Female pheromonal chorusing in an arctiid moth, Utetheisa ornatrix

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1 Behavioral Ecology doi: /beheco/arl069 Advance Access publication 6 November 2006 Female pheromonal chorusing in an arctiid moth, Utetheisa ornatrix Hangkyo Lim a and Michael D. Greenfield a,b a Department of Ecology and Evolutionary Biology, University of Kansas, 1200 Sunnyside Avenue, Lawrence, KS 66045, USA and b Institut de Recherche sur la Biologie de l Insecte, CNRS UMR 6035, Université Francxois Rabelais de Tours, Parc de Grandmont, Tours, France We report an unusual case of communal sexual display in the arctiid moth Utetheisa ornatrix that we designate female pheromonal chorusing. As in most moths, female U. ornatrix release a long-distance sexual advertisement pheromone during a nightly activity period. We arranged U. ornatrix females in 2 types of signaling conditions: grouped and solitary. When the females were grouped with neighboring signaling females (grouped), they initiated pheromone release sooner, continued release with less interruption and over a longer total period, and performed the release with faster abdominal pumping than observed in isolated females (solitary). This differs from the usual form of sexual communication in moths: female (chemical) signalers, male receivers, and a general lack of interaction among females. At mating, male U. ornatrix transfer a large spermatophore that may enhance female reproductive success and which represents either mating effort or paternal investment. This action results in an extended postmating male refractory period leading to a female-biased operational sex ratio. We argue that this biased sex ratio generates intrasexual competition among females, to which they respond by elevating signaling effort such that the likelihood of at least matching their neighbors signals is increased. In the field, U. ornatrix are clustered around patches of host plants, and we also explore the possibility that pheromonal chorusing is driven by cooperation among groups of related or nonrelated females. Key words: Lepidoptera, mating system, operational sex ratio, sexual selection, signal competition. [Behav Ecol 18: (2007)] Address correspondence to H. Lim. hlim@ku.edu. Received 20 January 2006; revised 25 August 2006; accepted 17 September Ó The Author Published by Oxford University Press on behalf of the International Society for Behavioral Ecology. All rights reserved. Downloaded from For permissions, please journals.permissions@oxfordjournals.org Females in various animal taxa emit sexual advertisement pheromones to attract mates and form pairs (Wyatt 2003). Among insects, female advertisement pheromones are best known and most thoroughly studied among Lepidoptera, namely moths (Wyatt 2003). Biochemical and physiological studies conducted in many species have identified the chemical structures, biosyntheses, and modes of emission of the pheromone molecules (Jurenka 2003; Rafaeli and Jurenka 2003; Cardé and Haynes 2004), whereas electrophysiological and behavioral tests have revealed the neural and orientation responses that males exhibit toward the compounds (Greenfield 2002; Blomquist and Vogt 2003). These studies have led biologists to accept a standard paradigm for sex pheromone communication in moths: female signalers and male receivers that track the female (chemical) signals via upwind flight, which is also influenced by visual input as well as self-steering mechanisms (Cardé and Haynes 2004). Male courtship pheromones, which males may release once they have oriented toward an advertising (pheromone emitting) female and established physical contact, often modify the basic communication protocol (Cardé and Haynes 2004). Here, too, various studies have identified the chemical structures and biosyntheses of these compounds (Cardé and Haynes 2004) as well as their role in sexual selection (Wyatt 2003; Cardé and Haynes 2004). Female responses to female sex pheromones have been generally overlooked, with females typically regarded as noninteractive signalers prior to pair formation. However, recent neurophysiological and behavioral evidence suggests that this view may not be accurate and that communication in moths may be more interactive and complex than previously thought. Researchers have found electrophysiological responses of female antennal neurons to conspecific female sex pheromone in 2 lepidopteran families: Arctiidae (Panaxia quadripunctaria, Schneider et al. 1998; Utetheisa ornatrix, Grant and O Connell 2000) and Noctuidae (Spodoptera littoralis, Ljungberg et al. 1993). Conspecific females and/or their sex pheromone have also been reported to induce signaling (Tortricidae; Choristoneura fumiferana, Palaniswamy and Seabrook 1978, 1985; Cydia fagiglandana and Cydia splendana, Den Otter et al. 1996), delay signaling in females (Tortricidae; Adoxophyes sp. and Homona magnanima, Noguchi and Tamaki 1985) and to attract (Tortricidae; C. fagiglandana and C. splendana; Den Otter et al. 1996) or repel females (Noctuidae; Heliothis armigera and Heliothis zea, Saad and Scott 1981). However, the ecological and evolutionary contexts of these neural and behavioral responses to conspecific females and their sex pheromones have not been analyzed fully, and the general subject of female female communication remains largely unexplored. Here, we investigated female female communication, including specific responses to conspecific female sex pheromone, in the New World arctiid moth U. ornatrix. In addition to the electrophysiological responses noted above for this species, our casual observations suggested that when multiple U. ornatrix females were caged together with multiple males, females were more likely to display signaling behavior and to mate than when a single female and male were held. Importantly, this event did not appear to result from forced mating by males when multiple males and females were caged together. Thus, we conducted a series of experiments designed to test the influence of neighboring females and their signals on the timing and intensity of female signaling. We also tested the mating frequency of males and females in our population in order to estimate operational sex ratio and the intrasexual competition that each sex may experience. We found that U. ornatrix females, clustered in space and within olfactory range, synchronized onsets of their daily advertisement periods and advertised with faster abdominal

2 166 Behavioral Ecology pumping. They were also more likely to advertise uninterruptedly and for a longer duration under such circumstances than when isolated. We name this phenomenon female pheromonal chorusing to indicate its possible analogy to the communal sexual displays commonly observed among males in acoustic insects and anurans and to the dawn chorus of birds. As in many moths, U. ornatrix females normally begin emitting their sexual advertisement pheromone at dusk and continue for several hours (Conner et al. 1980). Females pump their abdomen at s ÿ1, an activity found in many arctiid moths and easily recognizable by an observer, during pheromone emission. This rhythmic pumping entails extruding the terminal abdominal segments, which bear the sex pheromone glands (Conner et al. 1980). Females that are not pumping the abdomen do not attract males, and measurable quantities of sex pheromone a blend of several hydrocarbons (Conner et al. 1980; Jain et al. 1983) are not extractable from them. Presumably, extrusion of the terminal segments exposes the evaporative surface and elevates the pheromone release rate greatly. Several hypotheses explaining the adaptiveness of abdominal pumping have been proposed (Conner et al. 1980), including the possibility that it increases the peak pheromone release rate (Schal and Cardé 1985; Dusenbery 1989). Utetheisa ornatrix males that detect the female sex pheromone are stimulated to fly upwind and, on approaching, court the female with a pheromone, hydroxydanaidal, derived from host plant pyrrolizidine alkaloids obtained during larval feeding on seeds of their host plants (Crotalaria spp.; Conner et al. 1981; Eisner and Meinwald 1995). At mating, males transfer a large spermatophore that comprises as much as 10% of their body weight and contains sizable quantities of the pyrrolizidine alkaloids (Conner et al. 1990; LaMunyon and Eisner 1994; Iyengar et al. 2001). These alkaloids have toxic properties (Conner and Weller 2004), and female U. ornatrix may thereby derive added protection from predators on receiving a spermatophore. Because a female transfers some proportion of these alkaloids to her eggs, her offspring may also benefit (LaMunyon and Eisner 1993; Iyengar and Eisner 1999; Rossini et al. 2001; Bezzerides and Eisner 2002; del Campo et al. 2005). The opportunity to obtain these benefits may be responsible for the high level of multiple mating observed in female U. ornatrix, up to 23 times in the field (Bezzerides and Eisner 2002). Male U. ornatrix, however, may be expected to be refractory after mating because of the demands of spermatophore production. Extended refractory periods would reduce the male mating rate and establish a female-biased operational sex ratio conducive to female female competition for access to males. That is, a partial sex-role reversal of the typical situation in which males compete to be at least as attractive to females as their male neighbors may occur in U. ornatrix. In this paper, we examine male and female refractory periods and mating rates to explore the possibility that a female-biased operational sex ratio occurs in U. ornatrix and generates female competition and pheromonal chorusing. But, we also consider the possibility that other factors, such as intrasexual cooperation, may underlie this communal sexual display. METHODS Study population We used a laboratory population of U. ornatrix derived from moths collected in Highlands County, FL. The moths were reared on a standard pinto bean diet (Shorey and Hale 1965), to which we added seeds of Crotalaria mucronata, the host plant of U. ornatrix at the collection site; C. mucronata seeds comprised 20% of the diet by weight (Dussourd et al. 1991). We kept the moths in an environmental chamber maintained at 27 C and under a 14:10 h light:dark photoperiod. The experimental animals were from the fourth through sixth generations, measured from collection in the field. To ensure standard physiological and behavioral states among the experimental animals, we only used virgin females and tested them once on the second or third day after eclosion, that is, females aged h. Observation and analysis of signaling behavior The experiment was designed to compare sexual advertisement signaling by solitary and grouped females. On a given night, we compared the signaling of 6 solitary females with 6 females grouped together as an assemblage of potentially interacting individuals. The 6 solitary females were held individually within 100-ml glass jars, all placed within a covered, cm clear Plexiglas box (Figure 1). Neighboring jars were separated by 0.5 cm, the inside of each jar was lined with an opaque paper cylinder extending the full height, and the jars were closed with clear plastic lids. Thus, solitary females received no visual or chemical stimuli from their 5 neighbors. Separation of the jars minimized the (unlikely) possibility that substrate vibrations were transmitted between females, and a bat detector (UltraSound Advice model S-25, London, UK) did not record any acoustic, either sonic or ultrasonic, emissions from the moths (Weller et al on the absence of sound production in Utetheisa). Consequently, females would not have received mechanical stimuli from their neighbors either. The 6 grouped females were held similarly to the solitary ones save that glass jars were not used to enclose each individual within the Plexiglas box (Figure 1). Here, only the opaque paper cylinders separated each female from its neighbors. As above, the paper cylinders were separated by 0.5 cm and each extended the full height of the Plexiglas box; that is, the cylinder top was contiguous with the Plexiglas box cover. Each paper cylinder was perforated with many minute (diameter ¼ 0.5 mm) holes; these perforations were also used for the solitary females. Thus, grouped females received no visual or mechanical stimuli from their 5 neighbors, but unlike solitary females, they would have been able to receive chemical stimuli diffusing through the perforations in the paper cylinders. For both solitary and grouped females, we washed the jars and Plexiglas boxes daily with detergent to remove any residual pheromone and other odors from the moths. Females used for the solitary and grouped treatments were chosen randomly from the laboratory population. We made all observations of solitary and grouped females during the initial half of the night, the natural signaling and mating period in U. ornatrix. Temperature was kept at C, and illumination was provided by a 30-W incandescent red bulb. This lighting allowed us to make the observations but at the same time was treated as night by the moths. That is, the females signaled regularly while exposed to the red light. We observed a total of 198 solitary and 198 grouped U. ornatrix females on 33 nights; 9 solitary and 18 grouped females were not included in the analyses because they either died before or oviposited during the observation period. Solitary and grouped females were established in the observation room 2 h prior to the onset of night. Beginning at nightfall, we observed each of the 12 individuals from above for a 15-s interval every 3 min. During the 15-s observation, we noted 1) whether or not the female was signaling (pumping her abdomen), 2) the number of abdominal extrusions per 15 s if she was signaling, and 3) any other behavior. We maintained this schedule for the first 70 min after nightfall, the period during which many females initiated signaling. After

3 Lim and Greenfield Female pheromonal chorusing 167 Figure 2 A representation of data collected from observing 6 females in a given treatment during the initial 60 min after nightfall. X: signaling (pumping of abdomen) observed at a given observation cycle. Individual #2 was observed to begin signaling first, 7 min after nightfall, followed by individuals #6, #3, and #5; individual #1 was observed to begin signaling at 16 min, but the signal bout was shorter than 3 min and discarded from the indices; individual #4 was never observed signaling. The elapsed time between the cycle when the first signaler (#2) was observed signaling and the cycle when the second (#6) was observed was 6 min; elapsed times between the second and third, third and fourth signaler were 6 and 12 min, respectively. Horizontal bars above the X s indicate signaling bout lengths for the signaling individuals; bars with arrows pointing to the right indicate that signaling continued past 60 min and bouts of indeterminate length. Times along x axis indicate midpoints of 3-min observation cycles. Figure 1 Placement and separation of individual females in grouped and solitary treatments. Filled triangle, Utetheisa ornatrix female; solid circle, glass jar; dashed circle, perforated paper cylinder, extending to top lid of glass jar (solitary treatment) or top cover of Plexiglas box (grouped treatment); rectangle, Plexiglas box ( cm). this initial period, we switched to a slower schedule for another 230 min wherein we observed each of the 12 individuals for a 15-s interval only every 10 min. To ensure standardization, the same observer (H.L.) made each of these measurements for all individuals tested. We chose to measure the abdominal extrusion rate because it reflects the vigor with which signaling is performed and the possibility that it is related to energetic expenditure during signaling and the pheromone release rate. From the above observations, we determined the following 4 indices of each individual female s signaling: 1) the time when each individual was first observed signaling, with nightfall designated as 0 min; 2) the mean abdomen extrusion rate, in extrusionsmin ÿ1, of each individual that was observed signaling at least once during the 300-min observation period; 3) the mean length of each individual s signaling bouts, defined as periods during which she was seen signaling on all consecutive observations without interruption; for example, an individual observed signaling at 54 and 57 min but not at 51 or 60 min would have registered a signaling bout of 6 min for that period; 4) the total time that an individual spent signaling, defined as the sum of all of her signaling bouts (Figure 2). For all 4 indices, we listed the time of the reported event as the midpoint of the 3- or 10-min cycle of observations during which it was seen. In addition to the above measures of signaling by individual females, we also determined the following 3 collective measures: 1) the proportion of the 6 females in an assemblage of solitary or grouped individuals that were observed signaling at least once during the 300-min observation period, 2) the elapsed time between the observation when the first female in an assemblage of 6 solitary or grouped individuals was observed signaling and the observation when the second female was seen, and 3) the total number of females within an assemblage seen signaling during each of the regularly scheduled observations. Unless otherwise noted, variation in these indices is reported as mean 6 standard deviation. We treated the data obtained from an entire assemblage of 6 solitary or 6 grouped females as a single sample, in order to avoid problems arising from a lack of independence between behavior exhibited by individuals within an assemblage of grouped females, for example, pseudoreplication (Hurlbert 1984), from inflating the incidence of differences between the 2 treatments. Our determination of signaling indices 3 and 4 assumed that individuals did not briefly cease signaling between 2 consecutive observation cycles during which they were seen displaying the behavior (Conner et al. 1980; Itagaki and Conner 1987). Similarly, we assumed that individuals did not briefly signal between 2 consecutive observation cycles during which they were not seen displaying the behavior. Our general observations of U. ornatrix suggested that the incidence of such erratic events was low. Moreover, environmental conditions were constant throughout the 5-h observation period, and the act of our observations did not appear to affect their behavior unduly. When individuals were still signaling at the end of the 300-min nightly observation period, we designated 300 min as the end of that signaling bout and calculated

4 168 Behavioral Ecology indices 3 (mean signaling bout length) and 4 (total time spent signaling) accordingly. Intrasexual competition We compared the total number of matings that females completed during an extended period with the total number of matings that males completed during this period to estimate the expected levels of intrasexual competition in each sex. Thus, we tested the hypothesis that females mated as often as, or more than, males and thereby experienced elevated levels of competition potentially leading to pheromonal chorusing. We observed the mating success of 29 males and 25 females after each had mated once on their 2nd day after eclosion, which was designated as the first day of our observation period. On each night over a 9-day period after the first mating, we presented the test males, in groups of 6, with an equivalent number of 2-day-old virgin females; similarly, we presented the test females, in groups of 6, with an equivalent number of 2-day-old virgin males. Observed couples were isolated, and the hatching of deposited eggs was checked to confirm that mating was successful. On each night of the period, we calculated the proportion of individuals mating out of the total number of individuals tested for each sex. We also calculated the mean number of matings per individual for the observation period, for each sex, by determining the total number of matings observed throughout the observation period divided by the number of individuals. We interpreted the ratio of these female:male total numbers of mating as the amount of competition for mates that a female in the population might experience relative to that experienced by a male. We also calculated the mean remating interval of each test individual. We continued our observations over 9 days because of previous findings that regeneration of a full-sized spermatophore required at least 7 days under laboratory conditions (LaMunyon and Eisner 1994). Throughout the observation period, test individuals were provided with sugar water on a cotton ball. RESULTS Signaling behavior A majority of the test females signaled in the observation boxes under solitary and grouped treatments, and most of those that did signal began within 120 min of nightfall, often within 30 min (Figure 3). We found that on all 33 observation nights at least one female in the grouped treatment signaled. The incidence was slightly lower in the solitary treatment, where at least one female was observed signaling on 30 of the 33 observation nights. On a given observation night, we found that more than 4 of the 6 grouped females signaled during at least one observation cycle (mean proportion ¼ ; median ¼ 0.67), whereas fewer than 3 of the 6 solitary females did so (mean proportion ¼ ; median ¼ 0.50). Overall, the proportion of signaling females on a given observation night was significantly higher for grouped than solitary individuals (Wilcoxon matched-pairs signed-rank test, n ¼ 33, T ¼ 3.67, P, 0.001). Signaling began at approximately the same time after nightfall in both treatments (Figure 4A). Over the 30 observation nights, the first female in the solitary treatment began signaling min after nightfall, whereas the first female in the grouped treatment began at min (n ¼ 30, i.e., the 3 nights when no solitary female signaled were eliminated from analysis, Wilcoxon matched-pairs signed-rank test, T ¼ 1.30, P ¼ 0.10). Once one female began signaling, however, other females began significantly sooner in the Figure 3 Times of observed signaling onsets of grouped (A) and solitary (B) females. Data are taken from 126 grouped and 86 solitary females that were observed signaling during the initial 150 min after onset of darkness on 33 nights. grouped treatment than in the solitary one. The second grouped female began signaling min following the first signaler, whereas the second solitary female did not begin signaling until min following the first (Figure 4B: n ¼ 25, i.e., the 5 nights when fewer than 2 solitary females signaled were eliminated from analysis, Wilcoxon matched-pairs signed-rank test, T ¼ 3.11, P, 0.001). We continued this analysis by calculating the mean interval between the times when successive individuals began signaling in each treatment; that is, the mean time elapsing between the onset of signaling by individual i and individual i 1 1, i ¼ Again, the mean interval among grouped females ( min; n ¼ 25 observation nights) was significantly shorter than that among solitary females ( min; n ¼ 25, i.e., the 5 nights when fewer than 2 solitary females signaled were eliminated from analysis, Wilcoxon matched-pairs signed-rank test, T ¼ 2.22, P ¼ 0.01). In general, the number of females signaling in an assemblage of 6 solitary or 6 grouped individuals increased linearly over the initial 60 min after nightfall (Figure 5A). However, we found that the rate of increase was significantly greater for grouped females than solitary ones. For grouped females, the mean regression coefficient of increase over the period from the onset of signaling by the first individual until 60 min was individualsmin ÿ1 (Figure 5B: n ¼ 19

5 Lim and Greenfield Female pheromonal chorusing 169 Figure 4 (A) Difference between observed signaling onset times of the first solitary and the first grouped female on a given night; data are shown for the 30 nights when at least one female in each treatment was observed signaling. Negative value indicates that a solitary female started signaling before the first grouped female on that night. (B) Time elapsed between onsets of signaling by first and second individuals among grouped and solitary females on a given night. Data are taken from the 25 nights when at least 2 females in each treatment were observed signaling. Box plots show means (X), medians (horizontal line), 25 75% ranges (rectangle), 5 95% ranges (inner horizontal bar), and minimum/maximum values (outer horizontal bar) of time. observation nights, R 2 ¼ %, median ¼ 79.0%), whereas signaling among solitary females only increased at individualsmin ÿ1 (R 2 ¼ %, median ¼ 77.5%, n ¼ 19, i.e., the 14 nights when fewer than 3 females signaled during the initial 60 min were eliminated from analysis, sign test, n1 ¼ 12, nÿ ¼1, ties ¼ 6, P ¼ 0.002). Abdominal extrusion rates in both solitary and grouped females were moderate at first, peaked approximately at the 60th min after nightfall, and then decreased steadily throughout the remainder of the observation period (Figure 6). During the initial 30 min after nightfall, grouped females exhibited a significantly higher mean extrusion rate of min ÿ1 (n ¼ 11 observation nights) than solitary females exhibited ( min ÿ1, n ¼ 11, i.e., the 19 nights when no solitary female signaled during the initial 30 min were eliminated from analysis, sign test, n1 ¼ 9, nÿ ¼ 2, ties ¼ 0, P ¼ 0.03). During the period lasting from 50 to 90 min after nightfall, grouped females exhibited significantly higher peak mean extrusion rates ( min ÿ1, Figure 5 (A) Increase in the number of solitary (open circle) and grouped (filled triangle) females observed signaling during the 60 min after nightfall. Data are taken from the 19 nights when at least 2 females in each treatment were observed signaling during the initial 60 min. (B) Mean regression coefficient of the increase in the number of signaling females during the 60 min after nightfall from the 19 nights. Box plots show means (X), medians (horizontal line), 25 75% ranges (rectangle), 5 95% ranges (inner horizontal bar), and minimum/maximum values (outer horizontal bar) of regression coefficients. n ¼ 26 observation nights) than solitary females ( min ÿ1, n ¼ 26, i.e., the 4 nights when fewer than 2 solitary females signaled during the period from 50 to 90 min were eliminated from analysis, Wilcoxon matched-pairs signed-rank test, T ¼ 2.17, P ¼ 0.01). By the period extending from 100 to 200 min and from 200 to 300 min after nightfall, extrusion rates among grouped females ( min ÿ1, n ¼ 25; min ÿ1, n ¼ 15 observation nights, respectively) had become equivalent to those among solitary females ( min ÿ1, Wilcoxon matched-pairs signed-rank test, T ¼ 0.42, n ¼ 25, P ¼ 0.34; min ÿ1, n ¼ 15, sign test, n1 ¼ 10, nÿ ¼5, ties ¼ 0, P ¼ 0.15; i.e., the 5 and 15 nights when fewer than 2 solitary females signaled during the period from 100 to 200 min and from 200 to 300 min, respectively, were eliminated from analyses). Once signaling began, grouped females interrupted their signaling less often than solitary females did, and their total time spent signaling during the 300-min observation period was also higher. The mean length of signaling bouts in

6 170 Behavioral Ecology Figure 6 Mean abdominal extrusion frequencies observed among solitary and grouped females at 4 time windows after nightfall. Data are taken from 11, 26, 25, and 15 observation nights when at least 2 females in each treatment were observed signaling corresponding to each time window, respectively. Box plots show means (X), medians (horizontal line), 25 75% ranges (rectangle), 5 95% ranges (inner horizontal bar), and minimum/maximum values (outer horizontal bar) of frequencies. *P, grouped females (Figure 7A: min, n ¼ 30 nights) was significantly greater than the mean lengths in solitary females ( min, n ¼ 30, i.e., the 3 nights when no solitary females signaled were eliminated from analysis, Wilcoxon matched-pairs signed-rank test, T ¼ 3.30, P, 0.001). Similarly, the mean of the total time that grouped females spent signaling (Figure 7B: min, n ¼ 30 nights) was significantly greater than that solitary females spent ( min, n ¼ 30, i.e., the 3 nights when no solitary females signaled were eliminated from analysis; Wilcoxon matched-pairs signed-rank test, T ¼ 4.27, P, ). Figure 7 (A) Mean length of signaling bouts in the solitary and grouped treatments. Data are taken from 30 observation nights during which at least one individual was observed signaling in each treatment. (B) Mean length of the total time spent signaling in the solitary and grouped treatments. Data are taken from the same observation nights as above. Box plots show means (X), medians (horizontal line), 25 75% ranges (rectangle), 5 95% ranges (inner horizontal bar), and minmum/maximum values (outer horizontal bar). Intrasexual competition Of the 29 test males and 25 test females, 24 and 23 individuals, respectively, survived longer than the 9-day observation period. If we considered all 29 test males and 25 test females, the mean proportions of individuals mating on any given night of the 10-day period were for males and for females (Figure 8A). The mean numbers of matings for the period by the males ( mating/ individual/10 day) and that by the females ( ) were similar (Mann Whitney U test, W ¼ 740.5, P ¼ 0.33). If we considered only the 24 test males and 23 test females that survived until the end of the observation period, the mean proportions of individuals mating on any given night were for males and for females (Figure 8B). The mean numbers of matings for the period by the males ( ) and that by the females ( ) were also similar (Mann Whitney U test, W ¼ 508.5, P ¼ 0.15). The total female:male ratios of the mean numbers of matings were 1.19 and 1.27, respectively. The mean remating interval of males ( day, n ¼ 23, 6 males that did not remate during the 9-day observation period were eliminated from analysis) was significantly shorter (Mann Whitney U test, W ¼ 430.5, P, 0.01) than that of females ( day, n ¼ 24, one female that did not remate during the period was eliminated from analysis). These values indicate that, in a population at a 1:1 primary sex ratio and in which males and females experience comparable reproductive lifespans, the number of sexually active females would be expected to exceed the number of sexually active males at any given time. DISCUSSION Our findings on the signaling behavior indicate that when an U. ornatrix female perceives the odors of signaling conspecific females, she is 1) more likely to signal during that night, 2) begins signaling earlier, 3) exhibits a faster abdominal extrusion rate during signaling, and 4) continues signaling with fewer interruptions, and 5) over a longer total period than when she is isolated and cannot perceive neighboring conspecifics. Because female U. ornatrix appear to signal more vigorously in the presence of signaling neighbors and they time their signaling in relation to their neighbors signaling activity, we name this phenomenon female pheromonal chorusing to draw attention to its similarities with the well-known communal sexual displays shown by males in many acoustic species,

7 Lim and Greenfield Female pheromonal chorusing 171 Figure 8 (A) Proportions of mating individuals of 29 males (solid bars) and 24 females (empty bars) on each of the 9 days after an initial mating on day 0 (for 10 days totally). (B) Proportions of mating individuals of 25 males (solid bars) and 23 females (empty bars) that survived the entire 10-day observation period. Mating of test females and males was induced by presentation of comparable numbers of new virgin males and females on each night (see text). namely, acoustic insects and anurans (Gerhardt and Huber 2002) and the dawn chorus of birds (Todt and Naguib 2000; Naguib 2005). To be sure, female pheromonal chorusing in U. ornatrix does not include fine temporal interactions, such as the synchrony or alternation of song elements, that characterize many insect and anuran choruses (Gerhardt and Huber 2002). But, communally displaying U. ornatrix females do show an elevated level of signaling activity (Figure 6), and they concentrate that activity in time (Figures 4B and 5A). In fact, we could not expect more precisely timed temporal interactions in their pheromonal chorusing, as the velocity of (chemical) signal transmission between neighbors is comparatively slow and is largely dependent on the vagaries of wind speed and direction (Dusenbury 1989; Greenfield 2002). Our findings on the total numbers of matings by females and males over the 10-day observation periods suggest that the level of intrasexual competition among females is at least as great as among males, assuming a 1:1 primary sex ratio among adults and comparable reproductive lifespans in males and females (unpublished data from field collections and laboratory rearing experiences). The shorter remating interval observed among males reflects a greater propensity to remate early during adult life. However, males generally did not continue to remate after the first several days, whereas females typically remated throughout the observation period. We infer that the high rate of remating by females ultimately reflects the large male spermatophore transferred at mating. Whether spermatophore transfer corresponds with mating effort or parental investment (Vahed 1998), male U. ornatrix produce a large spermatophore that 1) females may seek to obtain as a means of enhancing their reproductive success (del Campo et al. 2005) and that 2) renders a male refractory for an extended period, which may exceed that in the female, particularly after a male s early matings. Although a precise determination of the operational sex ratio in a natural population is very difficult to obtain without information on eclosion dates, longevity, and changes in sexual attractiveness over age (Kokko and Monaghan 2001; Kokko and Johnstone 2002), it is probably safe to claim that the operational sex ratio in U. ornatrix is either 1:1 or female-biased during most of their reproductive lifespan in nature. Thus, we suggest that female pheromonal chorusing in this species represents a form of intrasexual competition related to the operational sex ratio (Trivers 1972; Emlen and Oring 1977; Parker and Simmons 1996), which might have selected females to at least match the signaling of their neighbors (Gerhardt and Huber 2002). Matching could be accomplished by initiating signaling when neighbors are detected signaling, by signaling over an extended period and by increasing signal intensity (Jia et al. 2001) which might be achieved via elevating the abdominal extrusion rate in U. ornatrix. Failure to do so could relegate a female to procuring fewer spermatophores than her neighbors and ultimately yield lower reproductive success. As such, sexual signaling in U. ornatrix females differs greatly from that typically seen in moths: In most species, females emit sexual advertisement pheromone at an extremely low rate, a rate so low that it might even function to filter out males that are poor searchers or that have lower sensitivity to pheromone (Greenfield 1981). Such screening of male quality remains a possibility in species where, unlike U. ornatrix, the female need only mate with one male, and males neither undergo a refractory period longer than one night nor provide spermatophore material critical for female reproductive success (Greenfield 2002). Here, the operational sex ratio is probably male biased, and female female competition would seldom arise. The ecology of U. ornatrix suggests that female female competition, and the female pheromonal chorusing that purportedly ensues, would occur in natural populations. At the Highlands County, Florida site, where our laboratory population had originally been collected, we observed that U. ornatrix sexual activity occurred on or near host plants, C. mucronata and C. spectabilis. Moreover, the restricted dispersal of Crotalaria seeds (Jacobi 2005) indicates that the host plants have generally occurred in dense patches, restricting U. ornatrix females to aggregated distributions. A thorough analysis of these spatial factors, and the movement responses of female U. ornatrix to conspecific females and their sex pheromone, will be treated in a future paper. Whereas our findings clearly support an increase in female signaling when neighboring female signalers are present, evidence that females are specifically responding to conspecific sex pheromone is circumstantial: Our experimental design ruled out responses to visual and mechanical stimuli, and the finding that the second, but not the first, female in the grouped treatment began signaling earlier than observed in the solitary treatment implies a specific response to stimuli associated with abdominal extrusion. The most likely such

8 172 Behavioral Ecology stimulus is the female advertisement pheromone, but genderspecific body odors may conceivably play a role (Grant and O Connell 2000). In another future paper, we will examine the specific responses of U. ornatrix females to conspecific female sex pheromone and its separate components. Likewise, our interpretations of female pheromonal chorusing in U. ornatrix have made the tacit assumption that females increase their signal intensity, and thereby their attractiveness, when elevating their abdominal extrusion rate. This relationship too is presently unconfirmed (but see Schal and Cardé 1985) and will be treated in the future paper. Is intrasexual competition the only explanation for the female female interactions and pheromonal chorusing observed in U. ornatrix? The spatial structure of populations of this species in the field suggests that cooperation may underlie these phenomena as well. Because U. ornatrix may cluster at host plant patches and members of these clusters may tend to be close kin, females that engage in pheromonal chorusing may be increasing the attractiveness of genetic relatives (Bezzerides et al. 2004): By raising the intensity, continuity, and total duration of signaling broadcast from a cluster, chorusing females may increase the overall attractiveness of their kinship group relative to others (Höglund 2003). Conceivably, this group effect may operate even when cluster members are not close relatives (Dugatkin 2002), although it may then be more susceptible to cheaters : Individual females may forgo signal amplification, thereby avoiding any energetic costs incurred by this heightened activity, while benefiting from the group s attractiveness (Barnard and Sibly 1981; Caraco and Giraldeau 1991 on producer scrounger games and the evolution of cooperative behavior). Importantly, the potential attractiveness of chorusing groups would not eliminate intrasexual competition from operating at the level of individual females once males arrive at the cluster. Rather, this phenomenon demonstrates how selection operating at several different levels may co-occur. We shall consider these alternative possibilities for the evolution of female pheromonal chorusing in the subsequent paper addressing spatial factors and movement responses. We thank Mark Deyrup of the Archbold Biological Station, Highlands County, Florida, for helping us collect U. ornatrix and its host plants. We also thank William E. Conner, Thomas E. Eisner, and Craig W. LaMunyon for providing critical information on U. ornatrix biology during various stages of the project. Kathleen M. 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