclusively by gregarious mature adult male desert locusts. Immature males, females, hoppers, and solitary mature adults do not produce this substance (Luber et al., 1993; ∗ Corresponding author. Tel.: +49-345-55-26476; fax: +49-34555-27152. E-mail address: [email protected] (K. Seidelmann).
0022-1910/02/$ - see front matter 2002 Published by Elsevier Science Ltd. PII: S 0 0 2 2 - 1 9 1 0 ( 0 2 ) 0 0 1 7 8 - 6
Obeng-Ofori et al., 1994). The site and control of its production are unknown. The restriction of PAN to the reproductive phase of gregarious male locusts led us to the assumption that it might play a role in sexual behavior and mate recognition in locust swarms. The mating behavior of gregarious desert locusts has been described in some detail (Popov, 1958; Loher, 1959; Uvarov, 1977). It includes a slow approach of the male to the female, interrupted by stops in a semi-stilted posture. But on the whole no complicated sequence of courtship behavior can be observed. Finally the interested male jumps on the back of the female from a short distance, though he may be rejected by the female. Females are receptive shortly before oviposition. Pairing, if successful, is quickly followed by copulation. After the sperm transfer the male remains on the female and only leaves at the termination of egg laying. If the pair is separated prior to egg laying, the female will mate immediately with other males. However, although in locusts swarms many mature males are around, the guarding male is usually not attacked by rivals (Popov, 1958). As mature males produce PAN only in the presence of other mature males (Seidelmann et al., 2000), we speculated that the repellent PAN might act like a courtship-inhibiting pheromone (Seidelmann et al.,
992
K. Seidelmann, H.-J. Ferenz / Journal of Insect Physiology 48 (2002) 991–996
2002). Thus we investigated courtship and mating in the presence and absence of PAN.
2. Materials and methods 2.1. Insects Our desert locust culture, Schistocerca gregaria (Orthoptera, Acrididae), originated from specimens collected in 1991 in Niger. The insects were reared in the laboratory under crowded (‘gregarious’) conditions as described previously (Seidelmann et al., 2000). 2.2. Behavioural assay Experiments were done with truly gregarious locusts taken directly from the culture. We used only locusts which were engaged in pairing to ensure the right stage of development and intensive sexual behavior. Each couple was placed individually into numbered small cages and the males were marked by a small dot of white water-soluble non-toxic paint on the pronotum. For experiments with an additional competing male the female of each separated couple was placed in an open field (660 cm2) together with the new test male. After pair formation the original male was introduced to monitor mating attempts. Arranging the pairs usually lasted about 30 min; the assay was terminated after 120 min. At least five independent attacks or pairing attempts had to be observed. Manipulations of the pheromone bouquet of the test males was done by keeping mature males isolated in small cages (10 x 10 x 20 cm) for 7 days. Gregarious mature male desert locusts cease PAN emission within a few days after isolation. After 1 week of isolation, male PAN release cannot be detected any more (Seidelmann et al., 2000). PAN (Sigma, Germany) was dissolved in dichloromethane (Merck, Germany). 1.0 µl of the 10% solution (v/v) was applied topically on the prothorax of the test male with a microliter syringe. In control experiments we applied dichloromethane only. For experiments without an additional competing male, separated pairs were introduced into small observation cages (10 x 10 x 20 cm). Re-pairing was monitored at 30 min intervals for 2 hrs. In these mate-choice experiments, we marked one of the females by clipping off the very tip of their forewings. This marking technique is commonly used and does not influence male mate choice.
described above and kept in a large cage. After 1, 30, 60, or 120 min each locust was put into a 40 ml airtight glass vial for 180 min at 30 °C. After 150 min a SPME fiber (PDMS 100 µm, SUPELCO) was introduced and evaporated PAN absorbed for 30 min. Then the PANloaded fiber was placed into the split/splitless injector (splittless mode, 220 °C) of a Hewlett Packard 5890A gas chromatograph equipped with a 30 m x 0.32 mm ID polar capillary column (EC-wax, 0.25 µm, Alltech). Helium was used as carrier gas, set at 1 ml min–1. A gradient of 10 °C min–1 from 40–240 °C was used. The PAN FID peak was identified by comparison with authentic reference material. The amount of PAN was calculated from a standard curve obtained by applying various amounts of PAN on a metal mesh under the same conditions. From the measured PAN doses a curve for PAN evaporation over time can be calculated. The evaporation curve was modeled as an exponential function (Fig. 1). The measured PAN amounts represent the area under this curve. After antidifferentiation the unknown parameters of the function were calculated from the data by the non-linear regression module of STATISTICA (StatSoft Inc. 2000). It can be estimated that during the bioassay time (30 to 120 min) the amount of PAN released from the treated locusts is in the range from 104 to 42 ng hr–1. These doses are in agreement with the PAN amounts normally released by mature gregarious males (Torto et al., 1994; Seidelmann et al., 2000).
3. Results A first experiment was designed to confirm that mating males are able to guard a gravid female. We took mating pairs and forcibly separated them. The female was placed in the small observation arena and offered a
2.3. Determination of PAN concentration The rate of PAN evaporation from the locust body surface was determined in the following way: Test locusts were treated with 1 µl of the PAN solution as
Fig. 1. Standard curve for the time-dependent release of topically applied PAN. Values are the means ± SD of 4–5 replicates for each dose.
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gregarious mature male (but not her original mate). As soon as pairing had taken place, the original male mate was added. In all experiments this first mate approached the couple but did not attack (Fig. 2 A). The male showed some interest in the pair but did not jump onto the couple or fight for possession of the female. No takeover attempts could be observed (Table 1). Obviously the copulating male was able to repel the competing male. Since PAN is a repellent (Seidelmann et al., 2002), the volatiles including PAN naturally emitted by a mature gregarious male might be responsible for this effect. We confirmed our interpretation by an experiment
Fig. 2. Mating experiments to demonstrate the role of PAN in mate guarding of S. gregaria. A, control experiment with 2 gregarious males; B, test of the guarding ability of an isolated male (PAN production ceased); C, as D but isolated male treated with PAN; D, mating after treatment of the female with PAN; E, mate choice test with one PAN-treated and one control female (females, gray; males, white; male from original couple with gray thorax; female from original couple with white thorax). For further explanations see text.
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Table 1 Effect of PAN on pairing attempts of male desert locusts
Experiment
A B C D E
Treatment
gregarious male isolated male isolated male + PAN female + PAN female + PAN (choice)
n
11 12 12 20 12
% attacks or pairing attempts Treated
Control
0 100 0 0 0
– – 100 80 92
with mature males which were kept isolated for 7 days and ceased PAN emission. Gregarious pairs were separated as in the first experiment and each female locust was placed in the arena (Fig. 2 B). An isolated mature male was added and, after successful pairing, the original gregarious male mate was introduced into the arena. In all experiments the added male quickly jumped onto the couple and made attempts to copulate as though not recognizing the presence of the copulating male competitor (Table 1). To demonstrate that PAN indeed keeps the rival male off the couple, we repeated this experiment but treated the isolated males prior to pairing with 1 µl of the PAN solution on the pronotum (Fig. 2 C). Under the experimental conditions the evaporation rate of the applied PAN is in the range of the natural emission of gregarious males. Now the added gregarious male tolerated the copulating couple and did not attack it or try to jump on top of it (Table 1). In the control experiment with application of dichloromethane only all males attacked the couple as in untreated isolated males (Fig. 2 B). These results demonstrate that indeed the PAN odor is necessary to repel rivals. In a further experiment we tested whether PAN can be effective if not presented in combination with other male characteristics or male defensive behaviors. Thus, again we separated copulating pairs. Both partners were placed in a small observation cage and re-pairing was monitored at 30 min intervals for 2 hrs (Fig. 2 D). In the control group we observed re-pairing in 80% of the tests within 30 min, and pairing then lasted for the 2 hr observation period. In the experimental group, 1 µl PAN solution was placed on the prothorax of each female prior to the re-pairing experiment. Following this treatment no pairing could be observed within the 2 hr observation period (Table 1, Fig. 2 D). Obviously, the PAN odor overrides all other attracting signals specific to mature females and prevents males from courting and pairing with the PAN-treated females. PAN alone and not male characteristics or male behavior repels rivals. A last experiment was designed to assure that PAN does not change the motivation of the test male to mate. Thus, we provided a male with two females, one of
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which was treated with PAN and the other served as control (Fig. 2 E). For this experiment copulating pairs were treated as described in experiment D. Then an extra female was added to each pair in the observation arena. This female was taken from another separated couple. All males avoided the PAN-treated female: within 30 min, 9 males (75%) were engaged in pairing with the control female, and within 60 min a total of 11 males (92%). One male made no mating attempts at all (Table 1). This choice experiment confirms the repellent properties of PAN overriding the attractiveness of a mature female.
4. Discussion The presence of the main component of the mature desert locust male pheromone bouquet, PAN, is strongly associated with both sexual maturity and gregarious phase (Luber et al., 1993; Seidelmann et al., 2000; Torto et al., 1994). These findings suggested that PAN has an important function in desert locust reproductive behavior. Our results of mating and take-over experiments provide evidence that PAN emitted by mating males prevents approach and copulatory behavior of rivals. Male desert locusts use PAN for a chemically enhanced mate guarding; it serves an important function to avoid sperm competition. We observed a significant last-male precedence in desert locusts when two males mate successively with a female before oviposition (unpublished results). This finding is in agreement with the earlier results of HunterJones (1960). If a female mates with two males prior to oviposition, the last male will father the majority of the offspring. Therefore, to ensure his reproductive success, a male desert locust must prevent his mate from accepting another male until she has laid eggs. We did not find a spermatophore or a mating plug deposited by the male that might physically block the female reproductive tract. Thus, desert locust males have to guard their inseminated mate to keep off rivals and to avoid sperm competition. However, postcopulatory mate guarding may be costly in terms of lost opportunities to mate with other females. In S. gregaria, pairing and copulation occur when the female carries well-developed eggs and is close to oviposition. Only at this stage does a female accept a courting male. It appears to signal readiness to accept a male by a certain behavior (Popov, 1958) or, perhaps, by a short-range sex pheromone (Torto et al., 1995). Therefore, the operational sex ratio is strongly male biased and the chance of finding an unguarded receptive female is rather low, while the probability of the current female mate being captured and copulated by a male rival is high. Costs of precopulatory mate guarding in terms of lost mating opportunities should be negligible in the light
of the fitness benefits due to ensured fatherhood of the actual mate’s offspring. Thus, mating desert locust males remain in physical contact with the female after the separation of the genitalia until oviposition has taken place (Popov, 1958; Stower, 1958). Under conditions of high local competition for mates, as in S. gregaria swarms, the most important precopulatory trait to avoid take-overs and sperm competition is mate concealment (Simmons and Siva-Jothy, 1998). When the female releases a mate-attracting pheromone, males should respond by releasing quickly a pheromone to mask or diminish the female’s attractant (Happ, 1969; Happ and Wheeler, 1969; Happ et al., 1970). PAN is such a concealing or courtship-inhibiting pheromone, a kind of olfactory camouflage that appears to hide the receptivity-signaling female and thus protects the couple from competing males. It acts as an antiaphrodisiac to conspecific males and prevents them from attempts to court and to mount a guarded female. Male-to-male repellents reducing the attractiveness of a receptive female are also known from some moth species (e.g. Jacobs and Adler, 1984; Hirai et al., 1978; Pliske, 1975) and beetles (e.g. Happ, 1969; Happ et al., 1970; Nijholt, 1973). PAN can also be called an abstinon (Schlein et al., 1981), because it marks mature gregarious males and prevents wasteful homosexual activity. In this respect PAN is a synergistic signal to the yellow coloration of gregarious males. However, PAN does not fully meet the common aspects of both a courtship-inhibition pheromone and an abstinon: a courtship-inhibition pheromone is usually placed on a female after copulation; abstinons are normally not used for mate guarding. PAN appears to be a new type of a male-specific courtshipinhibiting pheromone which combines both functions resulting in an efficient mechanism for mate guarding ensuring reproductive success. Desert locusts may occur in both extremes of the abundance scale: In the solitary phase individuals are present in very low population densities in their recession areas. However, during gradations and swarm formation, very high population densities can be reached. For adult male locusts these extreme situations require different tactics and adaptations in mating strategies and sperm competition. While under low population densities the ability to locate or attract a mate will be the key for reproductive success of a male locust, the avoidance of sperm competition becomes the main problem under crowded conditions. To ensure mating success, males must be able to guard a gravid female and to protect her from mating with other males until she has used his sperm to fertilize the egg batch. The production of a courtship-inhibiting pheromone only in the presence of male competitors (Seidelmann et al., 2000) appears to be a specific and useful adaptation to crowded conditions in a phase-polymorphic species. In another locust species, the African migratory locust
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Locusta migratoria, with a very similar phase polymorphism, a precopulatory mate guarding has been described (Parker and Smith, 1975). Males of L. migratoria deposit a spermatophore blocking the female’s spermathecal duct until oviposition. In this species there is no need to guard the female after successful copulation until oviposition in order to avoid sperm competition. However, males guard a mature female in order to be the first male mating after the spermatophore has been expelled during oviposition. The operational sex ratio is rather balanced as females accept guarding males, and all mature females are worth being guarded. Take-overs are therefore not so costly for the loser. Hoppers of L. migratoria release PAN (Niassy et al., 1999), but its function needs further clarification. Males of this species do not use this pheromone (personal observations). To date, courtship-inhibiting pheromones have not been detected for L. migratoria although in this locust phase polymorphism and aggregation pheromones have been studied intensively. Recently, the existence of an aggregation pheromone system in desert locusts has been questioned. First, because the attractance of PAN previously established by olfactometer experiments (Torto et al., 1994) could not be confirmed (Seidelmann et al., 2000; Seidelmann et al., 2002) and, secondly, because aggregation can be fully induced by various non-pheromonal factors (Ha¨ gele and Simpson, 2000; Simpson et al., 2001). Furthermore, the major component guaiacol has been shown to be unspecifically derived from gut bacteria which metabolize digestive waste material (Dillon et al., 2000). Our results show that PAN, the key component of the suggested desert locust aggregation pheromone bouquet, has a completely different and previously unsuspected function. To our knowledge it is the first identified courtship-inhibiting pheromone in grasshoppers.
Acknowledgements We thank S. Hertel for excellent technical assistance. Support from the Deutsche Forschungsgemeinschaft (Se 1043) is gratefully acknowledged.
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