Age-dependent fitness costs of alarm signaling in aphids - Canadian ...

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Age-dependent fitness costs of alarm signaling in aphids Edward B. Mondor and Bernard D. Roitberg

Abstract: For an alarm signal to evolve, the benefits to the signaler must outweigh the costs of sending the signal. Research has largely focused on the benefits of alarm signaling, and the costs to an organism of sending an alarm signal are not well known. When attacked by a predator, aphids secrete cornicle droplets, containing an alarm pheromone, for individual protection and to warn clonemates. As aphid alarm pheromone is synthesized de novo in a feedback loop with juvenile hormone, we hypothesized that the secretion of cornicle droplets may result in a direct fitness cost to the emitter. We show that the secretion of a single cornicle droplet by pre-reproductive (third- and fourth-instar) pea aphids, Acyrthosiphon pisum, directly altered the timing and number of offspring produced. Thirdinstar pea aphids delayed offspring production but produced more offspring overall than non-secreting aphids, demonstrating a life-history shift but no significant fitness cost of droplet secretion. Fourth-instar pea aphids also delayed offspring production but produced the same number of offspring as non-secretors, resulting in a direct fitness cost of droplet secretion. Offspring production by adult, reproductive pea aphids that secreted a cornicle droplet did not differ from that of non-secretors. Thus, the fitness costs of secreting cornicle droplets containing an alarm signal are agedependent. Résumé : Pour qu’un signal d’alerte évolue, il faut que les bénéfices qu’en tire l’émetteur soient supérieurs aux coûts qui s’y rattachent. Les bénéfices des signaux d’alerte ont été bien étudiés, mais les coûts rattachée à l’émission de signaux pour un organisme sont mal connus. Lors de l’attaque d’un prédateur, les pucerons sécrètent dans leurs cornicules des gouttelettes contenant une phéromone d’alerte pour leur protection individuelle et pour avertir les partenaires de leur clone. La phéromone d’alerte est synthétisée de novo dans un système de boucles de rétroaction avec l’hormone juvénile; nous posons donc en hypothèse que la sécrétion de gouttelettes dans les cornicules peut se faire directement au détriment du fitness de l’émetteur. Mous démontrons ici que la sécrétion d’une seule gouttelette de phéromone chez des pucerons du pois, Acyrthosiphon pisum, en phase préreproductive (3e et 4e stades) suffit pour retarder la production de rejetons et en modifier le nombre. Les pucerons de 3e stade retardent leur production de rejetons, mais, dans l’ensemble, ils en produisent plus que les pucerons qui ne sécrètent pas de phéromone, ce qui dénote que la production de sécrétions entraîne une modification des paramètres démographiques, sans aucune perte du fitness. Les pucerons de 4e stade retardent aussi leur production de rejetons, mais en produisent le même nombre que les pucerons qui ne sécrètent pas de phéromone, ce qui résulte en des pertes directes de fitness. Les pucerons reproducteurs adultes qui sécrètent des gouttelettes dans leurs cornicules produisent le même nombre de rejetons que les pucerons adultes qui ne le font pas. Donc, les coûts aux dépens du fitness de la production de gouttelettes contenant de la phéromone d’alerte dépendent de l’âge. [Traduit par la Rédaction]

Mondor and Roitberg

Introduction For alarm signals to evolve, the benefits of emitting a signal must outweigh the costs associated with the act of signaling itself (Maynard Smith 1965). Although the benefits of alarm signaling have been well documented for a number of organisms (Nault and Phelan 1984; Bradbury and Vehrencamp 1998; Chivers and Mirza 2001), it has been more difficult to quantify the costs associated with signal emission. Costs of alarm signaling have generally been regarded in terms of increased risk of injury and (or) death upon signal-

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ing (Sherman 1977, 1985; Rasa 1989); however, alarm signaling may also be costly to an organism if the signals are energetically expensive to produce. Olfactory (chemical) alarm signals may be physiologically costly to produce, especially if the compounds are synthesized de novo. For example, it has been argued that a large metabolic cost of producing alarm compounds (Wisenden and Smith 1997) may have been one factor in the evolution of damage-released substances, and not voluntary alarm signals, in fish (Mathis et al. 1995; Chivers and Smith 1998). It is not known whether other organisms experience similar

Received 19 December 2002. Accepted 11 March 2003. Published on the NRC Research Press Web site at http://cjz.nrc.ca on 15 May 2003. E.B. Mondor1,2 and B.D. Roitberg. Behavioural Ecology Research Group, Department of Biological Sciences, Simon Fraser University, Burnaby, BC V5A 1S6, Canada. 1

Corresponding author (e-mail: [email protected]). Present address: Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB T2N 1N4, Canada.

2

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doi: 10.1139/Z03-053

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physiological costs of pheromone production. Furthermore, it is unclear if putative physiological costs translate into reduced Darwinian fitness for the emitter via a reduction in either development or reproduction. Aphids are small phloem-feeding insects that often emit cornicle droplets containing a complex mixture of fatty acids and a volatile alarm pheromone (in the majority of aphid species it is (E)-β-farnesene (EBF); Nault and Montgomery 1979) when attacked by a predator (Dixon 1958; Strong 1967). This pheromone causes nearby clone mates to wave the antennae, cease feeding, walk from the area, and (or) drop off the host plant (Dahl 1971; Kislow and Edwards 1972; Roitberg and Myers 1978), thereby reducing predation. Aphids synthesize alarm pheromone and other cornicle-droplet constituents de novo (Gut and van Oosten 1985) in a pathway linked to juvenile-hormone production (Gut et al. 1987; van Oosten et al. 1990). As juvenile hormone controls insect functions such as nymph development, metamorphosis, and adult follicle development (Romoser 1981; Dingle and Winchell 1997), it is hypothesized that secretion of EBF may remove juvenile hormone precursors from their normal pathway, thereby directly altering an aphid’s subsequent development and reproduction. To test this hypothesis we evaluated whether the secretion of a cornicle droplet by pea aphids, Acyrthosiphon pisum, directly altered an individual’s fitness.

Materials and methods Insects and plants Pea aphids were collected from sweet pea, Lathyrus odoratus ‘Cuthbertson’, in Burnaby, British Columbia. Aphids were reared in the laboratory on broad bean plants, Vicia faba ‘Broad Windsor’, potted in standard garden soil. Bean plants used for colony maintenance and for the experiments were grown at 21–25°C, 45–55% RH, and a 16 h light (L) : 8 h dark (D) photoperiod. Aphids were maintained at 19– 23°C, 30–70% RH, and 16 h L : 8 h D. A synchronous colony of first instars was obtained by placing at least 40 mature apterous aphids, of a single clone, on a bean plant. The progeny that these mature aphids produced within 8 h were used for the experiments. Individual first instars were then transferred at 1 day of age onto individual bean leaf pairs. Bean leaves were cut from 3-weekold broad bean plants grown under the above conditions. Each leaf was placed in its own 8.5 cm i.d. × 2 cm high petri dish. The cut stem of the bean leaf was placed through a hole in the side of the petri dish and immersed in a waterfilled vial. Water bottles were refilled every 7 days and bean leaves were replaced every 14 days. Both aphids and bean leaf pairs were randomly assigned to petri dishes as well as randomly receiving one of the following experimental treatments. Experimental protocol Aphids were induced to produce one alarm signal at different stages of development: third instar (4 days old) (experiment 1), fourth instar (6 days old) (experiment 2), and adults (fourth day of reproduction; 10 days old) (experiment 3). To induce an aphid to emit alarm pheromone reliably, it was lightly stroked on the anterior portion of the thorax with a fine paintbrush, resulting in the secretion of visible cornicle droplets. Control

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aphids for each experiment were also brushed, but more posteriorly on the thorax to prevent secretion. Inducing aphids to emit droplets causes no physical damage to the organism, and does not cause higher levels of mortality than in control aphids (E.B. Mondor and B.D. Roitberg, unpublished data). Following manipulation, each aphid was allowed to settle and feed on its individual bean leaf pair. Aphids in the test group that did not produce cornicle droplets and aphids in the control group that produced droplets were eliminated. Every 24 h the progeny on each bean leaf pair were counted and removed. All experiments were conducted at 17–23°C, 33–64% RH, and 16 h L : 8 h D. Statistical analyses In all experiments the main factor was cornicle-droplet secretion versus no secretion (n = 13 vs. 16, n = 13 vs. 16, and n = 15 vs. 16 for experiments 1–3, respectively). For experiments 1 and 2, the lengths (in days) of the pre-reproductive, reproductive, and post-reproductive periods were compared using nonpaired t tests. For experiment 3, because adults were induced to secrete droplets, comparisons were restricted to the lengths of the reproductive and post-reproductive periods, using nonpaired t tests. For each experiment, multiple t tests were corrected with a Bonferroni adjustment (Lehner 1996) to control the experimentwise error rate. Numbers of offspring were compared using one-factor repeated-measures ANOVA, with cornicle-droplet secretion versus no secretion as the main factor and days of offspring production as the repeated measure. Individual fertility tables (Southwood 1978) were constructed for each aphid, from which its intrinsic rate of increase was calculated (for formulae see Begon et al. 1996). Intrinsic rates of increase for secretors versus nonsecretors were then compared using nonpaired t tests. For all experiments, tests of the assumptions underlying the parametric statistical tests (i.e., normality and homogeneity of variance) and the statistical analyses themselves were conducted using JMP IN 4.0.4 (SAS Institute Inc. 2001).

Results The lengths of the pre-reproductive, reproductive, and postreproductive periods did not differ for aphids secreting versus not secreting a cornicle droplet in any of the three experiments (Table 1). Third instars that emitted a single cornicle droplet had higher offspring production than non-secretors (F[1,27] = 11.47, P = 0.0022) (Fig. 1a). However, these offspring were produced later in the reproductive cycle than those of control aphids (F[18,486] = 2.73, P = 0.0002) (Fig. 2a). Increased offspring production offset the initial lower number of offspring produced, resulting in no significant difference in intrinsic rate of increase between secretors and non-secretors (t[27] = 0.28, P = 0.78) (Fig. 3a). Fourth instars had similar numbers of offspring, whether they secreted or did not secrete droplets (F[1,27] = 0.095, P = 0.76) (Fig. 1b), but as with third instars, secretors produced them later in the reproductive cycle than non-secretors (F[18,486] = 2.32, P = 0.0017) (Fig. 2b). Because of delaying offspring production and not producing additional offspring, secretors had lower intrinsic rates of increase than nonsecretors (t[27] = 2.32, P = 0.028) (Fig. 3b). © 2003 NRC Canada



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Table 1. Durations of developmental stages for pea aphids, Acyrthosiphon pisum, secreting versus not secreting cornicle droplets.

Exp. 1; third instar Pre-reproductive Reproductive Post-reproductive Exp. 2; fourth instar Pre-reproductive Reproductive Post-reproductive Exp. 3; adult Reproductive Post-reproductive

Mean no. of days (droplet vs. no droplet)

Significance

6.85 (0.13) vs. 6.56 (0.11) 14.38 (0.47) vs. 14.56 (0.43) 8.46 (0.74) vs. 9.06 (0.66)

t[27] = –1.66, P = 0.11 t[27] = 0.278, P = 0.78 t[27] = 0.606, P = 0.55

6.85 (0.15) vs. 6.88 (0.13) 14.00 (0.50) vs. 14.06 (0.45) 9.08 (0.73) vs. 8.19 (0.66)

t[27] = 0.147, P = 0.88 t[27] = 0.093, P = 0.93 t[27] = –0.90, P = 0.37

13.73 (0.49) vs. 13.63 (0.47) 10.67 (0.92) vs. 9.81 (0.89)

t[29] = –0.16, P = 0.88 t[29] = –0.67, P = 0.51

Note: Values in parentheses show the standard error.

Fig. 1. Numbers of offspring (mean + SE) produced per day by third-instar (a), fourth-instar (b), and adult pea aphids, Acyrthosiphon pisum (c), secreting versus not secreting cornicle droplets. Within each experiment, columns with different letters are significantly different (P < 0.05).

Adults secreting cornicle droplets had similar numbers of young to non-secretors (F[1,29] = 0.027, P = 0.87) (Fig. 1c). Secreting and non-secreting adults had similar numbers of young throughout the reproductive cycle (F[17,495] = 0.91, P = 0.56) (Fig. 2c). As a result, intrinsic rates of increase did not differ between secretors and non-secretors (t[29] = 0.46, P = 0.65) (Fig. 3c).

Discussion To understand the evolution of alarm signaling, it is imperative to understand both the costs and the benefits associated with a signal (Maynard Smith 1965). In many cases, the benefits associated with signaling are readily apparent, while the costs associated with the act are more difficult to discern. Furthermore, costs can be either physiological or ecological and either direct or indirect. In this paper we address the direct, physiological costs of cornicle-droplet secretion in pea aphids. Whether individuals experience a direct fitness cost (i.e., a reduced intrinsic rate of increase) of cornicle-droplet secretion is age-dependent. Pre-reproductive aphids that emit even a single cornicle droplet have life-history patterns that differ from those of non-secretors, while reproductive aphids do not experience these life-history changes. Early-instar pre-reproductive

aphids alter their life-history patterns but do not experience a direct fitness cost of cornicle-droplet secretion. Late-instar prereproductive aphids showed both altered life-history patterns and lower fitness than non-secretors. Reproductive aphids do not experience any changes in offspring production upon secreting cornicle droplets. Thus, the hypothesis that alarm signaling entails a fitness cost for aphids is supported only for late-instar pre-reproductive aphids. Aphids can alter the timing and number of offspring produced in response to a wide range of environmental stimuli, such as plant quality (Walters et al. 1988; Stadler 1995), ant attendance (Stadler and Dixon 1999; Yao et al. 2000), and the interacting effects of plant quality and ant attendance (Stadler et al. 2002). We suggest that there are two reasons why only late-instar pre-reproductive aphids experience fitness costs of alarm signaling. First, while juvenile aphids produce much smaller cornicle droplets than adult aphids, EBF levels in juvenile droplets are much greater (Mondor et al. 2000). Thus, as the most pronounced life-history changes occur during the juvenile stages, this suggests that (i) EBF is costly to secrete and (ii) the costs of secreting fatty acids, which are of similar composition but occur in greater quantities in adult droplets (Callow et al. 1973; Mondor et al. 2000), are less than those of secreting EBF. Second, it is © 2003 NRC Canada



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Fig. 2. Numbers of offspring (mean + SE) produced over the entire reproductive period by third-instar (droplet × day interaction; F[18,486] = 2.73, P = 0.0002) (a), fourth-instar (droplet × day interaction; F[18,486] = 2.32, P = 0.0017) (b), and adult aphids (droplet × day interaction; F[17,495] = 0.91, P = 0.56) (c) secreting versus not secreting cornicle droplets.

during the pre-reproductive period that aphids allocate resources to somatic and reproductive tissue (Kindlmann and Dixon 1989). When predation rates are high, it would be optimal to produce more offspring, even if they are smaller (Smith and Fretwell 1974; Winkler and Wallin 1987), to optimize the chances of clone survival. By reallocating resources, third-instar aphids can achieve similar fitness (i.e., intrinsic rates of increase) to non-signalers. Fourth instars, which are close to reaching adulthood, can reallocate a small amount of resources to reproduction before reaching adulthood, but not enough to offset the energy expenditure of signaling. Conversely, adult aphids, having already allocated

their resources to growth and reproduction, cannot adjust their reproductive output. In our experiments, we induced aphids to emit only a single cornicle droplet, and then evaluated subsequent changes in development and reproduction. However, two factors that may influence the overall fitness costs of alarm signaling are (1) the number of droplets emitted and (2) host-plant quality. Cornicle-droplet emission is highest in second-, third-, and fourth-instar aphids in response to both simulated (Mondor et al. 2000) and real (Mondor and Roitberg 2002) predation. Aphid species are also capable of emitting multiple (4–6) droplets (Strong 1967; Wynn and Boudreaux 1972). Thus, © 2003 NRC Canada



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Fig. 3. Intrinsic rates of increase for third-instar (a), fourth-instar (b), and adult aphids (c) secreting versus not secreting cornicle droplets. Within each experiment, columns with different letters are significantly different (P < 0.05).

emitting more than one droplet may further alter aphid lifehistory patterns. Furthermore, we raised our aphids on relatively high-quality leaves, i.e., excised leaves are of higher quality than intact leaves (Watt and Hales 1996). Low hostplant quality has often been shown to negatively affect offspring production (Walters et al. 1988; Stadler 1995). Emission of cornicle droplets may further exacerbate these negative effects, resulting in even greater physiological costs of cornicle-droplet secretion on low-quality host plants, though this remains to be tested. Of course, a substantial question remains unanswered: if aphids are capable of ovulating as adults, why do they not always bear these additional offspring? Clearly, natural selection would rapidly favor clones that produced additional offspring. This question is not unique to aphids, but has been addressed in a number of organisms, most notably birds (Ilmonen et al. 2002; Wallander and Andersson 2002). We did not follow the developmental trajectories of the aphids born in our experimental treatments; however, producing additional offspring may create trade-offs for the progeny, e.g., longer developmental times and reduced clutch sizes upon reaching adulthood (Traicevski and Ward 1994). Further experimentation is required to determine the adaptive significance of this age-dependent life-history shift in aphids.

Acknowledgments We thank J. Borden, M. Mackauer, B. Crespi, and L. Nault for discussions on this topic and comments on the manuscript. L. Takahashi provided insect-colony maintenance and laboratory assistance. Simon Fraser University and the Natural Sciences and Engineering Research Council of Canada provided financial support for this project.

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