Adult social environment alters female reproductive investment in the cricket Gryllus firmus

Adult social environment alters female reproductive investment in the cricket Gryllus firmus Abstract Phenotypically plastic responses have been increasingly documented in response to intraspecific, behavioral (often sexual) signals such as mating calls. We explored the effect of short-term exposure to male calling song on a reproductive life-history trade-off in adult females of the wing dimorphic cricket species, Gryllus firmus. In G. firmus, long-winged females possess flight muscles and small ovaries immediately after the adult molt, whereas short-winged females possess small, nonfunctional flight muscles and large ovaries at the same age. In long-winged females, flight muscles are histolyzed during ovary growth that occurs after the adult molt. Because of these differences in reproductive physiology, we predicted different responses to calling song exposure between the 2 morphs. We hypothesized that such exposure would boost ovary growth in both wing morphs, but also increase flight muscle histolysis in long-winged females, producing a greater relative response to exposure in this morph. As expected, we saw a significant increase in ovary mass in exposed females of both morphs, and a greater relative response in long-winged females. Calling song exposure did not have a strong effect on flight muscle histolysis, suggesting that the relatively larger ovary response in long-winged females was perhaps fueled instead by reallocation of flight fuels. Our study documents the rapid adult response of a fitness-linked trait to the adult social environment, a result with far-reaching implications, as the experience of mating signals during adulthood should be nearly universal across species. INTRODUCTION Phenotypic plasticity enables individuals to respond to environmental variability, both abiotic and biotic. Increasingly, interest has focused on plastic responses to variation in the intraspecific social environment (Bailey and Zuk 2008; Kasumovic and Brooks 2011; Rebar et al. 2016). The social environment of a given population contains individuals occupying a number of (nonmutually exclusive) roles: parent, offspring, sibling, competitor (for food or mates), and/or mate. Several broad patterns of plastic responses to the presence of individuals occupying these roles have begun to emerge across vertebrate and invertebrate taxa (see reviews in Kasumovic and Brooks 2011; Rodriguez et al. 2013; Vitousek et al. 2014; Groothuis and Taborsky 2015; Trillmich et al. 2015; Taborsky 2016). In this paper, we focus on plastic responses to variation in the mating environment. Effects of exposure to mating competitors The presence of intrasexual competitors for mates in the social environment induces several responses shared across taxa. Males placed with other males (or with signals or cues produced by males, e.g. pheromones, calling songs, etc.), either during the juvenile period or adulthood, tend to invest more in putatively sexually competitive traits than males housed with females or in isolation. For example, males housed with other males may exhibit higher levels of testosterone (blue-black grassquits, Volatinia jacarina: Lacava et al. 2011), increase elaboration of secondary sexual traits (Pacific field crickets, Teleogryllus oceanicus:Thomas et al. 2011; blue-black grassquits, Volatinia jacarina; Maia et al. 2012), or expend more effort in sperm competition (Pacific field crickets, Teleogryllus oceanicus: Bailey et al. 2011; Gray and Simmons 2013), than males housed with females or in isolation. Rearing juvenile males in the presence of adult males or adult male cues tends to retard male development rates to sexual maturity (guppies, Poecilia reticulata: Rodd et al. 1997; Magellan and Magurran 2009; green swordtails, Xiphophorus helleri: Walling et al. 2007; black field crickets, Teleogryllus commodus: Kasumovic et al. 2011; but see Maia et al. 2012), likely in an attempt to delay maturity until a time when fewer mating competitors might be present. When females are placed with other females, a striking and widespread effect that often occurs is the suppression or total inhibition of ovary development and function in subordinate females by dominant females, typically mediated by pheromones, in hymenopterans (de Groot and Voogd 1954; Nunes et al. 2014; Van Oystaeyen et al. 2014; Traynor et al. 2017), mammals (Wasser and Barash 1983; Faulkes et al. 1990) and western mosquitofish, Gambusia affinis (Lutnesky and Adkins 2003). Like males, juvenile females may also delay development to maturity if exposed to other females (house mice, Mus musculus: Drickamer 1975, 1977). Effects of exposure to potential mates The occurrence of potential mates in the social environment also has marked effects on behavioral, morphological, and life-history traits. The presence of adults of the opposite sex during focal individuals’ juvenile period may accelerate development to sexual maturity (house mice, Mus musculus [Drickamer 1974]; redback spiders, Latrodectus hasselti [Kasumovic and Andrade 2006; Stolz et al. 2012]; black field crickets, Teleogryllus commodus [Kasumovic et al. 2011]), such that focal individuals are more likely to mature when mates are present. Exposure to reproductively viable adult males or male signals during females’ juvenile or adult periods reduces female responsiveness to male courtship signals (Teleogryllus cricket spp.: Bailey and Zuk 2008; Bailey and Macleod 2014), increases choosiness (Pacific field crickets, T. oceanicus: Bailey and Zuk 2008; Bailey and Zuk 2009), and alters females’ preferences for male phenotypes and/or female preference functions during mating trials (wolf spiders, Schizocosa spp. [Hebets 2003; Stoffer and Uetz 2015]; highland swordtails, Xiphophorus malinche [Tudor and Morris 2009]; treehoppers, Enchenopa binotata [Fowler-Finn and Rodriguez 2012]; Pacific field crickets, T. oceanicus [Swanger and Zuk 2015]). Females may even attend to differences in the quality of males to which they are exposed, and alter their reproductive investment accordingly. Hert (1989) found that cichlid (Astatotilapia elegans) females exposed to males possessing intact breeding markings were more likely to lay clutches than females exposed to males with removed breeding markings, whereas Kasumovic et al. (2011) discovered that black field cricket (T. commodus) females exposed to a chorus of numerous, high quality males had larger ovaries (a proxy for pre-mating reproductive investment) than females exposed to fewer low quality males or no males. Study system and hypotheses To add to the growing collection of studies exploring phenotypic plasticity in response to the social environment, we tested the effect of adult-period exposure to a male mating signal (male calling song, a long distance attraction signal) on a life-history trade-off between reproduction and flight capability in females of the wing dimorphic sand cricket, Gryllus firmus. Gryllus firmus, a large (adult mass ca. 0.7 g) cricket inhabiting beaches and other sandy areas on the east coast of the United States, occurs in 2 morphs: a flight-capable, macropterous (long-winged, hereafter LW) morph and a flight-incapable, micropterous (short-winged, hereafter SW) morph. Gryllus firmus LW adult females undergo a life-history trade-off known as the “oogenesis-flight syndrome” (Johnson 1969). In this syndrome, after the molt to adulthood, LW females initially experience a flight-capable, non-reproductive phase (possessing large, functional flight muscles and small, immature ovaries) followed by a flight-incapable, reproductive phase (with small, histolyzed flight muscles and large, mature ovaries). During the switch from flight capability to flight incapability in LW females, histolyzed flight muscle tissue and stored flight fuels (primarily lipids) can be reallocated to ovary growth. This reallocation has been observed in the monomorphically macropterous cricket, Gryllus bimaculatus (Lorenz 2007) and the LW morph of G. firmus (Stirling et al. 2001). Meanwhile, SW females have small, vestigial flight muscles and rudimentary wings (rendering them incapable of flight), and possess large ovaries after eclosion (Roff 1984). Because SW females do not pass through a flying dispersal phase and do not allocate energy to flight muscle development and maintenance of flight fuels (Roff 1984; Mole and Zera 1994; Stirling et al. 2001, Zera and Larsen 2001; Crnokrak and Roff 2002), SW females of G. firmus begin reproduction earlier and have greater cumulative fecundity than LW females (Roff 1984). One of the consequences of this reproductive allocation pattern in LW females of G. firmus is a significant negative covariance between the mass of the main flight muscles (the dorso-longitudinal muscles, hereafter DLMs) and ovary mass, an index of fecundity (Roff 1994). Although SW females do not have functional DLMs, there is still a significant negative covariance between ovary mass and DLM mass (Zera et al. 1997; Stirling et al. 2001), which can be attributed to genetic correlations between wing morphs, DLM mass and ovary mass (Roff and Gelinas 2003; Roff and Fairbairn 2011, 2012). In this species (as in other Orthopteran species), male calling song advertises the presence of reproductively capable males. Hence, we hypothesized: 1) that adult females (of both wing morphs) exposed to male calling song would invest more energy in reproduction, as indicated by an increase in ovary mass, than unexposed females. Because of the trade-off between components of flight capability, such as DLM mass/ flight fuels, and ovary mass, we further hypothesized: 2) that the exposed females would reallocate flight-capability components (DLMs) to the ovaries. This response should be most evident in LW females but, because of the aforementioned genetic correlations, may also be present in SW females. Our last hypothesis pertains to differences in the relative ovary investment response between the wing morphs. Regardless of acoustic treatment, SW females should allocate more total energy to ovary growth than LW females (Roff 1984), and therefore should have fewer alternate sources of energy that they could reallocate to ovary growth in response to social signals such as calling song. Because LW females have larger additional energy stores that are initially uncommitted to reproduction (i.e. flight fuels and flight muscles), we predicted that in LW females, reallocation to ovary growth would likely include both the resources devoted to flight capability and those sources also used by SW females. Consequently, we hypothesized: 3) that the relative increase in ovary mass should be greater in exposed LW females than exposed SW females. We applied two different lengths of exposure (for either 3 days or 6 days after eclosion to adulthood) to determine if the length of exposure affected the magnitude of the responses of ovary mass and flight muscle mass. MATERIALS AND METHODS Experimental treatments We performed our acoustic exposure experiment in two blocks, which we shall refer to as Experiment 1 (performed in January 2016; total n = 115, with 98 LW females and 17 SW females) and Experiment 2 (performed in April–May 2016; total n = 159, with 64 LW females and 95 SW females). The total sample size for both experiments combined was 274 individuals. In both Experiments 1 and 2, individuals came from a lab population of G. firmus that was descended from a field sample collected in 2013 near Jacksonville, Florida. The field sample included 76 gravid females, ensuring that the subsequent lab colony did not suffer from founder effects. Individuals used in this study were the 6th generation (Experiment 1) and 7th generation (Experiment 2) descendants of the founding individuals. Because of logistical constraints, rearing densities differed slightly between Experiments 1 and 2, but the actual experimental protocol was identical. In Experiment 1, nymphs were raised in mixed-sex 63-L communal tubs (initial density ca. 1000–1200 individuals per tub) at 28 °C on a 15:9 h light:dark photoperiod and were supplied with ad libitum crushed Purina rabbit chow, cotton plugged-water vials, and egg cartons for shelter. In Experiment 2, conditions were identical except that starting nymphal density was ca. 1500 individuals in a single 63-L tub. In both experiments, when individuals neared the penultimate instar, we checked the tubs daily (in the morning) for the presence of emerging adults. Newly eclosed LW and SW females were removed from the communal tubs and placed in individual 500 mL plastic tubs with ad libitum rabbit chow, cotton plugged-water vials, and egg carton substrate. Newly eclosed males were also removed from the tub so that juvenile females would not be exposed to male calling (males do not call before Day 3 post-eclosion in this species; Roff, unpublished data). After removal from the communal tubs, adult females were randomly assigned to one of 4 experimental treatments (in a 2 × 2 factorial design): females were either exposed to male calling song chorus (exposure treatment) or not (silent treatment), and the length of the treatment (exposure or silence) lasted for either 3 or 6 days after the adult molt. We chose 2 different lengths of acoustic treatment (3 or 6 days) because we were uncertain about the minimum length of time necessary for female reproductive physiology to respond to acoustic cues. Previous work has shown that DLM histolysis and ovary growth occur during this time window (including days 3–6 after adult eclosion [Mole and Zera 1994; Stirling et al. 2001]), and that conditions experienced at the time of adult emergence can affect ovary growth (Roff 1989; Roff and Gelinas 2003). The exposure treatment and the silent treatment took place in two separate anechoic chambers that remained consistent within experiment but were switched between Experiments 1 and 2. Within each experiment, temperatures were highly uniform between chambers (mean ± S.E. temperatures in Experiment 1: exposure chamber = 28.90 ± 0.02 °C, silent chamber = 28.67 ± 0.02 °C; in Experiment 2: exposure chamber = 27.57 ± 0.02 °C, silent chamber = 27.89 ± 0.01 °C). In the exposure treatment, male calling song chorus consisted of multiple G. firmus males chirping (continuously looped every 9 min) and was broadcasted at ca. 70 decibels from a central source in the experimental chamber. After the assigned 3 or 6 days of acoustic treatment, females were freeze-killed and preserved in 70% ethanol. From each female, we dissected out the major flight muscles (DLMs) and ovaries (including all attached eggs). The person performing dissections was blind to the acoustic treatment and length of treatment of each female. A useful feature of G. firmus females is that virgin females develop eggs for at least seven days without resorption or laying infertile eggs: thus ovary mass is an excellent index of fecundity to age within this period (Roff 1994). We dried the DLMs and ovaries at 70 °C for 24 h and recorded the dry weights to the nearest 0.0001 g. Data analysis To test for a causal effect of acoustic exposure on DLM mass and ovary mass, we conducted a path analysis. Path analysis is an appropriate method of analysis because it can accommodate the non-independence of the two response variables, DLM mass and ovary mass (DLM mass has a significant negative causal effect on ovary mass; Roff and Fairbairn 2011). Path analysis has the ability to detect the effect of a main causal variable (i.e. acoustic treatment) on a dependent variable (e.g. ovary mass) while accounting for the effects of other intermediate causal variables (e.g. DLM mass) on the specified dependent variable. We ran the path analysis using maximum likelihood estimation in SPSS AMOS version 5.0.1. We performed separate path analyses for LW females (n = 162) and SW females (n = 112) to test for the effect of acoustic exposure on DLM mass and ovary mass within each wing morph, as the 2 morphs drastically differ in mean ovary and DLM masses (Roff 1984). A few factors other than acoustic exposure (yes or no) had the potential to explain variance in our 2 dependent variables (DLM mass and ovary mass): experiment (Experiment 1 or 2) and day [length of acoustic treatment (3 or 6 days), which was also the day on which crickets were preserved for measurement]. Therefore, we included these additional predictor variables in our a priori causal path model (Figure 1). We predicted the same a priori model for LW females and SW females but predicted that coefficients would vary between morphs (e.g. acoustic exposure would have a stronger effect on DLM mass in LW than SW females). Figure 1 View largeDownload slide Proposed path model. Arrows point from causal to dependent variables. The sign of the effect of each causal on each dependent variable is depicted as positive (+) or negative (1). Three variables have been coded as dummy variables: exposure (where “0” represents the silent treatment and “1” represents the calling song treatment), day (where “0” represents the 3-day treatment and “1” represents that 6-day treatment), and experiment (where “0” represents Experimental Block 1 and “1” represents Experimental Block 2). DLM mass and ovary mass are both continuous and measured in milligrams. See main text for description of predicted effects. Error terms have been omitted. Figure 1 View largeDownload slide Proposed path model. Arrows point from causal to dependent variables. The sign of the effect of each causal on each dependent variable is depicted as positive (+) or negative (1). Three variables have been coded as dummy variables: exposure (where “0” represents the silent treatment and “1” represents the calling song treatment), day (where “0” represents the 3-day treatment and “1” represents that 6-day treatment), and experiment (where “0” represents Experimental Block 1 and “1” represents Experimental Block 2). DLM mass and ovary mass are both continuous and measured in milligrams. See main text for description of predicted effects. Error terms have been omitted. In path analysis, arrows specify the direction of the relationship between two variables (pointing from causal to dependent variable), and signs (+/−) indicate the positive or negative effect of the causal on the dependent variable. In our model, DLM mass and ovary mass are continuous variables. Exposure was coded as “0” (silent treatment) or “1” (calling song playback). We hypothesized that exposure should have a negative effect on DLM mass (such that exposed females should have more greatly histolyzed, smaller DLM mass than unexposed females) and a positive effect on ovary mass (such that exposed females would amass larger ovaries than unexposed females). Day was coded as a dummy variable, “0” (Day 3) or “1” (Day 6); we predicted that with an increase in day, DLM mass should decrease and ovary mass should increase. Although SW females have small, nonfunctional flight muscles at eclosion, previous work has shown that these are nevertheless histolyzed over time as in LW females (see Introduction). Experiment was coded as a dummy variable, “0” (Experiment 1) or “1” (Experiment 2); we had no a priori predictions about the effect of experiment on DLM mass or ovary mass. Lastly, we predicted that larger DLM masses should result in smaller ovary masses, such that DLM mass should have a negative effect on ovary mass as previously described (see Introduction). For each wing morph, we assessed the fit of our a priori model to the data set using a χ2 test, where a nonsignificant χ2 value indicates a good fit. We also assessed model fit using an alternative index, the root mean square error of approximation (RMSEA). RMSEA values less than 0.07 denote a good fit and RMSEA values less than 0.03 signify an excellent fit (Steiger 2007). We used a χ2 difference test to determine whether a simpler model lacking the biologically inconsequential variable experiment fit the data as well as the full model including experiment (Tabachnick and Fidell 2007). In a χ2 difference test, if the difference in χ2 values between a complex model (e.g. including experiment) and a simpler model (e.g. lacking experiment) is not significant (based on adjusted degrees of freedom), the simpler model is preferred. We assessed multivariate normality, an assumption of path analysis, by evaluating Mardia’s multivariate kurtosis estimate. A perfectly normal multivariate distribution would have a Mardia’s multivariate kurtosis estimate of 0 (DeCarlo 1997; Arbuckle and Wothke 1999), but multivariate kurtosis estimates within ±7 units of 0 are acceptable for maximum likelihood estimation (West et al. 1995; Schepers 2004; Byrne 2010; Cohen et al. 2013). To illustrate the biological significance of the responses of ovary and DLM mass to song exposure, and to compare relative responses between LW and SW females, we computed the percent change in the response variables as a function of acoustic treatment. RESULTS Fit and selection of path model For both LW females and SW females, the a priori model (Figure 1) provided an excellent fit, as indicated by nonsignificant χ2 values and RMSEA values less than 0.07 (LW female model: χ2 = 1.335, df = 3, P = 0.721, RMSEA < 0.0001; SW female model: χ2 = 4.075, df = 3, P = 0.253, RMSEA = 0.057). Removing experiment from the model still resulted in nonsignificant χ2 values and lowered the RMSEA value for SW females (LW females: χ2 = 0.420, df = 1, P = 0.517, RMSEA < 0.0001; SW females: χ2 = 0.036, df = 1, P = 0.850, RMSEA < 0.0001). The full model (containing experiment) and reduced model (lacking experiment) differed by 2 degrees of freedom, so the χ2 difference (χ2reduced model minus χ2full model) would have to be +5.99 (at α = 0.05) to justify the selection of the full model over the reduced model. For LW females, the χ2 difference was −0.915, and for SW females, the χ2 difference was −4.039; therefore, for both wing morphs, we selected the reduced model (Figure 2) over the full model. Figure 2 View largeDownload slide Fitted path model. On each path, coefficients are presented as: LW path coefficient/SW path coefficient. Significant path coefficients are shown in bold. Solid path lines were significant for both wing morphs. Dashed lines show paths significant in LW only and dotted lines show paths that was not significant in either morph. Predicted directions of effects shown under each path. Figure 2 View largeDownload slide Fitted path model. On each path, coefficients are presented as: LW path coefficient/SW path coefficient. Significant path coefficients are shown in bold. Solid path lines were significant for both wing morphs. Dashed lines show paths significant in LW only and dotted lines show paths that was not significant in either morph. Predicted directions of effects shown under each path. The multivariate normality assumption was not violated in either data set (Mardia’s multivariate kurtosis estimates were within ±7 units of 0). For LW females, Mardia’s multivariate kurtosis estimate was −2.623. For SW females, Mardia’s multivariate kurtosis estimate was 0.051. Path model: LW females Our path analysis of LW females’ responses (Figure 2, Table 1) revealed a significant, positive effect of acoustic exposure on ovary mass (path coefficient = 0.086, P = 0.046), such that females exposed to male calling song during adulthood had larger ovaries than unexposed females. However, while the effect of acoustic exposure on DLM mass was negative as predicted, it was not statistically significant (path coefficient = −0.074, P = 0.167). Day (length of treatment and day on which females were preserved for measurement) had a significant negative effect on DLM mass (path coefficient = −0.226, P = 0.002), indicating that DLM histolysis occurred as time passed, as expected. Also as predicted, day had a significant positive effect on ovary mass (path coefficient = 0.588, P < 0.001), indicating that ovary mass increased as time passed. DLM mass had a significant negative effect on ovary mass (path coefficient = −0.364, P < 0.001), suggesting that DLM histolysis was linked to ovary growth in this sample of females (see Introduction). Table 1 Coefficients for the path analyses Path  Path coefficient      Standardized  Raw (SE)  P*  LW females: n = 162, Chi-square = 0.420, df = 1, P = 0.517, RMSEA = 0  Day ➔ DLM mass  −0.226  −1.492 (0.506)  0.002  Day ➔ ovary mass  0.588  55.149 (4.886)  <0.001  DLM mass ➔ ovary mass  −0.364  −5.169 (0.741)  <0.001  Exposure ➔ DLM mass  −0.074  −0.489 (0.505)  0.167  Exposure ➔ ovary mass  0.086  8.020 (4.765)  0.046  SW females: n = 112, Chi-square = 0.036, df = 1, P =0.85, RMSEA = 0  Day ➔ DLM mass  −0.176  −0.289 (0.152)  0.029  Day ➔ ovary mass  0.885  86.83 (4.138)  <0.001  DLM mass ➔ ovary mass  −0.055  −3.279 (2.538)  0.098  Exposure ➔ DLM mass  0.117  0.191 (0.152)  0.208§  Exposure ➔ ovary mass  0.079  7.751 (4.085)  0.029  Path  Path coefficient      Standardized  Raw (SE)  P*  LW females: n = 162, Chi-square = 0.420, df = 1, P = 0.517, RMSEA = 0  Day ➔ DLM mass  −0.226  −1.492 (0.506)  0.002  Day ➔ ovary mass  0.588  55.149 (4.886)  <0.001  DLM mass ➔ ovary mass  −0.364  −5.169 (0.741)  <0.001  Exposure ➔ DLM mass  −0.074  −0.489 (0.505)  0.167  Exposure ➔ ovary mass  0.086  8.020 (4.765)  0.046  SW females: n = 112, Chi-square = 0.036, df = 1, P =0.85, RMSEA = 0  Day ➔ DLM mass  −0.176  −0.289 (0.152)  0.029  Day ➔ ovary mass  0.885  86.83 (4.138)  <0.001  DLM mass ➔ ovary mass  −0.055  −3.279 (2.538)  0.098  Exposure ➔ DLM mass  0.117  0.191 (0.152)  0.208§  Exposure ➔ ovary mass  0.079  7.751 (4.085)  0.029  *P-values are 1-tailed, as the signs of the observed path coefficients were in the predicted direction except in one case (§), where, because the direction is opposite to that predicted, we show the 2-tailed P-value. View Large Path model: SW females Path analysis of SW females’ responses (Figure 2, Table 1) also indicated a significant, positive effect of acoustic exposure on ovary mass (path coefficient = 0.079, P = 0.029) but a nonsignificant effect of exposure on DLM mass (path coefficient = 0.117, P = 0.208). Day had a significant, positive effect on ovary mass (path coefficient = 0.885, P < 0.001) and a weaker but significant negative effect on DLM mass (path coefficient = −0.176, P = 0.029). This reduction in DLM response to day relative to the response observed in LW females is consistent with previous studies (see Introduction), and given the small size of the SW females’ flight muscles it is not surprising that there was no significant relationship between DLM mass and ovary mass (path coefficient = −0.055, P = 0.098). Biological significance of acoustic exposure and comparisons between morphs We hypothesized that the relative increase in ovary mass should be greater in exposed LW females than exposed SW females. To test this, we calculated the percent change in ovary mass in exposed versus unexposed females in each wing morph on either Day 3 or Day 6 post-eclosion. Among LW individuals, females exposed to calling song and measured at Day 3 had ovaries that were 30% heavier than females unexposed and measured at Day 3 (Table 2). While females exposed for 6 days had larger ovaries, the percent increase in ovary mass of 28% was nearly identical to that between exposed and unexposed females measured at Day 3. Thus, in LW females, acoustic exposure for 3 days was sufficient to evoke a relative response equivalent to that induced by 6 days of exposure. The observed 28–30% increase in ovary mass in LW females exposed to calling song translates into a similar increase in earlier fecundity, and therefore has a potentially large impact on fitness (Roff 1994). Table 2 Means (standard errors) and percent change in ovary mass and DLM mass as a function of acoustic exposure Wing morph  Day  Unexposed  Exposed  % Change in exposed Females  Ovary mass (mg)  LW  3  11.69 (1.73)  15.22 (1.88)  30%  LW  6  66.82 (8.01)  85.35 (7.85)  28%  SW  3  13.68 (2.71)  14.16 (1.75)  3%  SW  6  95.38 (4.80)  108.06 (5.11)  13%  DLM mass (mg)  LW  3  5.75 (0.48)  5.39 (0.48)  −6%  LW  6  4.40 (0.59)  3.77 (0.50)  −14%  SW  3  1.07 (0.14)  1.25 (0.15)  17%  SW  6  0.77 (0.14)  0.97 (0.17)  26%  Wing morph  Day  Unexposed  Exposed  % Change in exposed Females  Ovary mass (mg)  LW  3  11.69 (1.73)  15.22 (1.88)  30%  LW  6  66.82 (8.01)  85.35 (7.85)  28%  SW  3  13.68 (2.71)  14.16 (1.75)  3%  SW  6  95.38 (4.80)  108.06 (5.11)  13%  DLM mass (mg)  LW  3  5.75 (0.48)  5.39 (0.48)  −6%  LW  6  4.40 (0.59)  3.77 (0.50)  −14%  SW  3  1.07 (0.14)  1.25 (0.15)  17%  SW  6  0.77 (0.14)  0.97 (0.17)  26%  View Large As we predicted, acoustic exposure in SW females had a lower effect on the increase in ovary mass compare to LW females (Figure 3), with ovaries in exposed SW females being 3% larger than in unexposed SW females at Day 3 and 13% larger at Day 6 (Table 2, Figure 3). Thus, in SW females, the effect of acoustic exposure appeared to slightly increase with the length of exposure, but overall evoked a weaker relative response than in LW females. Despite the fact that SW females reacted less strongly to acoustic exposure than did LW females, the 13% increase in ovary growth in SW females exposed for six days still represents a potentially biologically impactful effect on fitness. Figure 3 View largeDownload slide Variation in ovary and DLM masses in relation to acoustic treatment, wing morph, and days since final molt. In addition to the path analysis, we ran Type III stepwise general linear models of each response variable (log-ovary mass, DLM mass) on acoustic treatment, wing morph, day, and all interactions. For ovary mass, the final significant model (F4,269 = 105.4, P < 0.0001, R2adjusted = 0.61) contained acoustic treatment (F1,269 = 8.95, P = 0.003), wing morph (F1,269 = 14.66, P < 0.001), day (F1,269 = 196.89, P < 0.001), and a wing morph*day interaction (F1,269 = 4.11, P = 0.044): log(y)=41.89+1.12E+0.10W+24.55D+0.51W*D where y is ovary mass, E is exposure (0=unexposed, 1=exposed), W is wing morph (0=SW, 1=LW) and D is day (Day 3 = 0, Day 6 = 1). For DLM mass, the final significant model (F2,271 = 80.92, P < 0.001, R2adjusted = 0.37) contained wing morph (F1,271 = 144.35, P < 0.001) and day (F1,271 = 10.74, P = 0.001): DLM=0.5432+3.78W−1.02D. Shown are means ± standard errors. Clockwise from the top left: ovary masses after 3 days of treatment, DLM masses of SW females, DLM masses of LW females, ovary masses after 6 days of treatment. Figure 3 View largeDownload slide Variation in ovary and DLM masses in relation to acoustic treatment, wing morph, and days since final molt. In addition to the path analysis, we ran Type III stepwise general linear models of each response variable (log-ovary mass, DLM mass) on acoustic treatment, wing morph, day, and all interactions. For ovary mass, the final significant model (F4,269 = 105.4, P < 0.0001, R2adjusted = 0.61) contained acoustic treatment (F1,269 = 8.95, P = 0.003), wing morph (F1,269 = 14.66, P < 0.001), day (F1,269 = 196.89, P < 0.001), and a wing morph*day interaction (F1,269 = 4.11, P = 0.044): log(y)=41.89+1.12E+0.10W+24.55D+0.51W*D where y is ovary mass, E is exposure (0=unexposed, 1=exposed), W is wing morph (0=SW, 1=LW) and D is day (Day 3 = 0, Day 6 = 1). For DLM mass, the final significant model (F2,271 = 80.92, P < 0.001, R2adjusted = 0.37) contained wing morph (F1,271 = 144.35, P < 0.001) and day (F1,271 = 10.74, P = 0.001): DLM=0.5432+3.78W−1.02D. Shown are means ± standard errors. Clockwise from the top left: ovary masses after 3 days of treatment, DLM masses of SW females, DLM masses of LW females, ovary masses after 6 days of treatment. Power analysis: acoustic exposure and flight muscle histolysis In the path analyses, the effect of acoustic exposure on DLM mass was statistically nonsignificant in both LW females and SW females. In LW females, acoustic exposure did reduce DLM mass by 6% and 14% at Days 3 and 6, respectively (Table 2, Figure 3). Because the direction of difference was as predicted, we conducted a simple power analysis based on a 2-sample t-test to determine, approximately, the required sample size for significance given the observed difference and the difference that could be detected given the sample size used for LW females. We set the alpha probability at 0.05 and power at 0.80 (Cohen 1988). The standard deviations for DLM masses on Days 3 and 6 ranged from 3.1 to 3.5: a sample size of greater than 400 individuals would be required to detect the observed differences. The sample size in the present data was 162 LW females, thus falling far short of the required number. The average difference between days 3 and 6 was approximately 0.5, and given the present sample size only a difference twice as large as this could be detected. In SW females, DLM mass was not significantly different between females, though those exposed to calling song had somewhat larger DLMs at Day 3 (+17%) and Day 6 (+26%). However, DLM masses in SW females were tiny, as expected, compared to those of LW females (Table 2, Figure 3) and therefore any small difference in DLM mass between exposed and unexposed females is overly emphasized when treated as a percent difference. DISCUSSION Here, we provide evidence that the social mating environment can induce a plastic response in female reproductive physiology, such that females exposed to male calling song invested more heavily in ovary mass (fecundity), a life-history trait directly linked to fitness, than unexposed females. Because LW females have larger energy stores that are initially uncommitted to reproduction, we predicted that LW females would respond more strongly to exposure than SW females. This prediction was upheld, with exposed LW females showing a 28–30% increase in ovary mass at both ages measured, whereas exposed SW females increased ovary mass by only 3% at day three and 13% at Day 6. In both wing morphs, the increase shown at Day 6 represents a substantial increase in early fecundity and hence would potentially have an impact on fitness. In the absence of acoustic cues indicating reproductively viable males, the maintenance of dispersal capability in LW females is favored. Given that SW females cannot disperse (at least by flight) there is less of a fitness advantage to altering the onset of reproduction. That we did observe an earlier onset of reproduction in exposed SW females (compared to unexposed SW females) suggests that there is a cost to the early development of eggs in the absence of mature males. One possibility is that agility is impeded by carrying eggs, which could make females more vulnerable to predation. Another possibility is that resources may potentially be scarce and the allocation of resources to eggs may place a physiological burden on females, thereby increasing their mortality rate. Females given reduced rations do in fact show a decrease in ovary mass indicating a plastic response to resource availability (Roff and Gelinas 2003). Exposure to calling song did not have a statistically significant effect on flight muscle histolysis in either wing morph. However, the decrease in DLM mass of 14% in LW females on Day 6 was potentially biologically meaningful. The power analysis showed that the lack of statistical significance, assuming that the decrease in DLM mass was real, could be attributed to relatively large standard errors. On the other hand, the increased ovary masses of LW females could also have been caused by reallocation of flight fuel reserves, which we did not measure in this study. Lipids serve as flight fuels in G. firmus and are negatively correlated with ovary mass (Zera et al. 1997), suggesting that the life-history trade-off between flight and reproduction involves multiple aspects of dispersal capability. While calling song exposure did not show a statistically significant effect on flight muscle histolysis, it nevertheless has the potential to alter the dispersal propensity of LW females via its significant effect on ovary mass. By accelerating the onset of reproduction (i.e. the growth of ovaries), calling song exposure may reduce the ability of LW females to fly, if possessing a heavier, egg-laden body limits flight capability. Flight ability in LW females might also be impaired by the reallocation of flight fuels to ovary growth in exposed females. Thus, if LW females mature to adulthood in an environment in which reproductively viable males are actively calling, female emigration out of the current habitat patch may decrease, subsequently reducing gene flow between populations. Our study joins a growing body of literature (see Introduction) documenting phenotypic plasticity in response to the experience of conspecific, social (primarily sexual) signals. Such plasticity should enable individuals to maximize their reproductive success in the current social environment (Kasumovic and Brooks 2011; Rodriguez et al. 2013). 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The metabolic basis of life history variation: Genetic and phenotypic differences in lipid reserves among life history morphs of the wing-polymorphic cricket, Gryllus firmus. J Insect Phys . 47: 1147– 1160. Google Scholar CrossRef Search ADS   © The Author(s) 2018. Published by Oxford University Press on behalf of the International Society for Behavioral Ecology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Behavioral Ecology Oxford University Press

Adult social environment alters female reproductive investment in the cricket Gryllus firmus

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Abstract

Abstract Phenotypically plastic responses have been increasingly documented in response to intraspecific, behavioral (often sexual) signals such as mating calls. We explored the effect of short-term exposure to male calling song on a reproductive life-history trade-off in adult females of the wing dimorphic cricket species, Gryllus firmus. In G. firmus, long-winged females possess flight muscles and small ovaries immediately after the adult molt, whereas short-winged females possess small, nonfunctional flight muscles and large ovaries at the same age. In long-winged females, flight muscles are histolyzed during ovary growth that occurs after the adult molt. Because of these differences in reproductive physiology, we predicted different responses to calling song exposure between the 2 morphs. We hypothesized that such exposure would boost ovary growth in both wing morphs, but also increase flight muscle histolysis in long-winged females, producing a greater relative response to exposure in this morph. As expected, we saw a significant increase in ovary mass in exposed females of both morphs, and a greater relative response in long-winged females. Calling song exposure did not have a strong effect on flight muscle histolysis, suggesting that the relatively larger ovary response in long-winged females was perhaps fueled instead by reallocation of flight fuels. Our study documents the rapid adult response of a fitness-linked trait to the adult social environment, a result with far-reaching implications, as the experience of mating signals during adulthood should be nearly universal across species. INTRODUCTION Phenotypic plasticity enables individuals to respond to environmental variability, both abiotic and biotic. Increasingly, interest has focused on plastic responses to variation in the intraspecific social environment (Bailey and Zuk 2008; Kasumovic and Brooks 2011; Rebar et al. 2016). The social environment of a given population contains individuals occupying a number of (nonmutually exclusive) roles: parent, offspring, sibling, competitor (for food or mates), and/or mate. Several broad patterns of plastic responses to the presence of individuals occupying these roles have begun to emerge across vertebrate and invertebrate taxa (see reviews in Kasumovic and Brooks 2011; Rodriguez et al. 2013; Vitousek et al. 2014; Groothuis and Taborsky 2015; Trillmich et al. 2015; Taborsky 2016). In this paper, we focus on plastic responses to variation in the mating environment. Effects of exposure to mating competitors The presence of intrasexual competitors for mates in the social environment induces several responses shared across taxa. Males placed with other males (or with signals or cues produced by males, e.g. pheromones, calling songs, etc.), either during the juvenile period or adulthood, tend to invest more in putatively sexually competitive traits than males housed with females or in isolation. For example, males housed with other males may exhibit higher levels of testosterone (blue-black grassquits, Volatinia jacarina: Lacava et al. 2011), increase elaboration of secondary sexual traits (Pacific field crickets, Teleogryllus oceanicus:Thomas et al. 2011; blue-black grassquits, Volatinia jacarina; Maia et al. 2012), or expend more effort in sperm competition (Pacific field crickets, Teleogryllus oceanicus: Bailey et al. 2011; Gray and Simmons 2013), than males housed with females or in isolation. Rearing juvenile males in the presence of adult males or adult male cues tends to retard male development rates to sexual maturity (guppies, Poecilia reticulata: Rodd et al. 1997; Magellan and Magurran 2009; green swordtails, Xiphophorus helleri: Walling et al. 2007; black field crickets, Teleogryllus commodus: Kasumovic et al. 2011; but see Maia et al. 2012), likely in an attempt to delay maturity until a time when fewer mating competitors might be present. When females are placed with other females, a striking and widespread effect that often occurs is the suppression or total inhibition of ovary development and function in subordinate females by dominant females, typically mediated by pheromones, in hymenopterans (de Groot and Voogd 1954; Nunes et al. 2014; Van Oystaeyen et al. 2014; Traynor et al. 2017), mammals (Wasser and Barash 1983; Faulkes et al. 1990) and western mosquitofish, Gambusia affinis (Lutnesky and Adkins 2003). Like males, juvenile females may also delay development to maturity if exposed to other females (house mice, Mus musculus: Drickamer 1975, 1977). Effects of exposure to potential mates The occurrence of potential mates in the social environment also has marked effects on behavioral, morphological, and life-history traits. The presence of adults of the opposite sex during focal individuals’ juvenile period may accelerate development to sexual maturity (house mice, Mus musculus [Drickamer 1974]; redback spiders, Latrodectus hasselti [Kasumovic and Andrade 2006; Stolz et al. 2012]; black field crickets, Teleogryllus commodus [Kasumovic et al. 2011]), such that focal individuals are more likely to mature when mates are present. Exposure to reproductively viable adult males or male signals during females’ juvenile or adult periods reduces female responsiveness to male courtship signals (Teleogryllus cricket spp.: Bailey and Zuk 2008; Bailey and Macleod 2014), increases choosiness (Pacific field crickets, T. oceanicus: Bailey and Zuk 2008; Bailey and Zuk 2009), and alters females’ preferences for male phenotypes and/or female preference functions during mating trials (wolf spiders, Schizocosa spp. [Hebets 2003; Stoffer and Uetz 2015]; highland swordtails, Xiphophorus malinche [Tudor and Morris 2009]; treehoppers, Enchenopa binotata [Fowler-Finn and Rodriguez 2012]; Pacific field crickets, T. oceanicus [Swanger and Zuk 2015]). Females may even attend to differences in the quality of males to which they are exposed, and alter their reproductive investment accordingly. Hert (1989) found that cichlid (Astatotilapia elegans) females exposed to males possessing intact breeding markings were more likely to lay clutches than females exposed to males with removed breeding markings, whereas Kasumovic et al. (2011) discovered that black field cricket (T. commodus) females exposed to a chorus of numerous, high quality males had larger ovaries (a proxy for pre-mating reproductive investment) than females exposed to fewer low quality males or no males. Study system and hypotheses To add to the growing collection of studies exploring phenotypic plasticity in response to the social environment, we tested the effect of adult-period exposure to a male mating signal (male calling song, a long distance attraction signal) on a life-history trade-off between reproduction and flight capability in females of the wing dimorphic sand cricket, Gryllus firmus. Gryllus firmus, a large (adult mass ca. 0.7 g) cricket inhabiting beaches and other sandy areas on the east coast of the United States, occurs in 2 morphs: a flight-capable, macropterous (long-winged, hereafter LW) morph and a flight-incapable, micropterous (short-winged, hereafter SW) morph. Gryllus firmus LW adult females undergo a life-history trade-off known as the “oogenesis-flight syndrome” (Johnson 1969). In this syndrome, after the molt to adulthood, LW females initially experience a flight-capable, non-reproductive phase (possessing large, functional flight muscles and small, immature ovaries) followed by a flight-incapable, reproductive phase (with small, histolyzed flight muscles and large, mature ovaries). During the switch from flight capability to flight incapability in LW females, histolyzed flight muscle tissue and stored flight fuels (primarily lipids) can be reallocated to ovary growth. This reallocation has been observed in the monomorphically macropterous cricket, Gryllus bimaculatus (Lorenz 2007) and the LW morph of G. firmus (Stirling et al. 2001). Meanwhile, SW females have small, vestigial flight muscles and rudimentary wings (rendering them incapable of flight), and possess large ovaries after eclosion (Roff 1984). Because SW females do not pass through a flying dispersal phase and do not allocate energy to flight muscle development and maintenance of flight fuels (Roff 1984; Mole and Zera 1994; Stirling et al. 2001, Zera and Larsen 2001; Crnokrak and Roff 2002), SW females of G. firmus begin reproduction earlier and have greater cumulative fecundity than LW females (Roff 1984). One of the consequences of this reproductive allocation pattern in LW females of G. firmus is a significant negative covariance between the mass of the main flight muscles (the dorso-longitudinal muscles, hereafter DLMs) and ovary mass, an index of fecundity (Roff 1994). Although SW females do not have functional DLMs, there is still a significant negative covariance between ovary mass and DLM mass (Zera et al. 1997; Stirling et al. 2001), which can be attributed to genetic correlations between wing morphs, DLM mass and ovary mass (Roff and Gelinas 2003; Roff and Fairbairn 2011, 2012). In this species (as in other Orthopteran species), male calling song advertises the presence of reproductively capable males. Hence, we hypothesized: 1) that adult females (of both wing morphs) exposed to male calling song would invest more energy in reproduction, as indicated by an increase in ovary mass, than unexposed females. Because of the trade-off between components of flight capability, such as DLM mass/ flight fuels, and ovary mass, we further hypothesized: 2) that the exposed females would reallocate flight-capability components (DLMs) to the ovaries. This response should be most evident in LW females but, because of the aforementioned genetic correlations, may also be present in SW females. Our last hypothesis pertains to differences in the relative ovary investment response between the wing morphs. Regardless of acoustic treatment, SW females should allocate more total energy to ovary growth than LW females (Roff 1984), and therefore should have fewer alternate sources of energy that they could reallocate to ovary growth in response to social signals such as calling song. Because LW females have larger additional energy stores that are initially uncommitted to reproduction (i.e. flight fuels and flight muscles), we predicted that in LW females, reallocation to ovary growth would likely include both the resources devoted to flight capability and those sources also used by SW females. Consequently, we hypothesized: 3) that the relative increase in ovary mass should be greater in exposed LW females than exposed SW females. We applied two different lengths of exposure (for either 3 days or 6 days after eclosion to adulthood) to determine if the length of exposure affected the magnitude of the responses of ovary mass and flight muscle mass. MATERIALS AND METHODS Experimental treatments We performed our acoustic exposure experiment in two blocks, which we shall refer to as Experiment 1 (performed in January 2016; total n = 115, with 98 LW females and 17 SW females) and Experiment 2 (performed in April–May 2016; total n = 159, with 64 LW females and 95 SW females). The total sample size for both experiments combined was 274 individuals. In both Experiments 1 and 2, individuals came from a lab population of G. firmus that was descended from a field sample collected in 2013 near Jacksonville, Florida. The field sample included 76 gravid females, ensuring that the subsequent lab colony did not suffer from founder effects. Individuals used in this study were the 6th generation (Experiment 1) and 7th generation (Experiment 2) descendants of the founding individuals. Because of logistical constraints, rearing densities differed slightly between Experiments 1 and 2, but the actual experimental protocol was identical. In Experiment 1, nymphs were raised in mixed-sex 63-L communal tubs (initial density ca. 1000–1200 individuals per tub) at 28 °C on a 15:9 h light:dark photoperiod and were supplied with ad libitum crushed Purina rabbit chow, cotton plugged-water vials, and egg cartons for shelter. In Experiment 2, conditions were identical except that starting nymphal density was ca. 1500 individuals in a single 63-L tub. In both experiments, when individuals neared the penultimate instar, we checked the tubs daily (in the morning) for the presence of emerging adults. Newly eclosed LW and SW females were removed from the communal tubs and placed in individual 500 mL plastic tubs with ad libitum rabbit chow, cotton plugged-water vials, and egg carton substrate. Newly eclosed males were also removed from the tub so that juvenile females would not be exposed to male calling (males do not call before Day 3 post-eclosion in this species; Roff, unpublished data). After removal from the communal tubs, adult females were randomly assigned to one of 4 experimental treatments (in a 2 × 2 factorial design): females were either exposed to male calling song chorus (exposure treatment) or not (silent treatment), and the length of the treatment (exposure or silence) lasted for either 3 or 6 days after the adult molt. We chose 2 different lengths of acoustic treatment (3 or 6 days) because we were uncertain about the minimum length of time necessary for female reproductive physiology to respond to acoustic cues. Previous work has shown that DLM histolysis and ovary growth occur during this time window (including days 3–6 after adult eclosion [Mole and Zera 1994; Stirling et al. 2001]), and that conditions experienced at the time of adult emergence can affect ovary growth (Roff 1989; Roff and Gelinas 2003). The exposure treatment and the silent treatment took place in two separate anechoic chambers that remained consistent within experiment but were switched between Experiments 1 and 2. Within each experiment, temperatures were highly uniform between chambers (mean ± S.E. temperatures in Experiment 1: exposure chamber = 28.90 ± 0.02 °C, silent chamber = 28.67 ± 0.02 °C; in Experiment 2: exposure chamber = 27.57 ± 0.02 °C, silent chamber = 27.89 ± 0.01 °C). In the exposure treatment, male calling song chorus consisted of multiple G. firmus males chirping (continuously looped every 9 min) and was broadcasted at ca. 70 decibels from a central source in the experimental chamber. After the assigned 3 or 6 days of acoustic treatment, females were freeze-killed and preserved in 70% ethanol. From each female, we dissected out the major flight muscles (DLMs) and ovaries (including all attached eggs). The person performing dissections was blind to the acoustic treatment and length of treatment of each female. A useful feature of G. firmus females is that virgin females develop eggs for at least seven days without resorption or laying infertile eggs: thus ovary mass is an excellent index of fecundity to age within this period (Roff 1994). We dried the DLMs and ovaries at 70 °C for 24 h and recorded the dry weights to the nearest 0.0001 g. Data analysis To test for a causal effect of acoustic exposure on DLM mass and ovary mass, we conducted a path analysis. Path analysis is an appropriate method of analysis because it can accommodate the non-independence of the two response variables, DLM mass and ovary mass (DLM mass has a significant negative causal effect on ovary mass; Roff and Fairbairn 2011). Path analysis has the ability to detect the effect of a main causal variable (i.e. acoustic treatment) on a dependent variable (e.g. ovary mass) while accounting for the effects of other intermediate causal variables (e.g. DLM mass) on the specified dependent variable. We ran the path analysis using maximum likelihood estimation in SPSS AMOS version 5.0.1. We performed separate path analyses for LW females (n = 162) and SW females (n = 112) to test for the effect of acoustic exposure on DLM mass and ovary mass within each wing morph, as the 2 morphs drastically differ in mean ovary and DLM masses (Roff 1984). A few factors other than acoustic exposure (yes or no) had the potential to explain variance in our 2 dependent variables (DLM mass and ovary mass): experiment (Experiment 1 or 2) and day [length of acoustic treatment (3 or 6 days), which was also the day on which crickets were preserved for measurement]. Therefore, we included these additional predictor variables in our a priori causal path model (Figure 1). We predicted the same a priori model for LW females and SW females but predicted that coefficients would vary between morphs (e.g. acoustic exposure would have a stronger effect on DLM mass in LW than SW females). Figure 1 View largeDownload slide Proposed path model. Arrows point from causal to dependent variables. The sign of the effect of each causal on each dependent variable is depicted as positive (+) or negative (1). Three variables have been coded as dummy variables: exposure (where “0” represents the silent treatment and “1” represents the calling song treatment), day (where “0” represents the 3-day treatment and “1” represents that 6-day treatment), and experiment (where “0” represents Experimental Block 1 and “1” represents Experimental Block 2). DLM mass and ovary mass are both continuous and measured in milligrams. See main text for description of predicted effects. Error terms have been omitted. Figure 1 View largeDownload slide Proposed path model. Arrows point from causal to dependent variables. The sign of the effect of each causal on each dependent variable is depicted as positive (+) or negative (1). Three variables have been coded as dummy variables: exposure (where “0” represents the silent treatment and “1” represents the calling song treatment), day (where “0” represents the 3-day treatment and “1” represents that 6-day treatment), and experiment (where “0” represents Experimental Block 1 and “1” represents Experimental Block 2). DLM mass and ovary mass are both continuous and measured in milligrams. See main text for description of predicted effects. Error terms have been omitted. In path analysis, arrows specify the direction of the relationship between two variables (pointing from causal to dependent variable), and signs (+/−) indicate the positive or negative effect of the causal on the dependent variable. In our model, DLM mass and ovary mass are continuous variables. Exposure was coded as “0” (silent treatment) or “1” (calling song playback). We hypothesized that exposure should have a negative effect on DLM mass (such that exposed females should have more greatly histolyzed, smaller DLM mass than unexposed females) and a positive effect on ovary mass (such that exposed females would amass larger ovaries than unexposed females). Day was coded as a dummy variable, “0” (Day 3) or “1” (Day 6); we predicted that with an increase in day, DLM mass should decrease and ovary mass should increase. Although SW females have small, nonfunctional flight muscles at eclosion, previous work has shown that these are nevertheless histolyzed over time as in LW females (see Introduction). Experiment was coded as a dummy variable, “0” (Experiment 1) or “1” (Experiment 2); we had no a priori predictions about the effect of experiment on DLM mass or ovary mass. Lastly, we predicted that larger DLM masses should result in smaller ovary masses, such that DLM mass should have a negative effect on ovary mass as previously described (see Introduction). For each wing morph, we assessed the fit of our a priori model to the data set using a χ2 test, where a nonsignificant χ2 value indicates a good fit. We also assessed model fit using an alternative index, the root mean square error of approximation (RMSEA). RMSEA values less than 0.07 denote a good fit and RMSEA values less than 0.03 signify an excellent fit (Steiger 2007). We used a χ2 difference test to determine whether a simpler model lacking the biologically inconsequential variable experiment fit the data as well as the full model including experiment (Tabachnick and Fidell 2007). In a χ2 difference test, if the difference in χ2 values between a complex model (e.g. including experiment) and a simpler model (e.g. lacking experiment) is not significant (based on adjusted degrees of freedom), the simpler model is preferred. We assessed multivariate normality, an assumption of path analysis, by evaluating Mardia’s multivariate kurtosis estimate. A perfectly normal multivariate distribution would have a Mardia’s multivariate kurtosis estimate of 0 (DeCarlo 1997; Arbuckle and Wothke 1999), but multivariate kurtosis estimates within ±7 units of 0 are acceptable for maximum likelihood estimation (West et al. 1995; Schepers 2004; Byrne 2010; Cohen et al. 2013). To illustrate the biological significance of the responses of ovary and DLM mass to song exposure, and to compare relative responses between LW and SW females, we computed the percent change in the response variables as a function of acoustic treatment. RESULTS Fit and selection of path model For both LW females and SW females, the a priori model (Figure 1) provided an excellent fit, as indicated by nonsignificant χ2 values and RMSEA values less than 0.07 (LW female model: χ2 = 1.335, df = 3, P = 0.721, RMSEA < 0.0001; SW female model: χ2 = 4.075, df = 3, P = 0.253, RMSEA = 0.057). Removing experiment from the model still resulted in nonsignificant χ2 values and lowered the RMSEA value for SW females (LW females: χ2 = 0.420, df = 1, P = 0.517, RMSEA < 0.0001; SW females: χ2 = 0.036, df = 1, P = 0.850, RMSEA < 0.0001). The full model (containing experiment) and reduced model (lacking experiment) differed by 2 degrees of freedom, so the χ2 difference (χ2reduced model minus χ2full model) would have to be +5.99 (at α = 0.05) to justify the selection of the full model over the reduced model. For LW females, the χ2 difference was −0.915, and for SW females, the χ2 difference was −4.039; therefore, for both wing morphs, we selected the reduced model (Figure 2) over the full model. Figure 2 View largeDownload slide Fitted path model. On each path, coefficients are presented as: LW path coefficient/SW path coefficient. Significant path coefficients are shown in bold. Solid path lines were significant for both wing morphs. Dashed lines show paths significant in LW only and dotted lines show paths that was not significant in either morph. Predicted directions of effects shown under each path. Figure 2 View largeDownload slide Fitted path model. On each path, coefficients are presented as: LW path coefficient/SW path coefficient. Significant path coefficients are shown in bold. Solid path lines were significant for both wing morphs. Dashed lines show paths significant in LW only and dotted lines show paths that was not significant in either morph. Predicted directions of effects shown under each path. The multivariate normality assumption was not violated in either data set (Mardia’s multivariate kurtosis estimates were within ±7 units of 0). For LW females, Mardia’s multivariate kurtosis estimate was −2.623. For SW females, Mardia’s multivariate kurtosis estimate was 0.051. Path model: LW females Our path analysis of LW females’ responses (Figure 2, Table 1) revealed a significant, positive effect of acoustic exposure on ovary mass (path coefficient = 0.086, P = 0.046), such that females exposed to male calling song during adulthood had larger ovaries than unexposed females. However, while the effect of acoustic exposure on DLM mass was negative as predicted, it was not statistically significant (path coefficient = −0.074, P = 0.167). Day (length of treatment and day on which females were preserved for measurement) had a significant negative effect on DLM mass (path coefficient = −0.226, P = 0.002), indicating that DLM histolysis occurred as time passed, as expected. Also as predicted, day had a significant positive effect on ovary mass (path coefficient = 0.588, P < 0.001), indicating that ovary mass increased as time passed. DLM mass had a significant negative effect on ovary mass (path coefficient = −0.364, P < 0.001), suggesting that DLM histolysis was linked to ovary growth in this sample of females (see Introduction). Table 1 Coefficients for the path analyses Path  Path coefficient      Standardized  Raw (SE)  P*  LW females: n = 162, Chi-square = 0.420, df = 1, P = 0.517, RMSEA = 0  Day ➔ DLM mass  −0.226  −1.492 (0.506)  0.002  Day ➔ ovary mass  0.588  55.149 (4.886)  <0.001  DLM mass ➔ ovary mass  −0.364  −5.169 (0.741)  <0.001  Exposure ➔ DLM mass  −0.074  −0.489 (0.505)  0.167  Exposure ➔ ovary mass  0.086  8.020 (4.765)  0.046  SW females: n = 112, Chi-square = 0.036, df = 1, P =0.85, RMSEA = 0  Day ➔ DLM mass  −0.176  −0.289 (0.152)  0.029  Day ➔ ovary mass  0.885  86.83 (4.138)  <0.001  DLM mass ➔ ovary mass  −0.055  −3.279 (2.538)  0.098  Exposure ➔ DLM mass  0.117  0.191 (0.152)  0.208§  Exposure ➔ ovary mass  0.079  7.751 (4.085)  0.029  Path  Path coefficient      Standardized  Raw (SE)  P*  LW females: n = 162, Chi-square = 0.420, df = 1, P = 0.517, RMSEA = 0  Day ➔ DLM mass  −0.226  −1.492 (0.506)  0.002  Day ➔ ovary mass  0.588  55.149 (4.886)  <0.001  DLM mass ➔ ovary mass  −0.364  −5.169 (0.741)  <0.001  Exposure ➔ DLM mass  −0.074  −0.489 (0.505)  0.167  Exposure ➔ ovary mass  0.086  8.020 (4.765)  0.046  SW females: n = 112, Chi-square = 0.036, df = 1, P =0.85, RMSEA = 0  Day ➔ DLM mass  −0.176  −0.289 (0.152)  0.029  Day ➔ ovary mass  0.885  86.83 (4.138)  <0.001  DLM mass ➔ ovary mass  −0.055  −3.279 (2.538)  0.098  Exposure ➔ DLM mass  0.117  0.191 (0.152)  0.208§  Exposure ➔ ovary mass  0.079  7.751 (4.085)  0.029  *P-values are 1-tailed, as the signs of the observed path coefficients were in the predicted direction except in one case (§), where, because the direction is opposite to that predicted, we show the 2-tailed P-value. View Large Path model: SW females Path analysis of SW females’ responses (Figure 2, Table 1) also indicated a significant, positive effect of acoustic exposure on ovary mass (path coefficient = 0.079, P = 0.029) but a nonsignificant effect of exposure on DLM mass (path coefficient = 0.117, P = 0.208). Day had a significant, positive effect on ovary mass (path coefficient = 0.885, P < 0.001) and a weaker but significant negative effect on DLM mass (path coefficient = −0.176, P = 0.029). This reduction in DLM response to day relative to the response observed in LW females is consistent with previous studies (see Introduction), and given the small size of the SW females’ flight muscles it is not surprising that there was no significant relationship between DLM mass and ovary mass (path coefficient = −0.055, P = 0.098). Biological significance of acoustic exposure and comparisons between morphs We hypothesized that the relative increase in ovary mass should be greater in exposed LW females than exposed SW females. To test this, we calculated the percent change in ovary mass in exposed versus unexposed females in each wing morph on either Day 3 or Day 6 post-eclosion. Among LW individuals, females exposed to calling song and measured at Day 3 had ovaries that were 30% heavier than females unexposed and measured at Day 3 (Table 2). While females exposed for 6 days had larger ovaries, the percent increase in ovary mass of 28% was nearly identical to that between exposed and unexposed females measured at Day 3. Thus, in LW females, acoustic exposure for 3 days was sufficient to evoke a relative response equivalent to that induced by 6 days of exposure. The observed 28–30% increase in ovary mass in LW females exposed to calling song translates into a similar increase in earlier fecundity, and therefore has a potentially large impact on fitness (Roff 1994). Table 2 Means (standard errors) and percent change in ovary mass and DLM mass as a function of acoustic exposure Wing morph  Day  Unexposed  Exposed  % Change in exposed Females  Ovary mass (mg)  LW  3  11.69 (1.73)  15.22 (1.88)  30%  LW  6  66.82 (8.01)  85.35 (7.85)  28%  SW  3  13.68 (2.71)  14.16 (1.75)  3%  SW  6  95.38 (4.80)  108.06 (5.11)  13%  DLM mass (mg)  LW  3  5.75 (0.48)  5.39 (0.48)  −6%  LW  6  4.40 (0.59)  3.77 (0.50)  −14%  SW  3  1.07 (0.14)  1.25 (0.15)  17%  SW  6  0.77 (0.14)  0.97 (0.17)  26%  Wing morph  Day  Unexposed  Exposed  % Change in exposed Females  Ovary mass (mg)  LW  3  11.69 (1.73)  15.22 (1.88)  30%  LW  6  66.82 (8.01)  85.35 (7.85)  28%  SW  3  13.68 (2.71)  14.16 (1.75)  3%  SW  6  95.38 (4.80)  108.06 (5.11)  13%  DLM mass (mg)  LW  3  5.75 (0.48)  5.39 (0.48)  −6%  LW  6  4.40 (0.59)  3.77 (0.50)  −14%  SW  3  1.07 (0.14)  1.25 (0.15)  17%  SW  6  0.77 (0.14)  0.97 (0.17)  26%  View Large As we predicted, acoustic exposure in SW females had a lower effect on the increase in ovary mass compare to LW females (Figure 3), with ovaries in exposed SW females being 3% larger than in unexposed SW females at Day 3 and 13% larger at Day 6 (Table 2, Figure 3). Thus, in SW females, the effect of acoustic exposure appeared to slightly increase with the length of exposure, but overall evoked a weaker relative response than in LW females. Despite the fact that SW females reacted less strongly to acoustic exposure than did LW females, the 13% increase in ovary growth in SW females exposed for six days still represents a potentially biologically impactful effect on fitness. Figure 3 View largeDownload slide Variation in ovary and DLM masses in relation to acoustic treatment, wing morph, and days since final molt. In addition to the path analysis, we ran Type III stepwise general linear models of each response variable (log-ovary mass, DLM mass) on acoustic treatment, wing morph, day, and all interactions. For ovary mass, the final significant model (F4,269 = 105.4, P < 0.0001, R2adjusted = 0.61) contained acoustic treatment (F1,269 = 8.95, P = 0.003), wing morph (F1,269 = 14.66, P < 0.001), day (F1,269 = 196.89, P < 0.001), and a wing morph*day interaction (F1,269 = 4.11, P = 0.044): log(y)=41.89+1.12E+0.10W+24.55D+0.51W*D where y is ovary mass, E is exposure (0=unexposed, 1=exposed), W is wing morph (0=SW, 1=LW) and D is day (Day 3 = 0, Day 6 = 1). For DLM mass, the final significant model (F2,271 = 80.92, P < 0.001, R2adjusted = 0.37) contained wing morph (F1,271 = 144.35, P < 0.001) and day (F1,271 = 10.74, P = 0.001): DLM=0.5432+3.78W−1.02D. Shown are means ± standard errors. Clockwise from the top left: ovary masses after 3 days of treatment, DLM masses of SW females, DLM masses of LW females, ovary masses after 6 days of treatment. Figure 3 View largeDownload slide Variation in ovary and DLM masses in relation to acoustic treatment, wing morph, and days since final molt. In addition to the path analysis, we ran Type III stepwise general linear models of each response variable (log-ovary mass, DLM mass) on acoustic treatment, wing morph, day, and all interactions. For ovary mass, the final significant model (F4,269 = 105.4, P < 0.0001, R2adjusted = 0.61) contained acoustic treatment (F1,269 = 8.95, P = 0.003), wing morph (F1,269 = 14.66, P < 0.001), day (F1,269 = 196.89, P < 0.001), and a wing morph*day interaction (F1,269 = 4.11, P = 0.044): log(y)=41.89+1.12E+0.10W+24.55D+0.51W*D where y is ovary mass, E is exposure (0=unexposed, 1=exposed), W is wing morph (0=SW, 1=LW) and D is day (Day 3 = 0, Day 6 = 1). For DLM mass, the final significant model (F2,271 = 80.92, P < 0.001, R2adjusted = 0.37) contained wing morph (F1,271 = 144.35, P < 0.001) and day (F1,271 = 10.74, P = 0.001): DLM=0.5432+3.78W−1.02D. Shown are means ± standard errors. Clockwise from the top left: ovary masses after 3 days of treatment, DLM masses of SW females, DLM masses of LW females, ovary masses after 6 days of treatment. Power analysis: acoustic exposure and flight muscle histolysis In the path analyses, the effect of acoustic exposure on DLM mass was statistically nonsignificant in both LW females and SW females. In LW females, acoustic exposure did reduce DLM mass by 6% and 14% at Days 3 and 6, respectively (Table 2, Figure 3). Because the direction of difference was as predicted, we conducted a simple power analysis based on a 2-sample t-test to determine, approximately, the required sample size for significance given the observed difference and the difference that could be detected given the sample size used for LW females. We set the alpha probability at 0.05 and power at 0.80 (Cohen 1988). The standard deviations for DLM masses on Days 3 and 6 ranged from 3.1 to 3.5: a sample size of greater than 400 individuals would be required to detect the observed differences. The sample size in the present data was 162 LW females, thus falling far short of the required number. The average difference between days 3 and 6 was approximately 0.5, and given the present sample size only a difference twice as large as this could be detected. In SW females, DLM mass was not significantly different between females, though those exposed to calling song had somewhat larger DLMs at Day 3 (+17%) and Day 6 (+26%). However, DLM masses in SW females were tiny, as expected, compared to those of LW females (Table 2, Figure 3) and therefore any small difference in DLM mass between exposed and unexposed females is overly emphasized when treated as a percent difference. DISCUSSION Here, we provide evidence that the social mating environment can induce a plastic response in female reproductive physiology, such that females exposed to male calling song invested more heavily in ovary mass (fecundity), a life-history trait directly linked to fitness, than unexposed females. Because LW females have larger energy stores that are initially uncommitted to reproduction, we predicted that LW females would respond more strongly to exposure than SW females. This prediction was upheld, with exposed LW females showing a 28–30% increase in ovary mass at both ages measured, whereas exposed SW females increased ovary mass by only 3% at day three and 13% at Day 6. In both wing morphs, the increase shown at Day 6 represents a substantial increase in early fecundity and hence would potentially have an impact on fitness. In the absence of acoustic cues indicating reproductively viable males, the maintenance of dispersal capability in LW females is favored. Given that SW females cannot disperse (at least by flight) there is less of a fitness advantage to altering the onset of reproduction. That we did observe an earlier onset of reproduction in exposed SW females (compared to unexposed SW females) suggests that there is a cost to the early development of eggs in the absence of mature males. One possibility is that agility is impeded by carrying eggs, which could make females more vulnerable to predation. Another possibility is that resources may potentially be scarce and the allocation of resources to eggs may place a physiological burden on females, thereby increasing their mortality rate. Females given reduced rations do in fact show a decrease in ovary mass indicating a plastic response to resource availability (Roff and Gelinas 2003). Exposure to calling song did not have a statistically significant effect on flight muscle histolysis in either wing morph. However, the decrease in DLM mass of 14% in LW females on Day 6 was potentially biologically meaningful. The power analysis showed that the lack of statistical significance, assuming that the decrease in DLM mass was real, could be attributed to relatively large standard errors. On the other hand, the increased ovary masses of LW females could also have been caused by reallocation of flight fuel reserves, which we did not measure in this study. Lipids serve as flight fuels in G. firmus and are negatively correlated with ovary mass (Zera et al. 1997), suggesting that the life-history trade-off between flight and reproduction involves multiple aspects of dispersal capability. While calling song exposure did not show a statistically significant effect on flight muscle histolysis, it nevertheless has the potential to alter the dispersal propensity of LW females via its significant effect on ovary mass. By accelerating the onset of reproduction (i.e. the growth of ovaries), calling song exposure may reduce the ability of LW females to fly, if possessing a heavier, egg-laden body limits flight capability. Flight ability in LW females might also be impaired by the reallocation of flight fuels to ovary growth in exposed females. Thus, if LW females mature to adulthood in an environment in which reproductively viable males are actively calling, female emigration out of the current habitat patch may decrease, subsequently reducing gene flow between populations. Our study joins a growing body of literature (see Introduction) documenting phenotypic plasticity in response to the experience of conspecific, social (primarily sexual) signals. Such plasticity should enable individuals to maximize their reproductive success in the current social environment (Kasumovic and Brooks 2011; Rodriguez et al. 2013). Our study demonstrates the rapidity of plastic responses and holds implications for a broad range of species, as the experience of social cues during adulthood should be nearly universal across animal species. This work was supported by a Newell Award from the University of California, Riverside (to L.P.C.), a fellowship from the Graduate Assistance in Areas of National Need program (to L.P.C.), and a National Science Foundation grant (NSF IOS-1353463 to D.A.R. and Dr. Daphne Fairbairn). We thank Drs. Daphne Fairbairn and Christopher Clark for comments on the manuscript, and Jaime Guzman for help maintaining the cricket colony. Suggestions from the editor and 2 anonymous reviewers improved the quality of the manuscript. Data accessibility: Analyses reported in this article can be reproduced using the data provided by Conroy and Roff (2017). REFERENCES Arbuckle J, Wothke W. 1999. AMOS 4.0 User’s Guide . Chicago (IL): Smallwaters Corporation, Inc. Bailey NW, Zuk M. 2008. 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Behavioral EcologyOxford University Press

Published: Mar 1, 2018

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