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Multiple web-borne pheromones in a spider Frontinella pyramitela (Araneae: Linyphiidae)
Anim. Behav., 1986, 34, 748-753
Multiple web-borne pheromones in a spider Frontinella pyramitela (Araneae: Linyphiidae) R O B E R T B. S U T E R & A N D R E A J. H I R S C H E I M E R
Biology Department, Vassar College, Poughkeepsie, New York 12601, U.S.A.
Abstract. The bowl-and-doily spider, Frontinella pyramitela, is a common inhabitant of low vegetation throughout most of temperate North America. All instars build concave-upward, bowl-shaped, nonviscid webs supported above and below by meshworks of silk. Previous studies of this species have revealed that chemical(s) on the silk of adult females elicit both courtship behaviour and positive geotaxis from adult males that contact the silk. This study demonstrates (1) that two different contact pheromones are responsible for the dual action of the silk of adult females and (2) that the webs of different age and sex classes of bowl-and-doily spiders (including the webs of adult males) contain functionally different mixtures of the two pheromones.
The growing literature on spider pheromones (Tietjen & Rovner 1982) suggests that the use of chemical signals by web-building spiders may be very common. In at least two families, pheromones that are not associated with the web itself have been demonstrated. Among social funnel-weavers (Agelenidae), Krafft (1965, 1969, 1975) showed that Agelena consociata exchange nutrients and possibly a colony pheromone during feeding, and that aggression among colony members is reduced by the presence of a cuticular contact pheromone. Among orb-weavers (Araneidae), female Crytophora cicatrosa (Blanke 1975a) and Argiope aurantia (Enders 1975) produce volatile, air-borne pheromones that attract conspecific males even before contact with females' webs. In a social cobweb weaver (Achaearanea Wau, Theridiidae), Lubin (personal communication) has observed male sexual attention to the moulted skin of a female, behaviour that suggests the presence of a cuticular contact sex pheromone. Web-borne pheromones have also been demonstrated in several spider taxa. In one araneid (Blanke 1975b) and in one diplurid (Hickman 1964), web-borne pheromones act as attractants. Among the Amaurobiidae, Agelenidae (Krafft 1978) and Dictynidae (Jackson 1978), web-borne pheromones release courtship behaviour in males. In contrast, Jackson (1978) has pointed out that for social web-builders, silk-borne chemicals may play little or no role in intraspecific communication. The bowl-and-doily spider, Frontinella pyramitela (Araneae: Linyphiidae), builds a relatively complex sheet web, the chemical constituents of
which alter the behaviour of conspecifics contacting the silk (Surer & Renkes 1982), A male, upon contact with the web of an adult female, quickly begins to court even in the absence of the female. At the same time, the male's orientation becomes positively geotactic even in the absence of webstructural cues to the direction of gravity. These responses to contact with female silk are obliterated (courtship) or reversed (geotaxis) when males contact a web that has been washed in methanol or made by a very young (less than three moults from adulthood) conspecific. Suter & Renkes (1982) speculated that the dual action of the web-borne pheromone(s) could arise from the presence of two web-borne chemicals, each with a separate function, or from the presence of a single chemical, the effects of which propagate along two central pathways in the nervous system of the recipient male. In this paper we demonstrate (1) that in F. pyramitela, immatures, penultimate males, penultimate females and adults of both sexes build webs that bear chemical signals, and (2) that the dual function of the pheromone produced by adult females is attributable to two distinct chemical components. M A T E R I A L S AND M E T H O D S
Spiders and Webs Bowl-and-doily spiders are common throughout much of temperate North America. The non-viscid web, built on low vegetation, consists of a bowlshaped (concave-upward) horizontal sheet, an
748
Suter & Hirscheimer: Web-borne pheromones underlying flat sheet, and a meshwork of silk above the bowl. Flying insects hit or land in the upper meshwork, fall or are shaken to the bowl, and are captured there by the spider who resides on the underside of the bowl. In addition to predation, courtship (Suter & Renkes 1984) and agonistic interactions (Suter & Keiley 1984) also occur on the web. Spiders used in this study were collected from webs in Poughkeepsie and Millbrook, New York, during May and June, 1984. In the laboratory, each spider was placed on a glass hexapod in a 3-8-1itre plastic aquarium. A layer of moist sand in the bottom of the aquarium kept the relative humidity around the spider at nearly 100%. Most spiders built webs within 24 h of capture. Adult males that did not build webs within 48 h of capture, ceased to build webs during captivity, or were never given the opportunity to build webs in captivity, were kept above a layer of moist sand in small glass vials. All spiders were fed a diet of vinegar flies (Drosophila
melanogaster). Adults of both sexes in this species are easily distinguished (visually) from each other and from juveniles. We could not, however, reliably distinguish between the sexes of juvenile instars nor between the late juvenile instars of a particular sex. Daily inspection for shed skins and careful recordkeeping permitted post hoc determination of the sex and age (instar) of each spider. We removed spiders from newly constructed, intact webs by gently blowing on the spider or, with particularly tenacious spiders, by driving them off the webs with a water gun. Both techniques left the web undamaged. Each web was labelled by date and the identity code of the spider that had constructed it. We stored the webs in dust-free cabinets before assaying. Webs were tested (see below), usually within 5 days of construction, although such promptness was probably not necessary; adult female webs stored for 30 days still elicit courtship behaviour in males (Suter & Renkes 1982). Webs constructed by adult males, because they were of particular interest, were first tested in their intact condition. Subsequently, these webs were washed in methanol (Suter & Renkes 1982) and tested again.
Behavioural Assays of Webs We mounted each glass hexapod containing a
749
test web in a clamp that held the web perpendicular to its usual orientation with respect to gravity; that is, the usually horizontal bowl was, after mounting, in a vertical plane (Suter & Renkes 1982). This new orientation of the web placed web-structural cues (as to the direction of gravity) perpendicular to direct gravitational cues, and permitted experimental determination of spider behaviour based only on gravity. For each assay, a male spider, suspended by his dragline, was swung into the centre of the top (now front) of the test web. The male's mean direction of movement (as viewed from the structural top of the web) from that initial position to some point on the periphery of the web was recorded as his orientation with respect to gravity (down = 0~ horizontal = 90~ Using a tally counter, we recorded the number of abdomen flexions during the 2 rain beginning when the male started locomotion on the web. (The male produces several distinct vibrations during courtship, one of which, the abdomen flexion, is generated when he flexes his abdomen sharply ventrad. See Suter & Renkes 1982 and 1984.) We used non-parametric statistical tests in all data analyses. During the course of this study, we used 38 males, each randomly assigned to webs in 279 bioassays of web quality. Because most of the males were used a number of times, we were concerned about (1) order effects within the data from each male and (2) reliability of male responses (i.e. did different webs of a particular class receive similar scores when tested with a single male?). The two concerns were evaluated in separate experiments in which (1) five males were tested serially on five adult females' webs and (2) four males were tested four times on one web and a fifth time on a different web of the same class. Again we used nonparametric tests in analyses of the data.
RESULTS Tests or order effects in the scores of individual males revealed that male abdomen flexion scores did not change systematically from early to late runs when tested repeatedly on webs of the same class (experiment 1, Kendall coefficient of concordance, W= 0.058, P > 0.05). Similarly, the mean of four scores from a single male on one web was a very good predictor of the score that male would generate on a different web of the same class
Animal Behaviour, 34, 3
750 180
z.
i OO
o
( N = 78) and females ( N = 67), and by adult males ( N = 68) and females ( N = 34). A Kruskal-Wallis one-way A N O V A on these data indicates that class differences account for significant amounts of the variation with respect to both orientation and abdomen flexion scores. We made pairwise comparisons between classes using M a n n - W h i t n e y U-tests. These showed that juvenile males' webs did not differ from juvenile females' webs in either orientation or abdomen flexion scores. Therefore, for subsequent tests, the data from these two classes were grouped ( N = 32).
9
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L_.....-'.'..
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IDOl@ ---'-"
IIID@III @ ~ l l l
r.'" I
I
260
130
Abdomen
Orientation Differences
Flexions
Figure 1. Scatterplot of the results of all web bioassays where both orientation and abdomen flexion data were available (N= 279). Non-parametric ANOVAs with respect to class (i.e. age and sex) of the web-builder reveal that much of the variation is attributable to class differences (Kruskal Wallis one-way ANOVA: geotaxis, H=11.21, P<0.05; abdomen flexions, H=338.9, P<0.0001).
(experiment 2, rs = 1'000, P < 0-05) but was a poor predictor of its behaviour on webs of another class. We are confident, then, that the data reported below are not biased by the multiple use of individual males as assay animals. Figure 1 shows the results of all web assays for which we had both orientation and abdomen flexion data. These data came from six classes of webs: those constructed by juvenile males ( N = 11) and females (N=21), by penultimate adult males
The results ofpairwise testing of orientation data from the five classes of webs by the M a n n - W h i t n e y U-test indicate that webs of adult males elicited significantly higher orientation scores than the webs of either adult females ( Z = 2-662, P < 0.01) or immature spiders (Z=2.080, P < 0 . 0 5 ) . N o other differences are significant and the median orientations in all five classes of webs were downward ( < 90~ In contrast, assays of methanol-washed male webs elicited upward orientations ( m e d i a n = 150~ Washed and chemically intact male webs differed significantly by the M a n n Whitney U-test ( Z = 4 . 2 9 t , P<0.0001). Table ] presents the orientation data divided into two classes of response (upward versus downward movement). Only methanol-washed webs, whether produced by females (Suter & Renkes 1982) or males, elicited significantly upward motion from male spiders. Figure 2, showing the frequency distributions of male orientations on both male and
Table I. Geotaxis by adult males on bowl-and-doily spider webs
Male web Median direction 30~ (0~= down) Number up 21 (>90 ~) Number down 40 (<90 ~) Pt <0.01
Penultimate male Immature web web 20 ~
12.5~
Penultimate female web 17.5~
Washed male web
Washed female web*
10~
150~
137~
Female web
13
4
6
1
18
50
18
26
21
1
<0.01
<0.0005
<0.001
<0.001
<0.0001
* Data taken from Suter & Renkes 1982. t Binomial test of the null hypothesis that 'up equals down'.
9 1
< 0-02
Suter & Hirscheimer: Web-borne pheromones
~
0
0-19
80-99
Orientation
(deg.,
751
0.35
0
160-180
0-1
10-11
O'=down)
Abdomen
Figure 2. Frequency distributions of male orientations on adult male (open bars, N=61) and adult female (filled bars, N = 22) webs that were mounted perpendicular to normal (see text). These distributions show the types of differences that account for the variation in the orientation data in Fig. 1. Medians of the two distributions (male webs, 30~ female webs, 10~ are indicated by arrows. The distributions are significantly different (Mann-Whitney U-test, P < 0.01).
female webs, characterize the data t h a t m a k e up the o r i e n t a t i o n p o r t i o n o f Fig. 1.
Abdomen Flexion Differences Table II shows the results of pairwise testing o f a b d o m e n flexion data f r o m the five classes o f webs using the M a n n W h i t n e y U-test. T h e results indicate t h a t webs o f adult males elicited significantly fewer a b d o m e n flexions t h a n the webs o f a n y other class of spider. In contrast, the webs o f penultimate females elicited significantly more a b d o m e n flexions t h a n the webs of either i m m a t u r e s or penulti-
-> $ 0
Flexions
Figure 3. Frequency distributions of male abdomen flexion scores on adult male (open bars, N = 68) and adult female (filled bars, N=22) webs. These distributions show the types of differences that account for the variation in the abdomen flexion data in Fig. 1. Medians of the two distributions (male webs, 0; female webs, 19.5) are indicated by arrows. The distributions are significantly different ( M a n , W h i t n e y U-test, P < 0.0001). mate males. N o other differences are significant. M e t h a n o l - w a s h e d male webs elicited as few a b d o m e n flexions ( m e d i a n = 0 , N = 19) as chemically intact male webs ( Z = 0 . 4 4 4 , P = 0 . 6 6 ) . Figure 3, showing the frequency distributions of male a b d o m e n flexion scores o n b o t h male a n d female webs, characterizes the d a t a t h a t m a k e u p the a b d o m e n flexion p o r t i o n of Fig. 1.
DISCUSSION
Demonstration of Multiple Pheromones Suter & Renkes (1982) implied that, a m o n g
Table II. Results of pairwise comparisons (by class) of abdomen flexion scores
Male web Median Comparisons (Mann Whitney U-test) Male web Penultimate male web Immature web Penultimate female web
0
Penultimate Penultimate male Immature female web web web 6.5 < 0.0002
8.0 < 0.001 NS
27 < 0.0001 < 0.02 <0.05
Female web 19.5 < 0.0001 NS NS NS
752
Animal Behaviour, 34, 3
bowl-and-doily spiders, there are two chemically different classes of webs: those that elicit both courtship behaviour (abdomen flexions) and positive geotaxis, and those that elicit only negative geotaxis. In the former group are the webs of adult females. In the latter group are the webs of early instar (more than three moults from adulthood, in contrast to the older but still immature spiders tested in the present study) F. pyramitela and methanol-washed webs of adult females. With only two chemically different classes of webs, it was possible to hypothesize the existence of only a single pheromone: when the pheromone was present, one set of behaviours (e.g. abdomen flexions and positive geotaxis) was stimulated; when no pheromone was present, the other set of behaviours (e.g. negative geotaxis and no courtship) was stimulated. Data presented here demonstrate that there is a third chemically distinct class of webs: those which elicit positive geotaxis but little or no courtship behaviour. These are the webs constructed by adult males (Figs 2, 3). The presence of this third class of webs eliminates the possibility that the web-borne pheromone on female webs has only one active chemical component but two roles (see Introduction). Three chemically different classes of webs cannot occur in the absence of at least two chemicals which have different functional roles. Our alternative hypothesis, that separate chemical components elicit the separate behavioural responses (geotaxis and courtship behaviours), is strongly supported by the data presented here. Webs constructed by adult males are qualitatively different from those constructed by all other classes of spiders tested; male webs elicit few or no abdomen flexions while the webs of adult females and of both sexes of penultimate adults and juveniles elicit many abdomen flexions (Table II, Fig. 3). Washing male-constructed webs in methanol does not alter their ability to elicit abdomen flexions (Table II), but similar washing of webs constructed by adult females renders those webs indistinguishable from adult male webs with respect to abdomen flexions (Table II; Suter & Renkes 1982). Thus, some chemical that is extracted or denatured by methanol elicits abdomen flexions when males contact it on a web. Neither adult males nor very young bowl-anddoily spiders (Suter & Renkes 1982) deposit that chemical on their webs. A second chemical, one that is also extracted or denatured by methanol,
elicits positive geotaxis from males. That chemical is found on all chemically intact webs tested in this study (Table I) but is absent from the webs of very young spiders (Suter & Renkes 1982).
Functions of a Dual System
A complete analysis of the courtship of F.
pyramitela (Suter & Renkes 1984) clarified the dual role of the pheromone(s) that the adult female deposits on her web. Not only does the chemical stimulate oriented searching in a way that minimizes the time between web contact and location of the female (because the bowl where the female resides is below most of the points of web-substrate contact, points where a male is apt to make first contact with the web), it also stimulates the one vibration-producing behaviour that is clearly involved in assuaging female predatory behaviour (Suter & Renkes 1984). In agonistic interactions between adult males on female webs, the abdomenflexion-stimulatingpheromone is also important in eliciting the initial vibratory behaviours of the intruding male, and these behaviours are probably used in the transmission of information about the resource-holding potential (i.e. the mass) of the intruder (Suter & Keiley 1984). Our demonstration that at least two pheromones are involved and that neither is a feature of all F. pyramitela webs (male webs lack one component, webs of very young spiders lack both) suggests that the known functions of the pheromones are beneficial only to certain age and sex classes of bowl-anddoily spiders. The roles of the two pheromones in the lives of juvenile bowl-and-doily spiders remain obscure. Because adult males attend penultimate females for many hours, on occasion, and mating of attended females occurs within minutes of the completion of the final moult, we speculate that penultimate females may increase the probability of early insemination by including both pheromones on webs they build. But we can see no such function for the pheromones of other juvenile classes of either sex. Further observation and experimentation may elucidate the other functions of these chemicals. ACKNOWLEDGMENTS We are indebted to two anonymous reviewers for their helpful comments on this manuscript. This
Surer & Hirscheimer: Web-borne pheromones study was supported in part by the Financial Aid Office and the Beadle F u n d o f Vassar College.
REFERENCES Blanke, R. 1975a. Untersuchungen zum Sexualverhalten von Cyrtophora cicatrosa (Stoliczka) (Araneae, Araneidae). Z. Tierpsychol., 37, 62-74. Blanke, R. 1975b. Das Sexualverhalten der Gattung Cyrtophora als Hilfsmittel ffir Phylogenetische Aussagen. Proc. Int. Arachnol. Congr., 6, 116-k119. Enders, F. 1975. Airborne pheromone probable in orb web spider Argiope aurantia (Araneidae). Br. Arachnol. Soc. News, 13, 5-6. Hickman, V. V. 1964. On Atrax infensus sp. n. (Araneida: Dipluridae), its habits and a method of trapping the males. Pap. Proc. R. Soc. Tasm., 98, 107 112. Jackson, R. R. 1978. Male mating strategies of dictynid spiders with differing types of social organization. Symp. Zool. Soc. Lond., 42, 79-88. Krafft, B. 1965. Sur une possibilit6 d'6changes de substance entre les individus chez l'araign6e sociale Agelena consoeiata Denis. C. R. Acad. Sci. Paris, 260, 5376,5378.
753
Krafft, B. 1969. Various aspects of the biology of Agelena consociata Denis when bred in the laboratory. Am. Zool., 9, 201-210. Krafft, B. 1975. La tol6rance r6ciproque chez l'araign6e sociale Agelena consociata Denis. Proc. Int. Arachnol. Congr., 6, 107 112. Krafft, B. 1978. The recording of vibratory signals performed by spiders during courtship. Syrup. Zool. Soc. Lond., 42, 59-67. Surer, R. B. & Kelley, M. 1984. Agonistic interactions between male Frontinella pyramitela (Araneae, Linyphiidae), Behav. Ecol. Sociobiol., 15, 1--7. Suter, R. B. & Renkes, G. 1982. Linyphiid spider courtship: releaser and attractant functions of a contact sex pheromone. Anita. Behav., 30, 714718. Surer, R. B. & Renkes, G. 1984. The courtship of Frontinella pyramitela (Araneae, Linyphiidae): patterns, vibrations and functions. J. Arachnol., 12, 37-54. Tietjen, W. J. & Rovner, J. S. 1982. Chemical communication in lycosids and other spiders. In: Chemical Communication." Mechanisms and Ecological Significance (Ed. by P. N. Witt & J. S. Rovner), pp 249 279. Princeton: Princeton University Press. (Received 19 November 1984; revised 9 April 1985; MS. number." A4422)
Multiple web-borne pheromones in a spider Frontinella pyramitela (Araneae: Linyphiidae) R O B E R T B. S U T E R & A N D R E A J. H I R S C H E I M E R
Biology Department, Vassar College, Poughkeepsie, New York 12601, U.S.A.
Abstract. The bowl-and-doily spider, Frontinella pyramitela, is a common inhabitant of low vegetation throughout most of temperate North America. All instars build concave-upward, bowl-shaped, nonviscid webs supported above and below by meshworks of silk. Previous studies of this species have revealed that chemical(s) on the silk of adult females elicit both courtship behaviour and positive geotaxis from adult males that contact the silk. This study demonstrates (1) that two different contact pheromones are responsible for the dual action of the silk of adult females and (2) that the webs of different age and sex classes of bowl-and-doily spiders (including the webs of adult males) contain functionally different mixtures of the two pheromones.
The growing literature on spider pheromones (Tietjen & Rovner 1982) suggests that the use of chemical signals by web-building spiders may be very common. In at least two families, pheromones that are not associated with the web itself have been demonstrated. Among social funnel-weavers (Agelenidae), Krafft (1965, 1969, 1975) showed that Agelena consociata exchange nutrients and possibly a colony pheromone during feeding, and that aggression among colony members is reduced by the presence of a cuticular contact pheromone. Among orb-weavers (Araneidae), female Crytophora cicatrosa (Blanke 1975a) and Argiope aurantia (Enders 1975) produce volatile, air-borne pheromones that attract conspecific males even before contact with females' webs. In a social cobweb weaver (Achaearanea Wau, Theridiidae), Lubin (personal communication) has observed male sexual attention to the moulted skin of a female, behaviour that suggests the presence of a cuticular contact sex pheromone. Web-borne pheromones have also been demonstrated in several spider taxa. In one araneid (Blanke 1975b) and in one diplurid (Hickman 1964), web-borne pheromones act as attractants. Among the Amaurobiidae, Agelenidae (Krafft 1978) and Dictynidae (Jackson 1978), web-borne pheromones release courtship behaviour in males. In contrast, Jackson (1978) has pointed out that for social web-builders, silk-borne chemicals may play little or no role in intraspecific communication. The bowl-and-doily spider, Frontinella pyramitela (Araneae: Linyphiidae), builds a relatively complex sheet web, the chemical constituents of
which alter the behaviour of conspecifics contacting the silk (Surer & Renkes 1982), A male, upon contact with the web of an adult female, quickly begins to court even in the absence of the female. At the same time, the male's orientation becomes positively geotactic even in the absence of webstructural cues to the direction of gravity. These responses to contact with female silk are obliterated (courtship) or reversed (geotaxis) when males contact a web that has been washed in methanol or made by a very young (less than three moults from adulthood) conspecific. Suter & Renkes (1982) speculated that the dual action of the web-borne pheromone(s) could arise from the presence of two web-borne chemicals, each with a separate function, or from the presence of a single chemical, the effects of which propagate along two central pathways in the nervous system of the recipient male. In this paper we demonstrate (1) that in F. pyramitela, immatures, penultimate males, penultimate females and adults of both sexes build webs that bear chemical signals, and (2) that the dual function of the pheromone produced by adult females is attributable to two distinct chemical components. M A T E R I A L S AND M E T H O D S
Spiders and Webs Bowl-and-doily spiders are common throughout much of temperate North America. The non-viscid web, built on low vegetation, consists of a bowlshaped (concave-upward) horizontal sheet, an
748
Suter & Hirscheimer: Web-borne pheromones underlying flat sheet, and a meshwork of silk above the bowl. Flying insects hit or land in the upper meshwork, fall or are shaken to the bowl, and are captured there by the spider who resides on the underside of the bowl. In addition to predation, courtship (Suter & Renkes 1984) and agonistic interactions (Suter & Keiley 1984) also occur on the web. Spiders used in this study were collected from webs in Poughkeepsie and Millbrook, New York, during May and June, 1984. In the laboratory, each spider was placed on a glass hexapod in a 3-8-1itre plastic aquarium. A layer of moist sand in the bottom of the aquarium kept the relative humidity around the spider at nearly 100%. Most spiders built webs within 24 h of capture. Adult males that did not build webs within 48 h of capture, ceased to build webs during captivity, or were never given the opportunity to build webs in captivity, were kept above a layer of moist sand in small glass vials. All spiders were fed a diet of vinegar flies (Drosophila
melanogaster). Adults of both sexes in this species are easily distinguished (visually) from each other and from juveniles. We could not, however, reliably distinguish between the sexes of juvenile instars nor between the late juvenile instars of a particular sex. Daily inspection for shed skins and careful recordkeeping permitted post hoc determination of the sex and age (instar) of each spider. We removed spiders from newly constructed, intact webs by gently blowing on the spider or, with particularly tenacious spiders, by driving them off the webs with a water gun. Both techniques left the web undamaged. Each web was labelled by date and the identity code of the spider that had constructed it. We stored the webs in dust-free cabinets before assaying. Webs were tested (see below), usually within 5 days of construction, although such promptness was probably not necessary; adult female webs stored for 30 days still elicit courtship behaviour in males (Suter & Renkes 1982). Webs constructed by adult males, because they were of particular interest, were first tested in their intact condition. Subsequently, these webs were washed in methanol (Suter & Renkes 1982) and tested again.
Behavioural Assays of Webs We mounted each glass hexapod containing a
749
test web in a clamp that held the web perpendicular to its usual orientation with respect to gravity; that is, the usually horizontal bowl was, after mounting, in a vertical plane (Suter & Renkes 1982). This new orientation of the web placed web-structural cues (as to the direction of gravity) perpendicular to direct gravitational cues, and permitted experimental determination of spider behaviour based only on gravity. For each assay, a male spider, suspended by his dragline, was swung into the centre of the top (now front) of the test web. The male's mean direction of movement (as viewed from the structural top of the web) from that initial position to some point on the periphery of the web was recorded as his orientation with respect to gravity (down = 0~ horizontal = 90~ Using a tally counter, we recorded the number of abdomen flexions during the 2 rain beginning when the male started locomotion on the web. (The male produces several distinct vibrations during courtship, one of which, the abdomen flexion, is generated when he flexes his abdomen sharply ventrad. See Suter & Renkes 1982 and 1984.) We used non-parametric statistical tests in all data analyses. During the course of this study, we used 38 males, each randomly assigned to webs in 279 bioassays of web quality. Because most of the males were used a number of times, we were concerned about (1) order effects within the data from each male and (2) reliability of male responses (i.e. did different webs of a particular class receive similar scores when tested with a single male?). The two concerns were evaluated in separate experiments in which (1) five males were tested serially on five adult females' webs and (2) four males were tested four times on one web and a fifth time on a different web of the same class. Again we used nonparametric tests in analyses of the data.
RESULTS Tests or order effects in the scores of individual males revealed that male abdomen flexion scores did not change systematically from early to late runs when tested repeatedly on webs of the same class (experiment 1, Kendall coefficient of concordance, W= 0.058, P > 0.05). Similarly, the mean of four scores from a single male on one web was a very good predictor of the score that male would generate on a different web of the same class
Animal Behaviour, 34, 3
750 180
z.
i OO
o
( N = 78) and females ( N = 67), and by adult males ( N = 68) and females ( N = 34). A Kruskal-Wallis one-way A N O V A on these data indicates that class differences account for significant amounts of the variation with respect to both orientation and abdomen flexion scores. We made pairwise comparisons between classes using M a n n - W h i t n e y U-tests. These showed that juvenile males' webs did not differ from juvenile females' webs in either orientation or abdomen flexion scores. Therefore, for subsequent tests, the data from these two classes were grouped ( N = 32).
9
II ~
90
.- - : :'.. 9 . W
~
:.
L_.....-'.'..
@
IDOl@ ---'-"
IIID@III @ ~ l l l
r.'" I
I
260
130
Abdomen
Orientation Differences
Flexions
Figure 1. Scatterplot of the results of all web bioassays where both orientation and abdomen flexion data were available (N= 279). Non-parametric ANOVAs with respect to class (i.e. age and sex) of the web-builder reveal that much of the variation is attributable to class differences (Kruskal Wallis one-way ANOVA: geotaxis, H=11.21, P<0.05; abdomen flexions, H=338.9, P<0.0001).
(experiment 2, rs = 1'000, P < 0-05) but was a poor predictor of its behaviour on webs of another class. We are confident, then, that the data reported below are not biased by the multiple use of individual males as assay animals. Figure 1 shows the results of all web assays for which we had both orientation and abdomen flexion data. These data came from six classes of webs: those constructed by juvenile males ( N = 11) and females (N=21), by penultimate adult males
The results ofpairwise testing of orientation data from the five classes of webs by the M a n n - W h i t n e y U-test indicate that webs of adult males elicited significantly higher orientation scores than the webs of either adult females ( Z = 2-662, P < 0.01) or immature spiders (Z=2.080, P < 0 . 0 5 ) . N o other differences are significant and the median orientations in all five classes of webs were downward ( < 90~ In contrast, assays of methanol-washed male webs elicited upward orientations ( m e d i a n = 150~ Washed and chemically intact male webs differed significantly by the M a n n Whitney U-test ( Z = 4 . 2 9 t , P<0.0001). Table ] presents the orientation data divided into two classes of response (upward versus downward movement). Only methanol-washed webs, whether produced by females (Suter & Renkes 1982) or males, elicited significantly upward motion from male spiders. Figure 2, showing the frequency distributions of male orientations on both male and
Table I. Geotaxis by adult males on bowl-and-doily spider webs
Male web Median direction 30~ (0~= down) Number up 21 (>90 ~) Number down 40 (<90 ~) Pt <0.01
Penultimate male Immature web web 20 ~
12.5~
Penultimate female web 17.5~
Washed male web
Washed female web*
10~
150~
137~
Female web
13
4
6
1
18
50
18
26
21
1
<0.01
<0.0005
<0.001
<0.001
<0.0001
* Data taken from Suter & Renkes 1982. t Binomial test of the null hypothesis that 'up equals down'.
9 1
< 0-02
Suter & Hirscheimer: Web-borne pheromones
~
0
0-19
80-99
Orientation
(deg.,
751
0.35
0
160-180
0-1
10-11
O'=down)
Abdomen
Figure 2. Frequency distributions of male orientations on adult male (open bars, N=61) and adult female (filled bars, N = 22) webs that were mounted perpendicular to normal (see text). These distributions show the types of differences that account for the variation in the orientation data in Fig. 1. Medians of the two distributions (male webs, 30~ female webs, 10~ are indicated by arrows. The distributions are significantly different (Mann-Whitney U-test, P < 0.01).
female webs, characterize the data t h a t m a k e up the o r i e n t a t i o n p o r t i o n o f Fig. 1.
Abdomen Flexion Differences Table II shows the results of pairwise testing o f a b d o m e n flexion data f r o m the five classes o f webs using the M a n n W h i t n e y U-test. T h e results indicate t h a t webs o f adult males elicited significantly fewer a b d o m e n flexions t h a n the webs o f a n y other class of spider. In contrast, the webs o f penultimate females elicited significantly more a b d o m e n flexions t h a n the webs of either i m m a t u r e s or penulti-
-> $ 0
Flexions
Figure 3. Frequency distributions of male abdomen flexion scores on adult male (open bars, N = 68) and adult female (filled bars, N=22) webs. These distributions show the types of differences that account for the variation in the abdomen flexion data in Fig. 1. Medians of the two distributions (male webs, 0; female webs, 19.5) are indicated by arrows. The distributions are significantly different ( M a n , W h i t n e y U-test, P < 0.0001). mate males. N o other differences are significant. M e t h a n o l - w a s h e d male webs elicited as few a b d o m e n flexions ( m e d i a n = 0 , N = 19) as chemically intact male webs ( Z = 0 . 4 4 4 , P = 0 . 6 6 ) . Figure 3, showing the frequency distributions of male a b d o m e n flexion scores o n b o t h male a n d female webs, characterizes the d a t a t h a t m a k e u p the a b d o m e n flexion p o r t i o n of Fig. 1.
DISCUSSION
Demonstration of Multiple Pheromones Suter & Renkes (1982) implied that, a m o n g
Table II. Results of pairwise comparisons (by class) of abdomen flexion scores
Male web Median Comparisons (Mann Whitney U-test) Male web Penultimate male web Immature web Penultimate female web
0
Penultimate Penultimate male Immature female web web web 6.5 < 0.0002
8.0 < 0.001 NS
27 < 0.0001 < 0.02 <0.05
Female web 19.5 < 0.0001 NS NS NS
752
Animal Behaviour, 34, 3
bowl-and-doily spiders, there are two chemically different classes of webs: those that elicit both courtship behaviour (abdomen flexions) and positive geotaxis, and those that elicit only negative geotaxis. In the former group are the webs of adult females. In the latter group are the webs of early instar (more than three moults from adulthood, in contrast to the older but still immature spiders tested in the present study) F. pyramitela and methanol-washed webs of adult females. With only two chemically different classes of webs, it was possible to hypothesize the existence of only a single pheromone: when the pheromone was present, one set of behaviours (e.g. abdomen flexions and positive geotaxis) was stimulated; when no pheromone was present, the other set of behaviours (e.g. negative geotaxis and no courtship) was stimulated. Data presented here demonstrate that there is a third chemically distinct class of webs: those which elicit positive geotaxis but little or no courtship behaviour. These are the webs constructed by adult males (Figs 2, 3). The presence of this third class of webs eliminates the possibility that the web-borne pheromone on female webs has only one active chemical component but two roles (see Introduction). Three chemically different classes of webs cannot occur in the absence of at least two chemicals which have different functional roles. Our alternative hypothesis, that separate chemical components elicit the separate behavioural responses (geotaxis and courtship behaviours), is strongly supported by the data presented here. Webs constructed by adult males are qualitatively different from those constructed by all other classes of spiders tested; male webs elicit few or no abdomen flexions while the webs of adult females and of both sexes of penultimate adults and juveniles elicit many abdomen flexions (Table II, Fig. 3). Washing male-constructed webs in methanol does not alter their ability to elicit abdomen flexions (Table II), but similar washing of webs constructed by adult females renders those webs indistinguishable from adult male webs with respect to abdomen flexions (Table II; Suter & Renkes 1982). Thus, some chemical that is extracted or denatured by methanol elicits abdomen flexions when males contact it on a web. Neither adult males nor very young bowl-anddoily spiders (Suter & Renkes 1982) deposit that chemical on their webs. A second chemical, one that is also extracted or denatured by methanol,
elicits positive geotaxis from males. That chemical is found on all chemically intact webs tested in this study (Table I) but is absent from the webs of very young spiders (Suter & Renkes 1982).
Functions of a Dual System
A complete analysis of the courtship of F.
pyramitela (Suter & Renkes 1984) clarified the dual role of the pheromone(s) that the adult female deposits on her web. Not only does the chemical stimulate oriented searching in a way that minimizes the time between web contact and location of the female (because the bowl where the female resides is below most of the points of web-substrate contact, points where a male is apt to make first contact with the web), it also stimulates the one vibration-producing behaviour that is clearly involved in assuaging female predatory behaviour (Suter & Renkes 1984). In agonistic interactions between adult males on female webs, the abdomenflexion-stimulatingpheromone is also important in eliciting the initial vibratory behaviours of the intruding male, and these behaviours are probably used in the transmission of information about the resource-holding potential (i.e. the mass) of the intruder (Suter & Keiley 1984). Our demonstration that at least two pheromones are involved and that neither is a feature of all F. pyramitela webs (male webs lack one component, webs of very young spiders lack both) suggests that the known functions of the pheromones are beneficial only to certain age and sex classes of bowl-anddoily spiders. The roles of the two pheromones in the lives of juvenile bowl-and-doily spiders remain obscure. Because adult males attend penultimate females for many hours, on occasion, and mating of attended females occurs within minutes of the completion of the final moult, we speculate that penultimate females may increase the probability of early insemination by including both pheromones on webs they build. But we can see no such function for the pheromones of other juvenile classes of either sex. Further observation and experimentation may elucidate the other functions of these chemicals. ACKNOWLEDGMENTS We are indebted to two anonymous reviewers for their helpful comments on this manuscript. This
Surer & Hirscheimer: Web-borne pheromones study was supported in part by the Financial Aid Office and the Beadle F u n d o f Vassar College.
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