Journal o f Chemical Ecology, Vol. 13, No. 10, 1987
CUTICULAR HYDROCARBONS REGULATE MATE RECOGNITION, MALE AGGRESSION, AND FEMALE CHOICE OF THE ROVE BEETLE, Aleochara curuda
K. PESCHKE Zoologisches lnst#ut tti der Universitdit Warzburg ROntgenring 10, D 8700 Wiirzburg, F.R.G.
(Received April 1, 1986; accepted November24, 1986) Abstract--Immature, starved, or multiply mated males of the staphylinid beetle, Aleochara cur_mla~ mimic their females chemically. The titer of the female sex pheromonecomponents(Z)-7-heneicoseneand (Z)-7-tricosenewas quantified for various physiologicaltypes and both sexes by gas chromatography and correlated with the sexual responseof males towards the cuticular hydrocarbon fractions. Modulationof intermate aggression by productionof the female pheromone was shown by (1) reduction of the alkene titer of females kept at elevated temperatures, (2) treating live males with the synthetic female pheromonemixture, and (3) gradual amputationof male antennalsegments. A. curtula males do not fight against members of other Aleochara species with a different hydrocarbon pattern. Contaminationof A. peschkei males with the hydrocarbonfraction ofA. curtula males, however, provoked the release of aggression. Choosy females reject mating attempts of males bearing the female sex pheromone. Key Words--Aleochara curtuta, Coieoptera, Staphylinidae, female sex pheromone, cuticular hydrocarbons, chemicalmimicry, male aggression, female choice.
INTRODUCTION
In many insect species particular components of the cuticular hydrocarbons are used as female sex pheromones (for review see Howard and Blomquist, 1982; Jallon, 1984). Other communicative functions of surface hydrocarbons like caste recognition in social insects are mediated by the complex pattern of many compounds that vary in chain length, methyl branching, or unsaturation (Lok et al., 1975; Howard et al., 1978, t982a; C~,ement and Lange, 1984). Deceptive in1993 0098-0331/87!1000-1993805.00/0 9 1987 Plenum Publishing Corporalion
1994
PESCHKE
formation is emitted by termitophilous or myrmecophilous beetles, mimicking the pattern of their hosts' cuticular hydrocarbons (Howard et al., 1980, 1982b; Vander Meer and Wojcik, 1982). Chemical female mimicry by hydrocarbon pheromones has been demonstrated for males of the carrion-inhabiting rove beetle, Aleochara curtula (Goeze) (Peschke, 1985, 1987). (Z)-7-Heneicosene and (Z)-7-tricosene were identified as main components of the female sex pheromone (Peschke and Metzler, 1986). As measured only in a bioassay (Peschke, 1985, 1987), various physiological types of A. curtula males also elicit homosexual responses of other males by means of aphrodisiacs, and such behavior correlates with the avoidance of severe intermale aggression, which may lead to mechanical injuries, contamination with the toxic defensive secretion (Peschke and Metzler, 1982), and expulsion from the carcass. By female mimicry, however, the males get access to the carrion as a feeding and mating site (Peschke et al., 1987). The chemical identity of male releasers of homosexual responses with the female sex pheromone has so far only been demonstrated for immature beetles (Peschke, 1985). In the present paper, we report the chemical quantification of the alkene pheromone titer for other A. curtula individuals of various sex and physiological status, such as age, nutrition, previous copulations, and body size. The evident positive correlation of the release of male homosexual responses by producing the female sex pheromone with the reduction of intermale aggression was confirmed by three sets of experiments: (1) The chemical information output of the females was affected by reducing their alkene pheromone content by keeping the beetles at elevated temperatures. (2) The chemical information output of the males was modified by contamination with the synthetic female pheromone mixture. (3) The sensory input of males was manipulated by gradual amputation of antennal segments. Although the modulation of male contest by the female sex pheromone is fairly well understood, the essential releasers of aggression are unknown. Therefore, experiments on the role of the species-specific hydrocarbon composition in the recognition of male competitors have been conducted. We investigated the behavioral responses ofA. curtula males towards other Aleochara species with different hydrocarbon patterns and the effect of their chemical manipulation. While males releasing homosexual responses are protected from assaults of other males, females repulse mating attempts of these mimicking males. In this way they choose physiologically competent, nonmimicking males, which are capable of withstanding the aggressive interactions at the mating site and of transferring a large spermatophore (Peschke, 1985, 1987). In order to prove the hypothesis that female sex pheromone components also act as releasers of female repulsion behavior, we manipulated the chemical information output of males by contamination with the synthetic pheromone mixture.
CUTICULAR HYDROCARBONSREGULATEROVEBEETLEBEHAVIOR
1995
METHODS AND MATERIALS
Field Collections and Laboratory Cultures A. curtula (Goeze) and Philonthus politus (L.) were collected at rabbit carcasses in deciduous forests near Ochsenfurt, Bavaria (Peschke et al., 1987). Aleochara (Euryodma) brevipennis (Grav.) originates from the "Zeubelrieder Moor" (Ochsenfurt) and the Bodensee (fish bait). The species co-occurs with A. curtula on carcasses in middle Europe (Peschke and Fuldner, 1977). A. peschkei (Likovsky, 1983) was collected from fish carcasses from the banks of the fiver Comoe (Ivory Coast, West Africa). These Aleochara species were continuously reared in the laboratory according to Fuldner (1968) and Pesehke (1978, 1987) with puparia of Calliphora erythrocephala (Meig.) or Lucilia sericata (Meig.) serving as hosts for the parasitoid larvae (Peschke and Fuldner, 1977). Small beetles were obtained by using small puparia as hosts. Males were kept in groups of 10 in plastic boxes of 10 • 10 x 7 cm. Individual beetles and single pairs were kept in boxes of 5.5 x 3.5 x 1.5 cm. Three times a week, the beetles were transferred to freshly prepared boxes with moist filter paper and cut third-instar Calliphora maggots as food. Sexually isolated and well-fed beetles of both sexes at an age of 3 weeks were termed "standard laboratory beetles." The normal temperature and light regime were 22~ and 16:8 hr light-dark. Starved males were kept individually in order to avoid cannibalism. Chemical Methods Extracts were prepared according to Peschke and Metzler (1986). Thirty to 100 beetles were killed by freezing and extracted in 200 ml methylene chloride in a Soxhlet apparatus for 24 hr. The hydrocarbon fraction was purified on a silica gel column. Gas chromatography was also conducted according to Peschke and Metzler (1986) on a Varian 3700 instrument with a 30-m DB-1701 capillary column (split 1 : 10, 1 ml He/min, 60~ to 300~ at 3~ flame ionization detector). Quantitative measurements were carried out with a Kontron-Anacomp 220 computer system. The concentrations of the two pheromone alkenes were determined by using the corresponding n-alkanes as internal standards of constant titer and then related to the pheromone content of standard laboratory females [1 female equivalent (FE) (Z)-7-heneicosene = 2 Izg, 1 FE (Z)-7-tricosene = 12 Izg; Peschke and Metzler, 1986]. Both alkenes were synthesized according to Peschke and Metzler (1986). Some samples were also obtained from Dr. L.L. Jackson (Bozeman, Montana). For comparison of hydrocarbon patterns, we calculated the equivalent chain length for methyl-branched alkanes and plot-
1996
PESCHKE
ted the cumulative distribution functions of compounds versus increasing chain length (Hadley, 1977; Toolson and Hadley, 1977).
Behavioral Responses At a distance of about 2.2 mm from the female, the male bends his mobile abdomen over his head and protrudes the genitalia with the tong-shaped parameres (grasping response; Peschke, 1978). Male aggression is characterized by pushing the opponent with head and mandibles and by drumming with the mobile abdomen on the other individual. Females that repulse the male grasping response do so by oscillating their mobile abdomen thereby avoiding the fixation of the mate genitalia. In neutral encounters of two individuals, no conspicuous behavior could be observed (Peschke, 1987).
Bioassays The sexual response of A. curtula males to the purified hydrocarbon fractions of beetles of various physiological status was tested in the model bioassay (Peschke, 1978). Soxhlet-extracted odorless beetles were glued to the tips of glass needles and treated with n-pentane solutions of 0.01 ml containing one beetle equivalent. The number of standard tester males responding to the model with the grasping response was specified in percentiles and statistically evaluated by the X2 test or the exact test of Fisher (Sachs, 1984). Confidence limits were calculated for the 95 % level. Beetles of both sexes and various physiological status were observed as single pairs in plastic boxes of 5.5 • 3.5 • 1.5 cm for a period of 30 rain. The individuals were marked by dots of enamel paint on pronotum and both elytra. We recorded whether a certain behavioral response was seen at least once during the observation period, or we registered all encounters of beetles and specified the percentage in which a certain behavior was shown. Living beetles were treated with extracts or synthetic chemicals: 0.01 ml pentane solutions with one beetle equivalent concentration were applied to the pronotum of a chilled individual (6~ As a control, beetles were treated with the pure solvent. One hour after contamination, the beetles were brought together and the pair was observed for 30 min as usual, Antennal segments or palps of males 7 days old were cut off using pincers which were sharpened like scissors. Scapus and pedicellus were counted as segments l and 2, the flagellar segments as 3-11. After a further week, males of normal agility were observed individually together with single females (30 min). For observation of interspecific reactions of beetles under natural conditions, we used an arena of 50 • 50 cm, in the center of which a small piece of carrion was placed (beef liver with Calliphora maggots, covered by a dish of
CUTICULAR HYDROCARBONS REGULATE ROVE BEETLE BEHAVIOR
1997
red glass; for details see Peschke, 1987). Individually marked A. curtula males and females (five each) were put together with five males and five females of another species (A. brevipennis, A. peschkei, and Philonthus politus). The beetles were observed at the carcass with a red-sensitive camera in a natural rhythm of white and dark red light (18:6 hr light-dark). The behavioral events at the carcass were recorded for 3 rain every 60 rain at normal speed over a period of 24 hr. For each encounter of two beetles, we recorded whether aggression, grasping, or neutral behavior was exhibited by A. curtula males. RESULTS Sex Pheromone Content in Beetles of Various Sex and Physiological Status In former investigations (Peschke, 1985, 1987), all bioassays for testing pheromone contents had been conducted with entire beetles freshly killed by freezing. In the present experiments, sexual responses were also elicited by the purified total hydrocarbon fraction from extracts of females and males of various physiological status (model bioassay; Figure 1). The cuticular hydrocarbon fraction from females collected in the field released the male sexual response at the same high rate as did extracts from standard laboratory females, from females just after copulation, and from those of small body size (Figure 1). Freshly emerged females yielded a somewhat lower grasping rate. The grasping response rate was drastically reduced by exposing the females to elevated temperatures (14 days at 27~ High behavioral release rates were also obtained with extracts from males captured in the field. The hydrocarbon fraction from young, starved, or multiply mated laboratory males also elicited homosexual grasping responses (Figure 1). On the other hand, hydrocarbons from sexually isolated, well-fed males reared in the laboratory did not trigger homosexual grasping responses, regardless of temperature (22~ or 27~ and body size (large standard beetles: breadth of pronotum 2.0 _+ 0.02 ram, small beetles: 1.4 _+ 0.03 mm). The quantitative determination of components of the hydrocarbon fractions used in the former bioassay gave a good correlation of grasping rates with the contents of (Z)-7-tricosene (Figure 1). Parallel to the determination of amounts (in micrograms), we preferred the scaling in terms of female equivalents in order to obtain a more lucid comparison of pheromone contents of mimicking individuals with that of standard laboratory females (defined as 1 FE). Females of various physiological status, apart from young beetles, had a constantly high tricosene content. In females kept at elevated temperatures, only trace amounts of this compound could be detected. Standard laboratory males had a concentration of 10 -3 FE tricosene irrespective of their body size and the temperature at which they had been reared. Young, starved, and multiply mated males, however, yielded tricosene contents ranging from 0.08 to 1 FE.
1998
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FiG. 1. Grasping response of A. curtula males towards models contaminated with one beetle equivalent of the total cuticular hydrocarbon fraction from extracts of males (triangles) and females (circles) of various physiological conditions: 1: laboratory standard females (sexually isolated, well fed, 22~ f: field-collected beetles; y: freshly emerged beetles; st: mature beetles starved for a period of 10 days; c: one copulation at the day of extraction; 27: beetles kept at elevated temperatures (27~ sm: beetles reared on a small host. Thirty to 100 beetles were used per extraction; at least 100 males were tested for grasping in five replicates per sample. The grasping rate is plotted against the concentrations of the two pheromone compounds (Z)-7-heneicosene (open symbols) and (Z)-tricosene (solid symbols) in the hydrocarbon fraction as determined by quantitative gas chromatography. The concentration is scaled in terms of female equivalents as related to the content of laboratory standard females (= 1 FE). Because the combined action of compounds was tested in the bioassay, one value of grasping rate refers to two values of concentration of both pheromone compounds, which are linked by a dotted line. The grasping rate is correlated with the concentration o f (Z)-7-heneicosene in a similar way (Figure 1), except in the cases of field-collected or starved males and of young females, which release male sexual responses but have a low or even undetectable titer o f the C21-alkene. In these cases, the tricosene alone seems to be responsible for the release o f the homosexual grasping response. Heneicosene could also not be detected in small males and males kept at 27~
1999
CUTICULAR HYDROCARBONS REGULATE ROVE BEETLE BEHAVIOR
Modulation of Male Aggression by Female Sex Pheromone In the experiments described above, we demonstrated a correlation of homosexual responses with the production of female sex pheromone components by males. In the following experiments, the sex-specific chemical information was manipulated in order to investigate the correlation of female pheromone production with the release of male aggressive behavior. Temperature Experiment. Males and females kept at 22~ or 27~ were observed as single pairs in various combinations (Table 1). As in the experiments with extracts, females kept at 27~ did not release male grasping at any encounter. On the other hand, the reduction of pheromone titer in females kept at elevated temperatures provoked a high aggression rate of males towards these females. The behavioral sequence was not different from that observed in intermale combats. Contamination of Males with Synthetic Female Pheromone Mixture. A reduction of the release of aggression was demonstrated in observations (30 min) of pairs of males, to one of which the hydrocarbon fraction of a female surface washing had been applied; to the other, the pure solvent (Table 2). The male bearing the female pheromone attacked the other male in a high percentage of encounters, whereas the male without the pheromone only occasionally behaved aggressively towards the treated male. The treated male, however, released a high rate of homosexual graspings. The same tendencies were observed when one male was contaminated with 1 FE of the synthetic female sex pheromone (Table 2). The rate of aggression is drastically reduced towards the treated male; however, more neutral encounters than homosexual grasping responses were observed in comparison to experiments with the total female hydrocarbon fraction. Amputation of Mate Antennal Segments. Males with their antennae amputated to various degrees were observed together with females during a 30min period. The number of males exhibiting the grasping response towards TABLE 1. AGGRESSION AND GRASPING RESPONSES DURING 30-MIN OBSERVATIONS OF SINGLE PAIRS OF Z. curtula WITH MALES AND FEMALES KEPT AT DIFFERENT TEMPERATURES
Response of males kept at
Towards females kept at
Response (%) Aggression
Neutral
Grasping
(Npalrs)
22~ 22~ 27~ 27~
22~ 27~ 22~ 27~
!1 52~ 0 33"
31 48 28 67
58 0~ 72 0n
160 (4) 84 (5) 93 (5) 30 (4)
Nencounters
"P < 0.001; X~ test: comparisonto the preceding line.
2000
PESCHKE
TABLE2. AGGRESSIONAND HOMOSEXUALGRASPING RESPONSES DURING 30-MIN OBSERVATIONS OF TWO A. CUFtula MALES, ONE CONTAMINATEDWITH HYDROCARBON FRACTION OF FEMALE EXTRACT (1 FE) OR SYNTHETIC FEMALE SEX PHEROMONE (1 FE: 12/~g (Z)-7-TRICOSENE + 2 /~g (Z)-7-HENEICOSENE),THE OTHERMALE (CONTROL) TREATED WITH SOLVENT (0.01 ml r/-PENTANE) Response (%) Contamination with Female hydrocarbon fraction
Synthetic female sex pheromone mixture
Response of control males towards contaminatedmales contaminated males towards control males controlmales towards contaminated males contaminatedmales towards control males
Aggression Neutral Grasping
Nencounters (Np~)
14
16
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93 (4)
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85 (5)
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73
17
60 (5)
946
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~P < 0.001; Xz test: comparison to the preceding line. females was not reduced as long as four antennal segments were left (Figure 2). The next step of amputation (three segments left), however, gave a significant decrease of the grasping rate, which was continued with removal of further antennal segments or palps. Coincidentally with the stepwise decrease of sexual responses, the percentage of males behaving aggressively towards females increased, with a peak rate obtained when only the scapus was exempted from amputation. The rate of aggression was then decreased with further steps of removal of antennal segments and palps.
Releasers of Aggression Sexual behavior as well as male aggression can be manipulated by the female sex pheromone titer. However, the simple absence of the female sex pheromone may be a signal for males to fight against another individual, or species-specific signals that may be other cuticular hydrocarbons may release male aggression. While in the former experiments the concentration of pheromone alkenes has been shown to vary considerably between the sexes, the saturated hydrocarbons have a very constant distribution (Peschke, 1985; Peschke and Metzler, t986). The cumulative frequency function of the equivalent chain length of saturated hydrocarbon components also shows a very similar pattern for both sexes of A. curtula (Figure 3). In contrast, cuticular hydrocarbons of male A. brevipennis or A. peschkei have a distribution very different from that
CUTICULAR HYDROCARBONS REG1LILATEROVE BEETLE BEHAVIOR rl
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FIG. 2. Behavioral responses (grasping, aggression) of A. curtula males (various steps of symmetrical amputation of antennal segments and pal~) to standard females during a 30-rain observation (percent pairs showing the response at least once). Steps of amputation: A: antenna (in parentheses number of segments exempted from amputation, A11: control), Mx: maxillar palps; Lb: labial palps. Vertical bars: 95% confidence limits; n: number of pairs).
of A. curtula. Most noteworthy is the shift to higher carbon numbers and the total absence of C 21 and C23 hydrocarbons. The identification of components of A. brevipennis and A. peschkei by mass spectrometry with the original gas chromatograms will be published elsewhere (Peschke and Metzler, in preparation). A. eurtula males and females were put together with individuals of other species in the video observation setup. A. brevipennis released a few grasping responses of A. curtula males (Table 3) which might be due to unsaturated C27 hydrocarbons. Aggression of A. curtula males against A. brevipennis individuals of both sexes was observed at an intermediate level. A. peschkei did not release interspecific grasping or aggressive responses of A. curtula males. Individuals of both sexes of Philonthus politus, a distantly related species from another subfamily of rove beetles (Staphylininae instead of Aleocharinae), also failed to provoke either aggression or sexual responses of A. curtula males (Table 3). In conclusion, male aggression of A. curtula seems to be released by species-specific chemical cues, probably involving the saturated hydrocarbons of the cuticle. This hypothesis is supported by experiments with A. peschkei males contaminated with the hydrocarbon fraction of male A. curtula extracts (Table
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CUTICULAR HYDROCARBONS REGULATE ROVE BEETLE BEHAVIOR
2003
TABLE 3. BEHAVIORAL RESPONSES OF A. curtula MALES TO MALES AND FEMALES OF OTHER ROVE BEETLE SPECIES AT LABORATORY-SIMULATED CARCASS (VIDEO
OBSERVATION). Response of males (%)~
Nencoua~er~ Response towards
Aggression
Neutral
Grasping
(Npa~,~)
0"**
1206 (15) 437 (15)
A. curtula Males
95
Females
15"**
66
19
38*** 34***
54 54
8 NS 12"*
13 (5) 143 (5)
6*** 0"** 1"**
94 100 99
0"** 0* 0"**
145 (5) 23 (5) 71 (5)
5
A. brevipennis Males Females
A. peschkei Males Females
Philonthus politus
~P < 0.05, **P < 0.01, ***P < 0.001; X2 test: comparison to A. curtula males (aggression) or to A. curtula females (grasping).
4). While A. curtula males did not behave aggressively towards A. peschkei individuals contaminated with the solvent in the 30-rain observation bioassay, they exhibited a conspicuous fighting behavior towards A. pesehkei specimens treated with male A. curtula hydrocarbons. Female Repulsion Behavior Males which release homosexual responses of other males may show the grasping response towards females. However, in comparison to nonmimicking TABLE 4-. AGGRESSION OF A. curtula MALES TOWARDS A. peschkei MALES CONTAMINATED WITH HYDROCARBON FRACTION OF EXTRACTS OF A. curtula MALES (1 M E , 0.01 ml) OR SOLVENT (n-PENTANE) DURING 30-M1N OBSERVATIONS OF S1NGEE PAIRS
Response of A. curtula males towards
Response (%)
Nencounters Aggression
Neutral
Grasping
(Npa~T~)
0 80 ~
100 20
0 0
19 (4) 45 (5)
A. peschkei males Control Contaminated
~P < 0.001; X2 test, comparison to control.
2004
PESCHKE
TABLE 5. GRASPING OF A. curtula MALES CONTAMINATED WITH SYNTHETIC FEMALE SEX PHEROMONE (1 FE: 12 # g (Z)-7-TRIcOSENE + 2 # g (Z)-7-HENEICOSENE) AND REPULSION OF GRASPING RESPONSES BY FEMALES DURING 30-MIN OBSERVATIONS OF SINGLE PAIRS
Males Contaminated with synthetic female pheromone Control
Grasping of males (%)
Repulse by females (%)
N, ..... ~r~ (Np,irs)
99 NS 94
80 a 32
285 (6) 83 (15)
~P < 0.001; 2 test: comparison to control.
males, they are more often repulsed by the female, who drums with her abdomen and thus avoids the fixation of male genitalia (Peschke, 1987). In order to investigate the releasers of female repulsion behavior, males were contaminated with 1 FE of the synthetic mixture of the female pheromone (Table 5). The treated males did not show an alteration in their own sexual response to females as measured by the grasping response rate during a 30-rain observation period. On the other hand, the repulsion behavior of females towards such treated males is significantly increased. DISCUSSION
Female mimicry in vertebrates using chemical (Mason and Crews, 1985) or behavioral and morphological cues (for review see Weldon and Burghardt, 1984) has been described for many species. Stealing sneaky copulations, avoidance of intermale aggression, and access to other males' territories are discussed as the main benefits of mimicking males. Behavioral transvestism in insects has only been described for scorpion flies where males behave like females in order to steal the nuptial gifts of other males (ThornhiU, 1979). Certain physiological types of males of the rove beetle, Aleochara curtula, mimic their females chemically in order to avoid intermale aggression. In this way they get access to a carcass and are able to feed on blow fly maggots in order to replenish their energy reserves for a forthcoming reproductive cycle (Peschke, 1987). On the other hand, females prefer males without the female pheromone. The adaptive significance of female coyness towards mimicking males seems to be the choice of an optimal mate: immature, starved, or multiply mating males, which all produce the female sex pheromone, need access to the
CUTICULAR HYDROCARBONS REGULATE ROVE BEETLE BEHAVIOR
2005
food resource; they transfer only small spermatophores and fertilize less eggs. On the other hand, physiologically competent males without the female sex pheromone have succeeded in numerous aggressive interactions with other males (Peschke, 1987). (Z)-Heneicosene and (Z)-7-tricosene have previously been isolated and identified from mature A. curtula females as the main sex pheromone components (Peschke and Metzler, 1986). The pheromone molecules are components of the epicuticular waxes and are spread over the entire surface of the beetles (Peschke, 1978, 1986). Male antagonistic pheromones, which might be involved in the regulation of sex specificity of the pheromone information, could not be detected (Peschke, 1986). In the present work the pheromone alkenes were also found in the cuticular hydrocarbons of those males which release homosexual responses, e.g., young, starved, or multiply mated beetles. All these physiological types of males are mixed up in field collections (Peschke et al., 1987), where they also bear the female sex pheromones. Small males, which perform an alternative mating tactic as satellites in the surroundings of carrion (Peschke, in preparation), do not produce the female sex pheromone for protection. The contents of the two pheromone components in various physiological types and both sexes of A. curtula range over three powers of 10 and are correlated with the release of male sexual responses. In most samples, both compounds contribute to the releasing effect of the total hydrocarbon fraction. In some cases, tricosene alone seems to be responsible for the release of sexual responses. In tests of individual synthetic substances, tricosene had its optimum at about 0.1 FE, whereas heneicosene released more sexual responses at the higher concentration of 1 FE (Peschke and Metzler, 1986). It is therefore difficult to quantify the contribution of the individual pheromone component to the releasing efficiency of the total hydrocarbon mixture of each physiological type. The combination of both compounds had no synergistic, only an additive, effect; the admixture of (Z)-9-heneicosene or (Z)-9-tricosene, which are produced together with the respective 7-isomers but have only little releasing efficiency (Peschke and Metzler, 1986), did not produce an antagonistic action (Peschke, in preparation). Differences in the release of grasping rates towards synthetic compounds or towards the total hydrocarbon fraction containing the same amounts of pheromone components may be due to retardation of evaporation by the accompanying saturated hydrocarbons in the latter ease (Peschke, 1986). This effect may also be responsible for different responses towards contaminated living males. Homosexual grasping is released at a lower rate after contamination with the synthetic pheromone mixture in comparison to males treated with the total hydrocarbon fraction of females. However, the aggression rate is reduced to the same extent in both cases. Individual (Z)-7-heneicosene and (Z)-7-tricosene
2006
PESCHKE
both contribute to the reduction of intermale aggression (Peschke, in preparation). The modulatory effect of the female sex pheromone on the release of male aggression was also demonstrated by manipulation of the chemical information output of the female. Females which have been exposed to elevated temperatures show a significantly reduced content of cuticular alkenes in comparison to that of saturated compounds. This may be due to differential evaporation of saturated and unsaturated compounds. However, it cannot be excluded that the beetles actively alter the pattern of hydrocarbon synthesis at different temperatures (see Hadley, 1977; Toolson and Hadley, 1977). Males behave aggressively towards these females in the same way as towards other males. A further indication of the modulatory effect of pheromone alkenes is the gradual increase of aggression accompanied by a decrease of sexual recognition with successive amputation of male antennal segments. In a preliminary investigation of the antennal morphology of A. curtula (Ungelenk and Altner, unpublished), no sex specific sensilla or striking quantitative difference in the distribution and number of various types could be found. The pattern of densely packed sensilla seems to be repeated in segments 5-11, whereas scapus, pedicellus, and the two proximal flagellar segments bear only a few sensilla. It seems likely that the sex pheromone information is perceived by numerous receptor organs sequentially distributed on the distal antennal segments. Removal of them gradually diminishes the input of sex pheromone information, whereas information on aggression-releasing chemicals, probably the saturated hydrocarbons, still can be perceived. Further steps of amputation reduce this input too. However, electrophysiological investigations of pheromone perception in A. curtula are urgently needed. Females of A. curtula are apparently able to perceive the female sex pheromone too. However, the female behavioral response elicited by these chemicals, the release of aggressive repulsion of male mating attempts, is the reverse of the male reaction where aggression is reduced. The releasers of intermale aggression of A. curtula may comprise components of the saturated hydrocarbon mixture that are present in both sexes. The shift to longer carbon chains in related species reduces aggression of A. curtula males; on the other hand, contamination of males of the other species with hydrocarbons of A. curtula males, which do not contain female pheromones, provoked intermale combats. At present, synthetic methyl-branched alkanes are not available, and therefore it is not possible to determine yet whether individual n-alkanes, methyl-branched alkanes, or the complex pattern of compounds of variable chain lengths serve as the releasers of male aggression. Saturated hydrocarbons modulate the information of alkenes acting as female sex pheromones in several muscid flies (e.g., Rogoff et al., 1980; Sonnet et al., 1975, 1977; Uebel et al., 1975a, b, 1976), or represent the major female pher-
CUTICULAR HYDROCARBONS REGULATE ROVE BEETLE BEHAVIOR
2007
o m o n e s , for e x a m p l e , in t s e t s e flies ( e . g . , C a r l s o n et al., 1978; H u y t o n et al., 1980a, b) o r b u t t e r f l i e s ( G r u l a et al., 1980). Acknowledgments--I am very grateful to Mrs. C. Gantert for technical assistance. Thanks are due to Prof. Dr. L.L. Jackson and Prof. Dr. M. Metzler for synthetic pheromone compounds. I wish to thank Dr. A.E. Jtirss for reading the English manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (Pe 231/3 and 4).
REFERENCES CARLSON, D.A., LANGLEY, P.A., and HUYTON, P.M. 1978. Sex pheromone of the tsetse fly: Isolation, identification and synthesis of contact aphrodisiacs. Science 201:750-753. CLEMENT, J.L., and LANCE, C. 1984. Variation of cuticular compounds and inter and intraspecific aggressive behaviour in termite species of the genus Reticulitermes. Abstract XVII International Congress on Entomology. Hamburg. p. 480. FULDNER, D. 1968. Experimentelle Analyse des Orientierungsverhaltens der Eilarve yon Aleochara curtula Goeze (Coleoptera: Staphylinidae) am Wirt. Z. Vergl. Physiol. 61:298-354. GRELA, J.W., MCCHESNEY, J.D., and TAYLOR, O.R., JR. 1980. Aphrodisiac of the sulfur butterflies Colias eurytheme and C. philodice (Lepidoptera, Pieridae). J. Chem. Ecol. 6:241-256. HADLEY, N.F. 1977. Epicuticutar lipids of the desert tenebrionid beetle, Eleodes armata: Seasonal and acclimatory effects on composition. Insect Biochem. 7:277-283. HOWARD, R.W., and BLOMQUIST,G.J. 1982. Chemical ecology of insect hydrocarbons. Annu. Rev. Entomol. 27:149-172. HOWARD, R.W., McDAMEL, C.A., and BLOMQUIST, G.J. 1978. Cuticular hydrocarbons of the eastern subterranean termite, Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae). J. Chem. Ecol. 4:233-245. HOWARD, R.W., MCDAMEL, C.A., and BLOMQEIST, G.J. 1980. Chemical mimicry as an integrating mechanism: cuticular hydrocarbons of a termitophile and its host. Science 210:431-433. HOWARD, R.W., MCDANIEL, C.A., NELSON, D.R., BLOMQUIST,G.J., GELBAUM,T., and ZALKOW, I.H. 1982a. Cuticular hydrocarbons of Reticulitermes virginicus (Banks) and their role as potential species and caste-recognition cues. J. Chem. Ecol. 8:1227-1239. HOWARD, R.W., MCDANIEL, C.A., and BLOMQUIST, G.J. 1982b. Chemical mimicry as an inte~ grating mechanism for three termitophiles associated with Reticulitermes virginicus (Banks). Psyche 89:157-167. HUYTON, P.M., LANGLEY, P.A., CARLSON, D.A., and COATES, T.W. 1980a. The role of sex pheromones in initiation of copulatory behaviour by male tsetse flies, Glossina morsitans morsitans. Physiol. Entomol. 5:243-252. HUYTON, P.M., LANGLEY, P.A., CARLSON, D.A., and SCHWARZ,M. 1980b. Specificity of contact sex pheromones in tsetse flies, Glossina spp. Physiol. Entomol. 5:253-264. JALLON, J.M. 1984. A few chemical words exchanged by Drosophila during courtship and mating. Behav. Genet. 14:441-478. LIKOVSKY, Z. 1983. Bemerkungen tiber Aleochara-Arten der afrikanischen Region (Coleoptera, Staphylinidae). Annot. Zool. Bot. 152:1-18. LOK, J.B., CuPP, E.W., and BLOMQUIST, G.J. 1975. Cuticular lipids of the imported fire ants, Solenopsis invicta and S. richteri. Insect Biochem. 5:821-829. MASON, R.T., and CREWS, D. 1985. Female mimicry in garter snakes. Nature 316:59-60. PESC;qKE, K. 1978. The female sex pheromone of the staphylinid beetle, Aleochara curtuta. J. Insect Physiol. 24:197-200.
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PESCI-IKE,K. 1985. Immature males ofAleochara curtula avoid intrasexual aggression by producing the female sex pheromone. Naturwissenschaften 72:274. PESCHKE,K. 1986. Development, sex specificity, and site of production of aphrodisiac pheromones in Aleochara curtula. J. Insect Physiol. 32:687-693. PESCHKE, K. 1987. Male aggression, female mimicry and female choice in the rove beetle, Aleochara curtula (Coteoptera, Staphytinidae). Ethology (In press). PESCHKE, K., and FULDNER, D. 1977. f0bersicht und neue Untersuchungen zur Lebensweise der parasitoiden Aleocharinae (Coleoptera; Staphylinidae). Zool. Jb. Syst. 104:242-262. PESCHKE, K., and METZLER, M. 1982. Defensive and pheromonal secretion of the tergal gland of Aleochara curtula. I. The chemical composition. J. Chem. Ecol. 8:773-783. PESCr~KE, K., and METZLER, M. 1986. Cuticular hydrocarbons and female sex pheromones of the rove beetle, A leochara curtula (Coleoptera, Staphylinidae). Insect Biochem. 17:167-178. PESCItKE, K. KRAPF, D., and FULDNER, D. 1987. Ecological separation, functional relationships, and limited resources in a carrion insect community. Zool, Jb. Syst. 114:241-265. ROGOFF, W.M., GRETZ, G.H., SONNET, P.E., and SCHWARZ,M. 1980. Responses of male house flies to muscalure and to combinations of hydrocarbons with and without muscalure. Environ. Entomol. 9:605-606. SACHS, L. 1984. Angewandte Statistik. Springer-Verlag, Heidelberg. SONNET,P.E., UEBEL, E.C., and MILLER,R.W. 1975. Sex pheromone of the face fly and compounds influencing pheromone activity. Environ. Entomol. 4:761-764. SONNET, P.E., UEBEL,E.C., HARRIS,R.L., and MILLER, R.W. 1977. Sex pheromone of the stable fly: evolution of methyl- and 1,5-dimethylalkanes as mating stimulants. J. Chem. Ecol. 3:245249. THORNHILL, R. 1979. Adaptive female-mimicking behavior in a scorpionfly. Science 205:412-414. TOOLSON, E.C., and HADLEY, N.F. 1977. Cuticular permeability and epicuticular lipid composition in two Arizona vej0vid scorpions. Physiol. Zool. 50:323-330. UEBEL, E.C., SONNET, P.E., MILLER, R.W., and BEROZA, M. 1975a. Sex pheromone of the face fly, Musca autumnalis De Geer (Diptera: Muscidae). J. Chem. Ecol. 1: 195-202. UEBEL, E.C., SONNET, P.E., BIZRL, B.A., and MmLER, R.W. 1975b. Sex pheromone of the stable fly: Isolation and preliminary identification of compounds that induce mating strike behavior. J. Chem. Ecol. 1:377-385. UEBEL, E.C., SONNET, P.E., and MILLER, R.W. 1976. House fly sex pheromones: Enhancement of mating strike activity by combination of (Z)-9-tricosene with branched saturated hydrocarbons. Environ. Entomol. 5:905-908. VANDE, MEER, R.K., and WOJCIK, D.P. 1982. Chemical mimicry in the myrmecophilous beetle Myrmecaphodius excavaticollis. Science 218:806-808. WELDON, P.J., and BURGHARDT, G. 1984. Deception divergence and sexual selection. Z. Tierpsychol. 65: 89-102.
CUTICULAR HYDROCARBONS REGULATE MATE RECOGNITION, MALE AGGRESSION, AND FEMALE CHOICE OF THE ROVE BEETLE, Aleochara curuda
K. PESCHKE Zoologisches lnst#ut tti der Universitdit Warzburg ROntgenring 10, D 8700 Wiirzburg, F.R.G.
(Received April 1, 1986; accepted November24, 1986) Abstract--Immature, starved, or multiply mated males of the staphylinid beetle, Aleochara cur_mla~ mimic their females chemically. The titer of the female sex pheromonecomponents(Z)-7-heneicoseneand (Z)-7-tricosenewas quantified for various physiologicaltypes and both sexes by gas chromatography and correlated with the sexual responseof males towards the cuticular hydrocarbon fractions. Modulationof intermate aggression by productionof the female pheromone was shown by (1) reduction of the alkene titer of females kept at elevated temperatures, (2) treating live males with the synthetic female pheromonemixture, and (3) gradual amputationof male antennalsegments. A. curtula males do not fight against members of other Aleochara species with a different hydrocarbon pattern. Contaminationof A. peschkei males with the hydrocarbonfraction ofA. curtula males, however, provoked the release of aggression. Choosy females reject mating attempts of males bearing the female sex pheromone. Key Words--Aleochara curtuta, Coieoptera, Staphylinidae, female sex pheromone, cuticular hydrocarbons, chemicalmimicry, male aggression, female choice.
INTRODUCTION
In many insect species particular components of the cuticular hydrocarbons are used as female sex pheromones (for review see Howard and Blomquist, 1982; Jallon, 1984). Other communicative functions of surface hydrocarbons like caste recognition in social insects are mediated by the complex pattern of many compounds that vary in chain length, methyl branching, or unsaturation (Lok et al., 1975; Howard et al., 1978, t982a; C~,ement and Lange, 1984). Deceptive in1993 0098-0331/87!1000-1993805.00/0 9 1987 Plenum Publishing Corporalion
1994
PESCHKE
formation is emitted by termitophilous or myrmecophilous beetles, mimicking the pattern of their hosts' cuticular hydrocarbons (Howard et al., 1980, 1982b; Vander Meer and Wojcik, 1982). Chemical female mimicry by hydrocarbon pheromones has been demonstrated for males of the carrion-inhabiting rove beetle, Aleochara curtula (Goeze) (Peschke, 1985, 1987). (Z)-7-Heneicosene and (Z)-7-tricosene were identified as main components of the female sex pheromone (Peschke and Metzler, 1986). As measured only in a bioassay (Peschke, 1985, 1987), various physiological types of A. curtula males also elicit homosexual responses of other males by means of aphrodisiacs, and such behavior correlates with the avoidance of severe intermale aggression, which may lead to mechanical injuries, contamination with the toxic defensive secretion (Peschke and Metzler, 1982), and expulsion from the carcass. By female mimicry, however, the males get access to the carrion as a feeding and mating site (Peschke et al., 1987). The chemical identity of male releasers of homosexual responses with the female sex pheromone has so far only been demonstrated for immature beetles (Peschke, 1985). In the present paper, we report the chemical quantification of the alkene pheromone titer for other A. curtula individuals of various sex and physiological status, such as age, nutrition, previous copulations, and body size. The evident positive correlation of the release of male homosexual responses by producing the female sex pheromone with the reduction of intermale aggression was confirmed by three sets of experiments: (1) The chemical information output of the females was affected by reducing their alkene pheromone content by keeping the beetles at elevated temperatures. (2) The chemical information output of the males was modified by contamination with the synthetic female pheromone mixture. (3) The sensory input of males was manipulated by gradual amputation of antennal segments. Although the modulation of male contest by the female sex pheromone is fairly well understood, the essential releasers of aggression are unknown. Therefore, experiments on the role of the species-specific hydrocarbon composition in the recognition of male competitors have been conducted. We investigated the behavioral responses ofA. curtula males towards other Aleochara species with different hydrocarbon patterns and the effect of their chemical manipulation. While males releasing homosexual responses are protected from assaults of other males, females repulse mating attempts of these mimicking males. In this way they choose physiologically competent, nonmimicking males, which are capable of withstanding the aggressive interactions at the mating site and of transferring a large spermatophore (Peschke, 1985, 1987). In order to prove the hypothesis that female sex pheromone components also act as releasers of female repulsion behavior, we manipulated the chemical information output of males by contamination with the synthetic pheromone mixture.
CUTICULAR HYDROCARBONSREGULATEROVEBEETLEBEHAVIOR
1995
METHODS AND MATERIALS
Field Collections and Laboratory Cultures A. curtula (Goeze) and Philonthus politus (L.) were collected at rabbit carcasses in deciduous forests near Ochsenfurt, Bavaria (Peschke et al., 1987). Aleochara (Euryodma) brevipennis (Grav.) originates from the "Zeubelrieder Moor" (Ochsenfurt) and the Bodensee (fish bait). The species co-occurs with A. curtula on carcasses in middle Europe (Peschke and Fuldner, 1977). A. peschkei (Likovsky, 1983) was collected from fish carcasses from the banks of the fiver Comoe (Ivory Coast, West Africa). These Aleochara species were continuously reared in the laboratory according to Fuldner (1968) and Pesehke (1978, 1987) with puparia of Calliphora erythrocephala (Meig.) or Lucilia sericata (Meig.) serving as hosts for the parasitoid larvae (Peschke and Fuldner, 1977). Small beetles were obtained by using small puparia as hosts. Males were kept in groups of 10 in plastic boxes of 10 • 10 x 7 cm. Individual beetles and single pairs were kept in boxes of 5.5 x 3.5 x 1.5 cm. Three times a week, the beetles were transferred to freshly prepared boxes with moist filter paper and cut third-instar Calliphora maggots as food. Sexually isolated and well-fed beetles of both sexes at an age of 3 weeks were termed "standard laboratory beetles." The normal temperature and light regime were 22~ and 16:8 hr light-dark. Starved males were kept individually in order to avoid cannibalism. Chemical Methods Extracts were prepared according to Peschke and Metzler (1986). Thirty to 100 beetles were killed by freezing and extracted in 200 ml methylene chloride in a Soxhlet apparatus for 24 hr. The hydrocarbon fraction was purified on a silica gel column. Gas chromatography was also conducted according to Peschke and Metzler (1986) on a Varian 3700 instrument with a 30-m DB-1701 capillary column (split 1 : 10, 1 ml He/min, 60~ to 300~ at 3~ flame ionization detector). Quantitative measurements were carried out with a Kontron-Anacomp 220 computer system. The concentrations of the two pheromone alkenes were determined by using the corresponding n-alkanes as internal standards of constant titer and then related to the pheromone content of standard laboratory females [1 female equivalent (FE) (Z)-7-heneicosene = 2 Izg, 1 FE (Z)-7-tricosene = 12 Izg; Peschke and Metzler, 1986]. Both alkenes were synthesized according to Peschke and Metzler (1986). Some samples were also obtained from Dr. L.L. Jackson (Bozeman, Montana). For comparison of hydrocarbon patterns, we calculated the equivalent chain length for methyl-branched alkanes and plot-
1996
PESCHKE
ted the cumulative distribution functions of compounds versus increasing chain length (Hadley, 1977; Toolson and Hadley, 1977).
Behavioral Responses At a distance of about 2.2 mm from the female, the male bends his mobile abdomen over his head and protrudes the genitalia with the tong-shaped parameres (grasping response; Peschke, 1978). Male aggression is characterized by pushing the opponent with head and mandibles and by drumming with the mobile abdomen on the other individual. Females that repulse the male grasping response do so by oscillating their mobile abdomen thereby avoiding the fixation of the mate genitalia. In neutral encounters of two individuals, no conspicuous behavior could be observed (Peschke, 1987).
Bioassays The sexual response of A. curtula males to the purified hydrocarbon fractions of beetles of various physiological status was tested in the model bioassay (Peschke, 1978). Soxhlet-extracted odorless beetles were glued to the tips of glass needles and treated with n-pentane solutions of 0.01 ml containing one beetle equivalent. The number of standard tester males responding to the model with the grasping response was specified in percentiles and statistically evaluated by the X2 test or the exact test of Fisher (Sachs, 1984). Confidence limits were calculated for the 95 % level. Beetles of both sexes and various physiological status were observed as single pairs in plastic boxes of 5.5 • 3.5 • 1.5 cm for a period of 30 rain. The individuals were marked by dots of enamel paint on pronotum and both elytra. We recorded whether a certain behavioral response was seen at least once during the observation period, or we registered all encounters of beetles and specified the percentage in which a certain behavior was shown. Living beetles were treated with extracts or synthetic chemicals: 0.01 ml pentane solutions with one beetle equivalent concentration were applied to the pronotum of a chilled individual (6~ As a control, beetles were treated with the pure solvent. One hour after contamination, the beetles were brought together and the pair was observed for 30 min as usual, Antennal segments or palps of males 7 days old were cut off using pincers which were sharpened like scissors. Scapus and pedicellus were counted as segments l and 2, the flagellar segments as 3-11. After a further week, males of normal agility were observed individually together with single females (30 min). For observation of interspecific reactions of beetles under natural conditions, we used an arena of 50 • 50 cm, in the center of which a small piece of carrion was placed (beef liver with Calliphora maggots, covered by a dish of
CUTICULAR HYDROCARBONS REGULATE ROVE BEETLE BEHAVIOR
1997
red glass; for details see Peschke, 1987). Individually marked A. curtula males and females (five each) were put together with five males and five females of another species (A. brevipennis, A. peschkei, and Philonthus politus). The beetles were observed at the carcass with a red-sensitive camera in a natural rhythm of white and dark red light (18:6 hr light-dark). The behavioral events at the carcass were recorded for 3 rain every 60 rain at normal speed over a period of 24 hr. For each encounter of two beetles, we recorded whether aggression, grasping, or neutral behavior was exhibited by A. curtula males. RESULTS Sex Pheromone Content in Beetles of Various Sex and Physiological Status In former investigations (Peschke, 1985, 1987), all bioassays for testing pheromone contents had been conducted with entire beetles freshly killed by freezing. In the present experiments, sexual responses were also elicited by the purified total hydrocarbon fraction from extracts of females and males of various physiological status (model bioassay; Figure 1). The cuticular hydrocarbon fraction from females collected in the field released the male sexual response at the same high rate as did extracts from standard laboratory females, from females just after copulation, and from those of small body size (Figure 1). Freshly emerged females yielded a somewhat lower grasping rate. The grasping response rate was drastically reduced by exposing the females to elevated temperatures (14 days at 27~ High behavioral release rates were also obtained with extracts from males captured in the field. The hydrocarbon fraction from young, starved, or multiply mated laboratory males also elicited homosexual grasping responses (Figure 1). On the other hand, hydrocarbons from sexually isolated, well-fed males reared in the laboratory did not trigger homosexual grasping responses, regardless of temperature (22~ or 27~ and body size (large standard beetles: breadth of pronotum 2.0 _+ 0.02 ram, small beetles: 1.4 _+ 0.03 mm). The quantitative determination of components of the hydrocarbon fractions used in the former bioassay gave a good correlation of grasping rates with the contents of (Z)-7-tricosene (Figure 1). Parallel to the determination of amounts (in micrograms), we preferred the scaling in terms of female equivalents in order to obtain a more lucid comparison of pheromone contents of mimicking individuals with that of standard laboratory females (defined as 1 FE). Females of various physiological status, apart from young beetles, had a constantly high tricosene content. In females kept at elevated temperatures, only trace amounts of this compound could be detected. Standard laboratory males had a concentration of 10 -3 FE tricosene irrespective of their body size and the temperature at which they had been reared. Young, starved, and multiply mated males, however, yielded tricosene contents ranging from 0.08 to 1 FE.
1998
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FiG. 1. Grasping response of A. curtula males towards models contaminated with one beetle equivalent of the total cuticular hydrocarbon fraction from extracts of males (triangles) and females (circles) of various physiological conditions: 1: laboratory standard females (sexually isolated, well fed, 22~ f: field-collected beetles; y: freshly emerged beetles; st: mature beetles starved for a period of 10 days; c: one copulation at the day of extraction; 27: beetles kept at elevated temperatures (27~ sm: beetles reared on a small host. Thirty to 100 beetles were used per extraction; at least 100 males were tested for grasping in five replicates per sample. The grasping rate is plotted against the concentrations of the two pheromone compounds (Z)-7-heneicosene (open symbols) and (Z)-tricosene (solid symbols) in the hydrocarbon fraction as determined by quantitative gas chromatography. The concentration is scaled in terms of female equivalents as related to the content of laboratory standard females (= 1 FE). Because the combined action of compounds was tested in the bioassay, one value of grasping rate refers to two values of concentration of both pheromone compounds, which are linked by a dotted line. The grasping rate is correlated with the concentration o f (Z)-7-heneicosene in a similar way (Figure 1), except in the cases of field-collected or starved males and of young females, which release male sexual responses but have a low or even undetectable titer o f the C21-alkene. In these cases, the tricosene alone seems to be responsible for the release o f the homosexual grasping response. Heneicosene could also not be detected in small males and males kept at 27~
1999
CUTICULAR HYDROCARBONS REGULATE ROVE BEETLE BEHAVIOR
Modulation of Male Aggression by Female Sex Pheromone In the experiments described above, we demonstrated a correlation of homosexual responses with the production of female sex pheromone components by males. In the following experiments, the sex-specific chemical information was manipulated in order to investigate the correlation of female pheromone production with the release of male aggressive behavior. Temperature Experiment. Males and females kept at 22~ or 27~ were observed as single pairs in various combinations (Table 1). As in the experiments with extracts, females kept at 27~ did not release male grasping at any encounter. On the other hand, the reduction of pheromone titer in females kept at elevated temperatures provoked a high aggression rate of males towards these females. The behavioral sequence was not different from that observed in intermale combats. Contamination of Males with Synthetic Female Pheromone Mixture. A reduction of the release of aggression was demonstrated in observations (30 min) of pairs of males, to one of which the hydrocarbon fraction of a female surface washing had been applied; to the other, the pure solvent (Table 2). The male bearing the female pheromone attacked the other male in a high percentage of encounters, whereas the male without the pheromone only occasionally behaved aggressively towards the treated male. The treated male, however, released a high rate of homosexual graspings. The same tendencies were observed when one male was contaminated with 1 FE of the synthetic female sex pheromone (Table 2). The rate of aggression is drastically reduced towards the treated male; however, more neutral encounters than homosexual grasping responses were observed in comparison to experiments with the total female hydrocarbon fraction. Amputation of Mate Antennal Segments. Males with their antennae amputated to various degrees were observed together with females during a 30min period. The number of males exhibiting the grasping response towards TABLE 1. AGGRESSION AND GRASPING RESPONSES DURING 30-MIN OBSERVATIONS OF SINGLE PAIRS OF Z. curtula WITH MALES AND FEMALES KEPT AT DIFFERENT TEMPERATURES
Response of males kept at
Towards females kept at
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(Npalrs)
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22~ 27~ 22~ 27~
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2000
PESCHKE
TABLE2. AGGRESSIONAND HOMOSEXUALGRASPING RESPONSES DURING 30-MIN OBSERVATIONS OF TWO A. CUFtula MALES, ONE CONTAMINATEDWITH HYDROCARBON FRACTION OF FEMALE EXTRACT (1 FE) OR SYNTHETIC FEMALE SEX PHEROMONE (1 FE: 12/~g (Z)-7-TRICOSENE + 2 /~g (Z)-7-HENEICOSENE),THE OTHERMALE (CONTROL) TREATED WITH SOLVENT (0.01 ml r/-PENTANE) Response (%) Contamination with Female hydrocarbon fraction
Synthetic female sex pheromone mixture
Response of control males towards contaminatedmales contaminated males towards control males controlmales towards contaminated males contaminatedmales towards control males
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~P < 0.001; Xz test: comparison to the preceding line. females was not reduced as long as four antennal segments were left (Figure 2). The next step of amputation (three segments left), however, gave a significant decrease of the grasping rate, which was continued with removal of further antennal segments or palps. Coincidentally with the stepwise decrease of sexual responses, the percentage of males behaving aggressively towards females increased, with a peak rate obtained when only the scapus was exempted from amputation. The rate of aggression was then decreased with further steps of removal of antennal segments and palps.
Releasers of Aggression Sexual behavior as well as male aggression can be manipulated by the female sex pheromone titer. However, the simple absence of the female sex pheromone may be a signal for males to fight against another individual, or species-specific signals that may be other cuticular hydrocarbons may release male aggression. While in the former experiments the concentration of pheromone alkenes has been shown to vary considerably between the sexes, the saturated hydrocarbons have a very constant distribution (Peschke, 1985; Peschke and Metzler, t986). The cumulative frequency function of the equivalent chain length of saturated hydrocarbon components also shows a very similar pattern for both sexes of A. curtula (Figure 3). In contrast, cuticular hydrocarbons of male A. brevipennis or A. peschkei have a distribution very different from that
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amputation
FIG. 2. Behavioral responses (grasping, aggression) of A. curtula males (various steps of symmetrical amputation of antennal segments and pal~) to standard females during a 30-rain observation (percent pairs showing the response at least once). Steps of amputation: A: antenna (in parentheses number of segments exempted from amputation, A11: control), Mx: maxillar palps; Lb: labial palps. Vertical bars: 95% confidence limits; n: number of pairs).
of A. curtula. Most noteworthy is the shift to higher carbon numbers and the total absence of C 21 and C23 hydrocarbons. The identification of components of A. brevipennis and A. peschkei by mass spectrometry with the original gas chromatograms will be published elsewhere (Peschke and Metzler, in preparation). A. eurtula males and females were put together with individuals of other species in the video observation setup. A. brevipennis released a few grasping responses of A. curtula males (Table 3) which might be due to unsaturated C27 hydrocarbons. Aggression of A. curtula males against A. brevipennis individuals of both sexes was observed at an intermediate level. A. peschkei did not release interspecific grasping or aggressive responses of A. curtula males. Individuals of both sexes of Philonthus politus, a distantly related species from another subfamily of rove beetles (Staphylininae instead of Aleocharinae), also failed to provoke either aggression or sexual responses of A. curtula males (Table 3). In conclusion, male aggression of A. curtula seems to be released by species-specific chemical cues, probably involving the saturated hydrocarbons of the cuticle. This hypothesis is supported by experiments with A. peschkei males contaminated with the hydrocarbon fraction of male A. curtula extracts (Table
0
10
20
30
40
50
60
f
20
,
,
21
/
,
22
2'3
2'4
2's equivalent
2'8 length
cfd'
2'7
chain
~'6
r,u,o
~
W-'-
3b
3~
~2
A. brevipennis cfcf
~
A.peschkei c~cf
FIG. 3. Cumulative distribution functions of cuticular hydrocarbons of various equivalent chain lengths of carrion-inhabiting Aleochara species (A. curtula males and females; males of A, brevipennis and A. peschkei).
,,.)
E -'1
.D
"6
>
i._
~3r
C:
(D
70
80
90
100
~v
to
CUTICULAR HYDROCARBONS REGULATE ROVE BEETLE BEHAVIOR
2003
TABLE 3. BEHAVIORAL RESPONSES OF A. curtula MALES TO MALES AND FEMALES OF OTHER ROVE BEETLE SPECIES AT LABORATORY-SIMULATED CARCASS (VIDEO
OBSERVATION). Response of males (%)~
Nencoua~er~ Response towards
Aggression
Neutral
Grasping
(Npa~,~)
0"**
1206 (15) 437 (15)
A. curtula Males
95
Females
15"**
66
19
38*** 34***
54 54
8 NS 12"*
13 (5) 143 (5)
6*** 0"** 1"**
94 100 99
0"** 0* 0"**
145 (5) 23 (5) 71 (5)
5
A. brevipennis Males Females
A. peschkei Males Females
Philonthus politus
~P < 0.05, **P < 0.01, ***P < 0.001; X2 test: comparison to A. curtula males (aggression) or to A. curtula females (grasping).
4). While A. curtula males did not behave aggressively towards A. peschkei individuals contaminated with the solvent in the 30-rain observation bioassay, they exhibited a conspicuous fighting behavior towards A. pesehkei specimens treated with male A. curtula hydrocarbons. Female Repulsion Behavior Males which release homosexual responses of other males may show the grasping response towards females. However, in comparison to nonmimicking TABLE 4-. AGGRESSION OF A. curtula MALES TOWARDS A. peschkei MALES CONTAMINATED WITH HYDROCARBON FRACTION OF EXTRACTS OF A. curtula MALES (1 M E , 0.01 ml) OR SOLVENT (n-PENTANE) DURING 30-M1N OBSERVATIONS OF S1NGEE PAIRS
Response of A. curtula males towards
Response (%)
Nencounters Aggression
Neutral
Grasping
(Npa~T~)
0 80 ~
100 20
0 0
19 (4) 45 (5)
A. peschkei males Control Contaminated
~P < 0.001; X2 test, comparison to control.
2004
PESCHKE
TABLE 5. GRASPING OF A. curtula MALES CONTAMINATED WITH SYNTHETIC FEMALE SEX PHEROMONE (1 FE: 12 # g (Z)-7-TRIcOSENE + 2 # g (Z)-7-HENEICOSENE) AND REPULSION OF GRASPING RESPONSES BY FEMALES DURING 30-MIN OBSERVATIONS OF SINGLE PAIRS
Males Contaminated with synthetic female pheromone Control
Grasping of males (%)
Repulse by females (%)
N, ..... ~r~ (Np,irs)
99 NS 94
80 a 32
285 (6) 83 (15)
~P < 0.001; 2 test: comparison to control.
males, they are more often repulsed by the female, who drums with her abdomen and thus avoids the fixation of male genitalia (Peschke, 1987). In order to investigate the releasers of female repulsion behavior, males were contaminated with 1 FE of the synthetic mixture of the female pheromone (Table 5). The treated males did not show an alteration in their own sexual response to females as measured by the grasping response rate during a 30-rain observation period. On the other hand, the repulsion behavior of females towards such treated males is significantly increased. DISCUSSION
Female mimicry in vertebrates using chemical (Mason and Crews, 1985) or behavioral and morphological cues (for review see Weldon and Burghardt, 1984) has been described for many species. Stealing sneaky copulations, avoidance of intermale aggression, and access to other males' territories are discussed as the main benefits of mimicking males. Behavioral transvestism in insects has only been described for scorpion flies where males behave like females in order to steal the nuptial gifts of other males (ThornhiU, 1979). Certain physiological types of males of the rove beetle, Aleochara curtula, mimic their females chemically in order to avoid intermale aggression. In this way they get access to a carcass and are able to feed on blow fly maggots in order to replenish their energy reserves for a forthcoming reproductive cycle (Peschke, 1987). On the other hand, females prefer males without the female pheromone. The adaptive significance of female coyness towards mimicking males seems to be the choice of an optimal mate: immature, starved, or multiply mating males, which all produce the female sex pheromone, need access to the
CUTICULAR HYDROCARBONS REGULATE ROVE BEETLE BEHAVIOR
2005
food resource; they transfer only small spermatophores and fertilize less eggs. On the other hand, physiologically competent males without the female sex pheromone have succeeded in numerous aggressive interactions with other males (Peschke, 1987). (Z)-Heneicosene and (Z)-7-tricosene have previously been isolated and identified from mature A. curtula females as the main sex pheromone components (Peschke and Metzler, 1986). The pheromone molecules are components of the epicuticular waxes and are spread over the entire surface of the beetles (Peschke, 1978, 1986). Male antagonistic pheromones, which might be involved in the regulation of sex specificity of the pheromone information, could not be detected (Peschke, 1986). In the present work the pheromone alkenes were also found in the cuticular hydrocarbons of those males which release homosexual responses, e.g., young, starved, or multiply mated beetles. All these physiological types of males are mixed up in field collections (Peschke et al., 1987), where they also bear the female sex pheromones. Small males, which perform an alternative mating tactic as satellites in the surroundings of carrion (Peschke, in preparation), do not produce the female sex pheromone for protection. The contents of the two pheromone components in various physiological types and both sexes of A. curtula range over three powers of 10 and are correlated with the release of male sexual responses. In most samples, both compounds contribute to the releasing effect of the total hydrocarbon fraction. In some cases, tricosene alone seems to be responsible for the release of sexual responses. In tests of individual synthetic substances, tricosene had its optimum at about 0.1 FE, whereas heneicosene released more sexual responses at the higher concentration of 1 FE (Peschke and Metzler, 1986). It is therefore difficult to quantify the contribution of the individual pheromone component to the releasing efficiency of the total hydrocarbon mixture of each physiological type. The combination of both compounds had no synergistic, only an additive, effect; the admixture of (Z)-9-heneicosene or (Z)-9-tricosene, which are produced together with the respective 7-isomers but have only little releasing efficiency (Peschke and Metzler, 1986), did not produce an antagonistic action (Peschke, in preparation). Differences in the release of grasping rates towards synthetic compounds or towards the total hydrocarbon fraction containing the same amounts of pheromone components may be due to retardation of evaporation by the accompanying saturated hydrocarbons in the latter ease (Peschke, 1986). This effect may also be responsible for different responses towards contaminated living males. Homosexual grasping is released at a lower rate after contamination with the synthetic pheromone mixture in comparison to males treated with the total hydrocarbon fraction of females. However, the aggression rate is reduced to the same extent in both cases. Individual (Z)-7-heneicosene and (Z)-7-tricosene
2006
PESCHKE
both contribute to the reduction of intermale aggression (Peschke, in preparation). The modulatory effect of the female sex pheromone on the release of male aggression was also demonstrated by manipulation of the chemical information output of the female. Females which have been exposed to elevated temperatures show a significantly reduced content of cuticular alkenes in comparison to that of saturated compounds. This may be due to differential evaporation of saturated and unsaturated compounds. However, it cannot be excluded that the beetles actively alter the pattern of hydrocarbon synthesis at different temperatures (see Hadley, 1977; Toolson and Hadley, 1977). Males behave aggressively towards these females in the same way as towards other males. A further indication of the modulatory effect of pheromone alkenes is the gradual increase of aggression accompanied by a decrease of sexual recognition with successive amputation of male antennal segments. In a preliminary investigation of the antennal morphology of A. curtula (Ungelenk and Altner, unpublished), no sex specific sensilla or striking quantitative difference in the distribution and number of various types could be found. The pattern of densely packed sensilla seems to be repeated in segments 5-11, whereas scapus, pedicellus, and the two proximal flagellar segments bear only a few sensilla. It seems likely that the sex pheromone information is perceived by numerous receptor organs sequentially distributed on the distal antennal segments. Removal of them gradually diminishes the input of sex pheromone information, whereas information on aggression-releasing chemicals, probably the saturated hydrocarbons, still can be perceived. Further steps of amputation reduce this input too. However, electrophysiological investigations of pheromone perception in A. curtula are urgently needed. Females of A. curtula are apparently able to perceive the female sex pheromone too. However, the female behavioral response elicited by these chemicals, the release of aggressive repulsion of male mating attempts, is the reverse of the male reaction where aggression is reduced. The releasers of intermale aggression of A. curtula may comprise components of the saturated hydrocarbon mixture that are present in both sexes. The shift to longer carbon chains in related species reduces aggression of A. curtula males; on the other hand, contamination of males of the other species with hydrocarbons of A. curtula males, which do not contain female pheromones, provoked intermale combats. At present, synthetic methyl-branched alkanes are not available, and therefore it is not possible to determine yet whether individual n-alkanes, methyl-branched alkanes, or the complex pattern of compounds of variable chain lengths serve as the releasers of male aggression. Saturated hydrocarbons modulate the information of alkenes acting as female sex pheromones in several muscid flies (e.g., Rogoff et al., 1980; Sonnet et al., 1975, 1977; Uebel et al., 1975a, b, 1976), or represent the major female pher-
CUTICULAR HYDROCARBONS REGULATE ROVE BEETLE BEHAVIOR
2007
o m o n e s , for e x a m p l e , in t s e t s e flies ( e . g . , C a r l s o n et al., 1978; H u y t o n et al., 1980a, b) o r b u t t e r f l i e s ( G r u l a et al., 1980). Acknowledgments--I am very grateful to Mrs. C. Gantert for technical assistance. Thanks are due to Prof. Dr. L.L. Jackson and Prof. Dr. M. Metzler for synthetic pheromone compounds. I wish to thank Dr. A.E. Jtirss for reading the English manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (Pe 231/3 and 4).
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