Chem. Senses 40: 109–124, 2015
doi:10.1093/chemse/bju065 Advance Access publication December 23, 2014
Phenotypic Plasticity in a Willow Leaf Beetle Depends on Host Plant Species: Release and Recognition of Beetle Odors Nadine Austel1, Andreas Reinecke2,5, Christer Björkman3, Monika Hilker1 and 1,4 Torsten Meiners 1
5
Present address: Department of Behavioural Ecology and Evolutionary Genetics, Max Planck Institute for Ornithology, Eberhard-Gwinner-Straße 4, 82319 Seewiesen, Germany Correspondence to be sent to: Torsten Meiners, Freie Universitaet Berlin, Dahlem Centre of Plant Sciences, Institute of Biology, Haderslebener Straße 9, 12163 Berlin, Germany. e-mail: [email protected] Accepted November 17, 2014
Abstract Aggregation behavior of herbivorous insects is mediated by a wide range of biotic and abiotic factors. It has been suggested that aggregation behavior of the blue willow leaf beetle Phratora vulgatissima is mediated by both host plant odor and by odor released by the beetles. Previous studies show that the beetles respond to plant odors according to their prior host plant experiences. Here, we analyzed the effect of the host plant species on odor released and perceived by adult P. vulgatissima. The major difference between the odor of beetles feeding on salicin-rich and salicin-poor host plants was the presence of salicylaldehyde in the odor of the former, where both males and females released this compound. Electrophysiological studies showed that the intensity of responses to single components of odor released by beetles was sex specific and dependent on the host plant species with which the beetles were fed. Finally, behavioral studies revealed that males feeding on salicin-rich willows were attracted by salicylaldehyde, whereas females did not respond behaviorally to this compound, despite showing clear antennal responses to it. Finally, the ecological relevance of the influence of a host plant species on the plasticity of beetle odor chemistry, perception, and behavior is discussed. Key words: aggregation, Chrysomelidae, EAG, feces, GC–MS analysis, pheromone
Introduction Herbivorous insects can benefit from aggregation on their host plants since feeding in groups helps to overcome host defenses, reduces predation pressure from natural enemies, or enhances the probability of finding a mating partner (Fryxell 1991 and references therein; Harari et al. 1994; Alcock 1998; Ruxton and Sherratt 2006). Despite these advantages, the expression of aggregation behavior is often plastic and depends on factors such as the climate or the season, host availability, predator density, or the host plant of the herbivore; these factors may singly or in combination affect the timing, intensity, and site of aggregation (Prokopy and Roitberg 2001; Aukema and Raffa 2004; Wertheim et al. 2005).
Chemical stimuli eliciting aggregation behavior from herbivorous insects may be emitted by the herbivores themselves and/or by the noncolonized or colonized host plants (reviewed in Loughrin et al. 1996; Wertheim et al. 2005; Fernandez and Hilker 2007). Aggregation pheromones and host plant volatiles may act in concert and, thereby, efficiently mediate aggregation of herbivorous insects (Tinzaara et al. 2003; Dickens 2006; and reviewed in Fernandez and Hilker 2007). Variation in insect aggregation behavior may be caused by plasticity occurring on different levels. It is known that the biogenesis and release of aggregation pheromones depends on a multitude of factors such as the photoperiod (reviewed
© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]
Downloaded from http://chemse.oxfordjournals.org/ at KTH Royal Institute of Technology on August 16, 2015
Freie Universitaet Berlin, Dahlem Centre of Plant Sciences, Institute of Biology, Haderslebener 2 Straße 9, 12163 Berlin, Germany, Department of Neuroethology, Max Planck Institute for Chemical 3 Ecology, Hans-Knöll-Straße 8, 07745 Jena, Germany, Department of Ecology, Swedish University 4 of Agricultural Sciences, Box 7044, 750 07 Uppsala, Sweden and Department of Chemical Biology, Helmholtz-Centre for Infection Research, Inhoffen-Straße 7, 38124 Braunschweig, Germany
110 N. Austel et al.
plants is mediated by both olfactory (Peacock et al. 2001a) and visual cues (Björkman and Eklund 2006). Olfactory preferences for host plant volatiles are plastic and the willow leaf beetle orientates preferably toward S. dasyclados volatiles when having already experienced this host plant (Austel et al. 2014). Peacock et al. (2001a) showed in a field study that S. dasyclados trees with beetles (feeding or not feeding) on them were more attractive than feeding-damaged trees from which beetles were removed. Hence, the combination of host plant cues and odor of conspecifics may induce attraction of P. vulgatissima to infested plants and thus promote aggregation. The aim of this study is to elucidate the phenotypic plasticity in the release of, and responses to, odor released by adult P. vulgatissima. We investigated 1) the chemical composition of the beetles’ body odor and how this odor is influenced by the host plant upon which the beetles are feeding. Furthermore, we studied 2) which components of the odor released by the beetles elicit electrophysiological responses in female and male antennae and whether these electrophysiological responses depend on the food plant species upon which the beetles are feeding. Because our chemical analysis revealed that only beetles that fed on salicin-rich S. dasyclados released salicylaldehyde and that these beetles were electrophysiologically more sensitive to salicylaldehyde, we investigated 3) whether the plasticity in odor release and physiological response is reflected by the beetles’ behavioral response to salicylaldehyde.
Materials and methods Plants and insects
Plant cuttings (20 cm) of S. viminalis (clone 78183) and S. dasyclados (clone Loden) were harvested, and P. vulgatissima adults were collected in early spring 2009 at hibernation sites in the surroundings of Uppsala, Sweden (59°51′N, 17°37′E). The tree cuttings were incubated for 2 weeks in water after being supplemented via a 1-min bath with pyrethroids (1:100 Spruzid, Neudorff). After development of roots, the saplings were potted in standardized soil (Einheitserde Classic Typ-T, Einheitserde). Plants were grown under constant conditions (20 °C, 70%, relative humidity, 16 h light). After reaching at least 60 cm height, shoots of both species were used for feeding adult beetles and after being defoliated, used to produce new cuttings. A continuous laboratory rearing of P. vulgatissima was established on S. viminalis. Treatments of beetles
To investigate the influence of the host plant species on the beetles’ odor emission and the electrophysiological sensitivity of their antennae to their own body odor, pupae of P. vulgatissima from the laboratory rearing were divided into 2 groups, one of which was kept on S. viminalis, and the other of which was transferred to S. dasyclados. Hence,
Downloaded from http://chemse.oxfordjournals.org/ at KTH Royal Institute of Technology on August 16, 2015
in McNeil 1991; Gemeno and Haynes 2000; Bean et al. 2007), the juvenile hormone status (Dickens et al. 2002; Blomquist et al. 2010 and references therein), the quality of diet (McNeil 1991; Landolt and Phillips 1997; Edgar et al. 2007; Blomquist et al. 2010; Zhao et al. 2011; Geiselhardt et al. 2012), contact or exposure to the host plant (reviewed in Reddy and Guerrero 2004; Bartelt et al. 2008; RuizMontiel et al. 2009), individual insect density (reviewed in Applebaum and Heifetz 1999; Bashir et al. 2003; Edde and Phillips 2010), or experience had by insects with their conspecific pheromones (McNeil 1991; Groot et al. 2010). The plasticity of behavior-mediating sensory responses observed in herbivorous insects to chemical stimuli may be determined by a wide range of factors, for example, age and/or juvenile hormone level (Palaniswamy et al. 1979; Ignell et al. 2001; Greiner et al. 2002; Anton et al. 2007), mating status (Anton et al. 2007; Martel et al. 2009; Barrozo et al. 2010; Barrozo et al. 2011; Saveer et al. 2012), population density (Applebaum and Heifetz 1999), experience with conspecific pheromones (McNeil 1991; Stelinski et al. 2003a; Judd et al. 2005; Stelinski et al. 2006; Anderson et al. 2007; Guerrieri et al. 2012), host plant volatiles (Barron and Corbet 1999; Stelinski et al. 2003b; Tinzaara et al. 2003; Party et al. 2009; Trona et al. 2010), or host plant diet (Harari and Landolt 1999; Rietdorf and Steidle 2002; Coyle et al. 2011; Radžiutė and Būda 2012; Austel et al. 2014). Moreover, herbivory may induce the release of plant volatiles that contribute to insect attraction to pheromone-releasing conspecifics feeding upon plants (Giblin-Davis et al. 1996; Ruther et al. 2001; Reinecke et al. 2002; Reddy and Guerrero 2004; Cossé et al. 2006; Dickens 2006; Beran et al. 2011). Here, we studied the phenotypic plasticity of odor released by adults of the blue willow leaf beetle Phratora vulgatissima (Coleoptera: Chrysomelidae) and the plasticity of responses to this odor exhibited by the beetle. We hypothesized that the composition of the odor released by the beetles as well as the beetles’ ability to respond to the components of this odor depend on the host plant species upon which the beetles are feeding. Phratora vulgatissima is one of the main pest insects in willow short rotation coppice (Sage and Tucker 1998), where it occurs in high population densities. Adult beetles show aggregation behavior when colonizing host plants in spring and when entering hibernation sites in leaf litter on the soil in late autumn (Kendall and Wiltshire 1998; Peacock et al. 1999; Karp and Peacock 2004). Choice of food plants by P. vulgatissima is mediated by the presence of phenolic glycosides in host leaves: the beetle prefers willows with low levels of phenolic glycosides (Salix viminalis) over species with higher levels (Salix dasyclados) (Julkunen-Titto 1986, 1989; Kendall et al. 1996) and shows improved performance among plants with low concentrations of these secondary metabolites (Kelly and Curry 1991; Peacock et al. 2004; Lehrman et al. 2012). However, a study by Peacock et al. (2001b) indicated plasticity in host plant choice and even feeding preferences for S. dasyclados over S. viminalis. Orientation of P. vulgatissima to host
Phenotypic Plasticity in a Willow Leaf Beetle 111
the emerging adults were fed S. viminalis (V-beetles), whereas another group was fed S. dasyclados (D-beetles). Each group comprised 100–120 beetles. Groups were kept in cages of 30 × 30 × 70 cm and supplied with potted willow plants. We used 1-month-old P. vulgatissima adults for the chemical and electroantennographic (EAG) analyses. Males and females were separated briefly prior to analysis. Chemical analysis of beetle odor
Table 1 Relative peak areas of headspace compounds of odor released by adult Phratora vulgatissima fed upon Salix viminalis (V-beetles) or Salix dasyclados (D-beetles) Compounds
RIa
V-Beetlesb
D-Beetlesb
Median (25%/75% percentiles)
Median (25%/75% percentiles)
P levelc
Carboxylic acids Hexanoic acid
965
62.5 (52.4/69.2)
30.3 (10.7/43.5)
***
Heptanoic acid
1092
5.0 (4.3/8.1)
2.9 (0.0/4.9)
n.s.
Octanoic acid
1165
14.7 (7.9/21.9)
12.7 (0.0/16.5)
n.s.
Nonanoic acid
1261
5.8 (0.0/8.3)
4.7 (0.0/6.4)
n.s.
Heptanal
904
1.6 (0.0/4.2)
0.3 (0.0/6.4)
n.s.
Nonanal
1101
4.1 (2.3/6.3)
2.3 (1.5/10.8)
n.s.
Benzaldehyde
962
4.9 (3.1/7.7)
4.0 (2.0/6.4)
n.s.
Salicylaldehyde
1040
0.0 (0.0/0.0)
42.5 (14.5/60.4)
***
Aldehydes
a
RI: retention index. Odor of 25 beetles was sampled in a single sampling vial. For each sampling (in total, N = 12 for each type of beetles), relative peak areas (%) were calculated by dividing the peak area of a compound by the total area of all compounds detected. c Asterisks indicate significant differences between V- and D-beetles, Mann–Whitney U test, exception hexanoic acid compared by Welch test. n.s.: P > 0.05; ***P 0.05
Dose
5
64.498
doi:10.1093/chemse/bju065 Advance Access publication December 23, 2014
Phenotypic Plasticity in a Willow Leaf Beetle Depends on Host Plant Species: Release and Recognition of Beetle Odors Nadine Austel1, Andreas Reinecke2,5, Christer Björkman3, Monika Hilker1 and 1,4 Torsten Meiners 1
5
Present address: Department of Behavioural Ecology and Evolutionary Genetics, Max Planck Institute for Ornithology, Eberhard-Gwinner-Straße 4, 82319 Seewiesen, Germany Correspondence to be sent to: Torsten Meiners, Freie Universitaet Berlin, Dahlem Centre of Plant Sciences, Institute of Biology, Haderslebener Straße 9, 12163 Berlin, Germany. e-mail: [email protected] Accepted November 17, 2014
Abstract Aggregation behavior of herbivorous insects is mediated by a wide range of biotic and abiotic factors. It has been suggested that aggregation behavior of the blue willow leaf beetle Phratora vulgatissima is mediated by both host plant odor and by odor released by the beetles. Previous studies show that the beetles respond to plant odors according to their prior host plant experiences. Here, we analyzed the effect of the host plant species on odor released and perceived by adult P. vulgatissima. The major difference between the odor of beetles feeding on salicin-rich and salicin-poor host plants was the presence of salicylaldehyde in the odor of the former, where both males and females released this compound. Electrophysiological studies showed that the intensity of responses to single components of odor released by beetles was sex specific and dependent on the host plant species with which the beetles were fed. Finally, behavioral studies revealed that males feeding on salicin-rich willows were attracted by salicylaldehyde, whereas females did not respond behaviorally to this compound, despite showing clear antennal responses to it. Finally, the ecological relevance of the influence of a host plant species on the plasticity of beetle odor chemistry, perception, and behavior is discussed. Key words: aggregation, Chrysomelidae, EAG, feces, GC–MS analysis, pheromone
Introduction Herbivorous insects can benefit from aggregation on their host plants since feeding in groups helps to overcome host defenses, reduces predation pressure from natural enemies, or enhances the probability of finding a mating partner (Fryxell 1991 and references therein; Harari et al. 1994; Alcock 1998; Ruxton and Sherratt 2006). Despite these advantages, the expression of aggregation behavior is often plastic and depends on factors such as the climate or the season, host availability, predator density, or the host plant of the herbivore; these factors may singly or in combination affect the timing, intensity, and site of aggregation (Prokopy and Roitberg 2001; Aukema and Raffa 2004; Wertheim et al. 2005).
Chemical stimuli eliciting aggregation behavior from herbivorous insects may be emitted by the herbivores themselves and/or by the noncolonized or colonized host plants (reviewed in Loughrin et al. 1996; Wertheim et al. 2005; Fernandez and Hilker 2007). Aggregation pheromones and host plant volatiles may act in concert and, thereby, efficiently mediate aggregation of herbivorous insects (Tinzaara et al. 2003; Dickens 2006; and reviewed in Fernandez and Hilker 2007). Variation in insect aggregation behavior may be caused by plasticity occurring on different levels. It is known that the biogenesis and release of aggregation pheromones depends on a multitude of factors such as the photoperiod (reviewed
© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]
Downloaded from http://chemse.oxfordjournals.org/ at KTH Royal Institute of Technology on August 16, 2015
Freie Universitaet Berlin, Dahlem Centre of Plant Sciences, Institute of Biology, Haderslebener 2 Straße 9, 12163 Berlin, Germany, Department of Neuroethology, Max Planck Institute for Chemical 3 Ecology, Hans-Knöll-Straße 8, 07745 Jena, Germany, Department of Ecology, Swedish University 4 of Agricultural Sciences, Box 7044, 750 07 Uppsala, Sweden and Department of Chemical Biology, Helmholtz-Centre for Infection Research, Inhoffen-Straße 7, 38124 Braunschweig, Germany
110 N. Austel et al.
plants is mediated by both olfactory (Peacock et al. 2001a) and visual cues (Björkman and Eklund 2006). Olfactory preferences for host plant volatiles are plastic and the willow leaf beetle orientates preferably toward S. dasyclados volatiles when having already experienced this host plant (Austel et al. 2014). Peacock et al. (2001a) showed in a field study that S. dasyclados trees with beetles (feeding or not feeding) on them were more attractive than feeding-damaged trees from which beetles were removed. Hence, the combination of host plant cues and odor of conspecifics may induce attraction of P. vulgatissima to infested plants and thus promote aggregation. The aim of this study is to elucidate the phenotypic plasticity in the release of, and responses to, odor released by adult P. vulgatissima. We investigated 1) the chemical composition of the beetles’ body odor and how this odor is influenced by the host plant upon which the beetles are feeding. Furthermore, we studied 2) which components of the odor released by the beetles elicit electrophysiological responses in female and male antennae and whether these electrophysiological responses depend on the food plant species upon which the beetles are feeding. Because our chemical analysis revealed that only beetles that fed on salicin-rich S. dasyclados released salicylaldehyde and that these beetles were electrophysiologically more sensitive to salicylaldehyde, we investigated 3) whether the plasticity in odor release and physiological response is reflected by the beetles’ behavioral response to salicylaldehyde.
Materials and methods Plants and insects
Plant cuttings (20 cm) of S. viminalis (clone 78183) and S. dasyclados (clone Loden) were harvested, and P. vulgatissima adults were collected in early spring 2009 at hibernation sites in the surroundings of Uppsala, Sweden (59°51′N, 17°37′E). The tree cuttings were incubated for 2 weeks in water after being supplemented via a 1-min bath with pyrethroids (1:100 Spruzid, Neudorff). After development of roots, the saplings were potted in standardized soil (Einheitserde Classic Typ-T, Einheitserde). Plants were grown under constant conditions (20 °C, 70%, relative humidity, 16 h light). After reaching at least 60 cm height, shoots of both species were used for feeding adult beetles and after being defoliated, used to produce new cuttings. A continuous laboratory rearing of P. vulgatissima was established on S. viminalis. Treatments of beetles
To investigate the influence of the host plant species on the beetles’ odor emission and the electrophysiological sensitivity of their antennae to their own body odor, pupae of P. vulgatissima from the laboratory rearing were divided into 2 groups, one of which was kept on S. viminalis, and the other of which was transferred to S. dasyclados. Hence,
Downloaded from http://chemse.oxfordjournals.org/ at KTH Royal Institute of Technology on August 16, 2015
in McNeil 1991; Gemeno and Haynes 2000; Bean et al. 2007), the juvenile hormone status (Dickens et al. 2002; Blomquist et al. 2010 and references therein), the quality of diet (McNeil 1991; Landolt and Phillips 1997; Edgar et al. 2007; Blomquist et al. 2010; Zhao et al. 2011; Geiselhardt et al. 2012), contact or exposure to the host plant (reviewed in Reddy and Guerrero 2004; Bartelt et al. 2008; RuizMontiel et al. 2009), individual insect density (reviewed in Applebaum and Heifetz 1999; Bashir et al. 2003; Edde and Phillips 2010), or experience had by insects with their conspecific pheromones (McNeil 1991; Groot et al. 2010). The plasticity of behavior-mediating sensory responses observed in herbivorous insects to chemical stimuli may be determined by a wide range of factors, for example, age and/or juvenile hormone level (Palaniswamy et al. 1979; Ignell et al. 2001; Greiner et al. 2002; Anton et al. 2007), mating status (Anton et al. 2007; Martel et al. 2009; Barrozo et al. 2010; Barrozo et al. 2011; Saveer et al. 2012), population density (Applebaum and Heifetz 1999), experience with conspecific pheromones (McNeil 1991; Stelinski et al. 2003a; Judd et al. 2005; Stelinski et al. 2006; Anderson et al. 2007; Guerrieri et al. 2012), host plant volatiles (Barron and Corbet 1999; Stelinski et al. 2003b; Tinzaara et al. 2003; Party et al. 2009; Trona et al. 2010), or host plant diet (Harari and Landolt 1999; Rietdorf and Steidle 2002; Coyle et al. 2011; Radžiutė and Būda 2012; Austel et al. 2014). Moreover, herbivory may induce the release of plant volatiles that contribute to insect attraction to pheromone-releasing conspecifics feeding upon plants (Giblin-Davis et al. 1996; Ruther et al. 2001; Reinecke et al. 2002; Reddy and Guerrero 2004; Cossé et al. 2006; Dickens 2006; Beran et al. 2011). Here, we studied the phenotypic plasticity of odor released by adults of the blue willow leaf beetle Phratora vulgatissima (Coleoptera: Chrysomelidae) and the plasticity of responses to this odor exhibited by the beetle. We hypothesized that the composition of the odor released by the beetles as well as the beetles’ ability to respond to the components of this odor depend on the host plant species upon which the beetles are feeding. Phratora vulgatissima is one of the main pest insects in willow short rotation coppice (Sage and Tucker 1998), where it occurs in high population densities. Adult beetles show aggregation behavior when colonizing host plants in spring and when entering hibernation sites in leaf litter on the soil in late autumn (Kendall and Wiltshire 1998; Peacock et al. 1999; Karp and Peacock 2004). Choice of food plants by P. vulgatissima is mediated by the presence of phenolic glycosides in host leaves: the beetle prefers willows with low levels of phenolic glycosides (Salix viminalis) over species with higher levels (Salix dasyclados) (Julkunen-Titto 1986, 1989; Kendall et al. 1996) and shows improved performance among plants with low concentrations of these secondary metabolites (Kelly and Curry 1991; Peacock et al. 2004; Lehrman et al. 2012). However, a study by Peacock et al. (2001b) indicated plasticity in host plant choice and even feeding preferences for S. dasyclados over S. viminalis. Orientation of P. vulgatissima to host
Phenotypic Plasticity in a Willow Leaf Beetle 111
the emerging adults were fed S. viminalis (V-beetles), whereas another group was fed S. dasyclados (D-beetles). Each group comprised 100–120 beetles. Groups were kept in cages of 30 × 30 × 70 cm and supplied with potted willow plants. We used 1-month-old P. vulgatissima adults for the chemical and electroantennographic (EAG) analyses. Males and females were separated briefly prior to analysis. Chemical analysis of beetle odor
Table 1 Relative peak areas of headspace compounds of odor released by adult Phratora vulgatissima fed upon Salix viminalis (V-beetles) or Salix dasyclados (D-beetles) Compounds
RIa
V-Beetlesb
D-Beetlesb
Median (25%/75% percentiles)
Median (25%/75% percentiles)
P levelc
Carboxylic acids Hexanoic acid
965
62.5 (52.4/69.2)
30.3 (10.7/43.5)
***
Heptanoic acid
1092
5.0 (4.3/8.1)
2.9 (0.0/4.9)
n.s.
Octanoic acid
1165
14.7 (7.9/21.9)
12.7 (0.0/16.5)
n.s.
Nonanoic acid
1261
5.8 (0.0/8.3)
4.7 (0.0/6.4)
n.s.
Heptanal
904
1.6 (0.0/4.2)
0.3 (0.0/6.4)
n.s.
Nonanal
1101
4.1 (2.3/6.3)
2.3 (1.5/10.8)
n.s.
Benzaldehyde
962
4.9 (3.1/7.7)
4.0 (2.0/6.4)
n.s.
Salicylaldehyde
1040
0.0 (0.0/0.0)
42.5 (14.5/60.4)
***
Aldehydes
a
RI: retention index. Odor of 25 beetles was sampled in a single sampling vial. For each sampling (in total, N = 12 for each type of beetles), relative peak areas (%) were calculated by dividing the peak area of a compound by the total area of all compounds detected. c Asterisks indicate significant differences between V- and D-beetles, Mann–Whitney U test, exception hexanoic acid compared by Welch test. n.s.: P > 0.05; ***P 0.05
Dose
5
64.498
