1. Pea aphids (Acyrthosiphon pisum Harris; Hemiptera: Aphididae) exhibit transgenerational wing polyphenism, in which unwinged females produce genetically identical winged offspring in response to environmental cues such as overcrowding and predation risk that indicate poor habitat quality.
2. Laboratory experiments were carried out to explore the intensity of the wing polyphenic response of pea aphids exposed to cues from ladybird predators and crowding, and their response was compared with pea aphids that were not exposed to any cues (control).
3. The study used cues from two different ladybird species—Coccinella septempunctata L. (Coleoptera: Coccinellidae) and Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae)—to investigate whether the wing polyphenic response of pea aphids to predator cues can be generalized.
4. The intensity of the wing polyphenic response of pea aphids to crowding was found to be much stronger than their response to predator cues. There was no response to H. convergens cues and the response to C. septempunctata cues was mixed.
Includes 2 supplementary tables

Brisson, Jennifer A.

Purandare, Swapna R.

Tenhumberg, Brigitte

English

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Brigitte Tenhumberg Papers Papers in the Biological Sciences 
2014 
Comparison of the wing polyphenic response of pea aphids 
(Acyrthosiphon pisum) to crowding and predator cues 
Swapna R. Purandare 
swapna.purandare@huskers.unl.edu 
Brigitte Tenhumberg 
University of Nebraska - Lincoln, btenhumberg2@unl.edu 
Jennifer A. Brisson 
University of Nebraska - Lincoln, jennifer.brisson@rochester.edu 
Follow this and additional works at: https://digitalcommons.unl.edu/bioscitenhumberg 
Purandare, Swapna R.; Tenhumberg, Brigitte; and Brisson, Jennifer A., "Comparison of the wing polyphenic 
response of pea aphids (Acyrthosiphon pisum) to crowding and predator cues" (2014). Brigitte 
Tenhumberg Papers. 1. 
https://digitalcommons.unl.edu/bioscitenhumberg/1 
This Article is brought to you for free and open access by the Papers in the Biological Sciences at 
DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Brigitte Tenhumberg 
Papers by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 
Introduction
Polyphenism is an extreme form of phenotypic plasticity 
in which alternate, discrete phenotypes are produced from 
the same genotype as a response to environmental varia-
tion (Nijhout, 1999). The pea aphid (Acyrthosiphon pisum) 
exhibits a transgenerational wing polyphenism, in which 
unwinged asexual females produce genetically identical 
winged offspring. Winged morphs engage in long-range 
dispersal and thus can escape declining habitat quality, 
but long-range dispersal is risky because it mainly occurs 
passively through wind (Dieckmann et al., 1999; Comp-
ton, 2002), and consequently a large proportion of dispers-
ers die before reaching a new host plant (Ward et al., 1998). 
Furthermore, the costs for producing winged morphs in-
clude an extended development time and reduced fecun-
dity (Dixon, 1998).
Crowding conditions induce the production of winged 
offspring in aphids because host plant quality deteriorates 
when the number of feeding aphids is high (Sutherland, 
1969a,b). Aphids also experience elevated mortality risk if 
the density of natural enemies and/or pathogens is high, 
and hence transgenerational wing polyphenism in aphids 
can be induced by the presence of parasitoids (Sloggett & 
Weisser, 2002), pathogens (Hatano et al., 2012), and aphid 
predators (Weisser et al., 1999; Kunert & Weisser, 2003). For 
wing induction via predation, it is sufficient for aphids to 
encounter cues associated with high predation risk such 
as predator tracks (Dixon & Agarwala, 1999; Mondor et al., 
2005).
Aphid predators are mobile and may leave an area 
soon after depositing cues. Thus, the presence of predatory 
cues may not predict poor habitat quality with the same 
certainty as crowding, in which case we would expect a 
weaker intensity of the wing polyphenic response. In order 
to understand the costs and benefits of dispersal, it is im-
portant to evaluate the intensity of wing polyphenism as-
sociated with different habitat quality indicators. We com-
pared the intensity of the wing polyphenic response of pea 
aphids to crowding and cues of two different ladybird spe-
cies, Coccinella septempunctata and Hippodamia convergens. 
Our results provide an insight into understanding the in-
teractions between two fundamental ecological processes, 
predation and dispersal.
Published in Ecological Entomology 39 (2014), pp. 263–266; doi 10.1111/een.12080
Copyright © 2013 Royal Entomological Society. Used by permission.
Accepted September 12, 2013; published online November 18, 2013.
Supporting information for this article follows the References.
Comparison of the wing polyphenic response of  
pea aphids (Acyrthosiphon pisum) to crowding  
and predator cues
Swapna R. Purandare,1 Brigitte Tenhumberg,1,2 and Jennifer A. Brisson1
1. School of Biological Sciences, University of Nebraska–Lincoln, Lincoln, Nebraska, USA
2. Department of Mathematics, University of Nebraska–Lincoln, Lincoln, Nebraska, USA
Corresponding author – Swapna R. Purandare, School of Biological Sciences, University of Nebraska–Lincoln,  
Lincoln, NE 68588-0118, USA, email swapna.purandare@huskers.unl.edu
Abstract
1. Pea aphids (Acyrthosiphon pisum Harris; Hemiptera: Aphididae) exhibit transgenerational wing polyphenism, in which un-
winged females produce genetically identical winged offspring in response to environmental cues such as overcrowding and 
predation risk that indicate poor habitat quality.
2. Laboratory experiments were carried out to explore the intensity of the wing polyphenic response of pea aphids exposed to 
cues from ladybird predators and crowding, and their response was compared with pea aphids that were not exposed to any 
cues (control).
3. The study used cues from two different ladybird species—Coccinella septempunctata L. (Coleoptera: Coccinellidae) and Hippo-
damia convergens Guérin-Méneville (Coleoptera: Coccinellidae)—to investigate whether the wing polyphenic response of pea 
aphids to predator cues can be generalized.
4. The intensity of the wing polyphenic response of pea aphids to crowding was found to be much stronger than their response to 
predator cues. There was no response to H. convergens cues and the response to C. septempunctata cues was mixed.
Keywords: Crowding, dispersal, polyphenism, predation risk, wing induction
263
digitalcommons.unl.edu
264 Pu r a n d a r e,  Te n h u m b e r g,  a n d br i s s o n,  Ec o l o g i c a l En t o m o l o g y  39  (2014) 
Materials and methods
Insect collection and rearing
Predators. Adult C. septempunctata were collected from an 
alfalfa field in Lincoln, Nebraska, in July 2011, while adult 
H. convergens were purchased from commercial suppliers 
(Hirt’s Gardens, Wadsworth, Ohio) in May 2011. Both coc-
cinellid species were reared in chiffon-netted aluminum 
cages (44×51×61 cm) in growth chambers at approximately 
25°C and LD 16:8 h on Vicia faba L. plants infested with pea 
aphids.
Aphids. Three pea aphid clones were used to increase the 
generalization of our results because different aphid clones 
vary in their sensitivity to environmental cues and in their 
wing polyphenic response (Sutherland, 1969a; Weisser & 
Braendle, 2001). To prevent mixing of clones, all aphids 
were caged by enclosing the host plant V. faba using Plexi-
glass tubes (21.5 cm high and 6.5 cm diameter) with a mesh 
on top. The aphids were maintained in the laboratory at 17 
± 1.5°C, RH 35–45%, and LD 16:8 h.
Experimental procedure
The experiments were carried out in growth chambers at 
17 ± 1.5°C, RH 35–45%, and LD 16:8 h. Pea aphids were 
maintained on V. faba plants at low density (six individuals 
per plant) until they started reproducing. Aphids reared on 
the same plant were divided randomly between the treat-
ments to avoid differences in the maternal environment 
that could influence their offspring phenotype. Pea aphids 
are viviparous, and therefore adult asexual females have 
embryos in their ovaries. Since the winged/unwinged 
morph determination in pea aphids is prenatal (Suther-
land, 1969a,b), all experimental treatments were applied to 
adult asexual females within the first 3 days of the begin-
ning of their reproductive period.
The experimental arena consisted of a plastic Petri plate 
(60mm×15 mm, Fisher) with two Medicago arborea leaves in-
serted in 3ml of 2% bactoagar mixed with Miracle-Gro (The 
Scotts Miracle-Gro Company, Marysville, Ohio) (plate). 
Leaves, rather than plants, were used in order to restrict 
the predator cues to a small area, which increased the prob-
ability that aphids encountered the cues during the experi-
ment. Adult unwinged asexual female pea aphids were di-
vided randomly between the following treatments:
• Predator cues. One well-fed adult of either H. convergens 
or C. septempunctata was released in the experimental 
arena. The predators were allowed to search and de-
posit cues (eggs, feces, and tracks) at 22°C and LD 16:8 
h under fluorescent light for 24 h. As the response of 
aphids to ladybird tracks can depend on the predator 
sex (Ninkovic et al., 2013), we included both sexes in 
our experiments and randomly distributed them be-
tween the treatments. After removal of the predator, 
one adult, unwinged aphid female was placed in each 
plate.
• Crowding. Ten adult unwinged aphid females were 
placed in a small empty plastic Petri dish (32.5mm×15 
mm) for 24 h, and then each individual was transferred 
to its own plate. In our statistical analysis, we included 
only one randomly chosen aphid per Petri dish.
• Control. A single adult, unwinged aphid female was 
placed in a plate; predator cues were absent.
The response of aphids to predator tracks is known 
to decrease with the age of the tracks (Ninkovic et al., 
2013). Therefore, female aphids were removed from all 
plates after 24 h, the offspring produced during that du-
ration were reared until they reached adulthood (14 ± 
2 days), and the phenotype of the offspring after reach-
ing maturity was recorded. We recorded the proportion 
of pea aphid females producing at least one winged off-
spring (“induced aphids”) and the proportion of winged 
offspring produced by induced pea aphid females. The 
experiments were replicated for each of the three aphid 
clones (Hf-alf-07, seven replicates; Roc-1, six replicates; 
HF-74, four replicates).
All analyses were performed using r (v. 2.15.0; R De-
velopment Core Team, 2012). Backward model selection 
was performed and likelihood ratio tests were used to de-
cide which model fitted the data best. A generalized lin-
ear mixed model (GLMM) with a binomial error distribu-
tion was used to analyze the proportion of induced aphids, 
treating clone and treatment as fixed effects and starting 
date as a random effect. The same statistical model was 
used to analyze the proportion of winged offspring pro-
duced by each induced aphid.
Results
Aphid clone had no significant effect on the proportion 
of aphids induced (Hf-alf-07, P = 0.17; Roc-1, P = 0.33; ta-
ble S1 in supporting information) and the proportion of 
winged offspring produced by the induced aphids (Hf-
alf-07, P = 0.30; Roc-1, P = 0.92; table S2 in supporting 
information). Crowding produced a strong wing poly-
phenic response. In comparison to the control treatment 
(no cues), the proportion of induced aphids (P = 0.001, 
fig. 1) and the proportion of winged offspring produced 
by induced aphids (P = 0.0001, fig. 2) were significantly 
higher than in the control. Exposure to C. septempunc-
tata cues elicited a weak but significant response. Com-
pared with the control, the proportion of induced aphids 
increased significantly (P = 0.003), but the proportion of 
winged offspring produced by induced aphids decreased 
(P = 0.03). By contrast, exposure to H. convergens cues had 
no effect on the proportion of induced aphids (P = 0.14) 
and winged offspring (P = 0.87).
Discussion
We found a strong wing polyphenic response of pea aphids 
to crowding (roughly twice as high as the control; fig. 1), 
Co m P a r i s o n o f T h e w i n g P o l y P h e n i C r e s P o n s e o f P e a a P h i d s   265
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
which is consistent with previous studies (Sutherland, 
1969a,b). We found no response of pea aphids to H. con-
vergens cues and a mixed response to C. septempunc-
tata cues. Even though the proportion of induced aphids 
(Pinduced) was significantly higher in aphids exposed to C. 
septempunctata cues, the proportion of winged offspring 
(Poffspring) was lower than the control aphids, suggest-
ing that the total dispersal response to predator cues is 
negligible [PinducedPoffspring = 12% (Control), 13% (H. conver-
gens), and 14% (C. septempunctata)]. We suggest two poten-
tial reasons for the weak response to predator cues. First, 
the wing polyphenic response is transgenerational in pea 
aphids, and the delay between the time the females are in-
duced and the offspring dispersal response is substantial. 
Thus, the predatory ladybird that induces the production 
of winged offspring in an aphid colony is unlikely to be 
present when the winged offspring mature (Minoretti & 
Weisser, 2000). The evolution of delayed predator-induced 
dispersal is only adaptive under special circumstances. Ac-
cording to a model by Poethke et al. (2010), a substantial 
proportion (more than 80%) of the population should dis-
perse only if the predation risk is high (≥ 0.8), predators 
revisit patches (return probability ≥ 0.8), and predator-in-
duced mortality increases after the first visit of a preda-
tor. These conditions may not apply to ladybirds foraging 
for aphids. Secondly, it is possible that our predator treat-
ment did not provide a sufficient number of cues to in-
duce a strong wing polyphenic response. In our predator 
treatment, we used isolated aphids to separate the effect of 
predator cues on wing induction from cues (such as tactile 
cues) associated with crowding (Sutherland, 1969a). Only 
a weak response by isolated pea aphids to predator tracks 
has been observed in a different aphid-coccinellid system 
(pea aphids–Adalia bipunctata; Dixon & Agarwala, 1999).
Acknowledgments – We would like to thank two anon-
ymous reviewers for their helpful comments, Emily Zahn 
for her help with aphid rearing and conducting the experi-
ments, and Travis Hinkelman for the useful discussions. This 
work was supported by the University of Nebraska–Lincoln 
School of Biological Sciences Startup Funds and NIEHS award 
4R00ES017367 (JAB) and University of Nebraska–Lincoln 
School of Biological Sciences Special Funds (SRP).
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Figure 2. Proportion of winged offspring produced by in-
duced pea aphids for each treatment. The bars indicate the 
means, and the lines show the 95% confidence interval. Con-
trol, no cues; H. conv., exposure to Hippodamia convergens cues; 
C. sept, exposure to Coccinella septempunctata cues; crowding, 
exposure to crowding. All comparisons are made with respect 
to the control.
Figure 1. Proportion of induced pea aphids for each treat-
ment. The bars indicate the mean values, and the lines show 
the 95% confidence interval. Control, no cues; H. conv., expo-
sure to Hippodamia convergens cues; C. sept, exposure to Cocci-
nella septempunctata cues; crowding, exposure to crowding. All 
comparisons are made with respect to the control.
266 Pu r a n d a r e,  Te n h u m b e r g,  a n d br i s s o n,  Ec o l o g i c a l En t o m o l o g y  39  (2014) 
interactions a case study in aphid wing polymorphism. 
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opment. Bioscience, 49, 181–192.
Ninkovic, V., Feng, Y., Olsson, U., & Pettersson, J. (2013) Lady-
bird footprints induce aphid avoidance behavior. Biological 
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Supplementary Table S1: Proportion of pea aphids induced.  The predicted proportion 
of induced aphids for each of the treatment is depicted in Figure 1.  
SE = standard error. 
 
Fixed Effects Estimate SE P Value 
Control and Clone HF-74 (Intercept) –0.72 0.38 0.054 
Predator Treatment-C7 0.66 0.21 0.0017** 
Predator Treatment-HC 0.31 0.21 0.14 
Crowding Treatment-CR 1.81 0.56 0.001** 
Clone HF-alf-07 –0.61 0.45 0.17 
Clone Roc-1 0.4 0.44 0.33 
 
 
      
Supplementary Table S2: Proportion of winged offspring produced by induced pea 1 
aphids. The predicted proportion of induced aphids for each of the treatment is 2 
depicted in Figure 2. SE = standard error. 3 
 4 
Fixed Effects Estimate SE P Value 
Control and Clone HF-74 (Intercept) –0.55 0.31 0.0795 
Predator Treatment-C7 –0.26 0.12 0.03 * 
Predator Treatment-HC –0.02 0.13 0.87 
Crowding Treatment-CR 0.78 0.19 4.97e-05 *** 
Clone HF-alf-07 0.39 0.38 0.30 
Clone Roc-1 –0.04 0.37 0.92 
 5 
 6 


Comparison of the wing polyphenic response of
pea aphids (\u3ci\u3eAcyrthosiphon pisum\u3c/i\u3e) to crowding
and predator cues

1. Pea aphids (Acyrthosiphon pisum Harris; Hemiptera: Aphididae) exhibit transgenerational wing polyphenism, in which unwinged females produce genetically identical winged offspring in response to environmental cues such as overcrowding and predation risk that indicate poor habitat quality.
2. Laboratory experiments were carried out to explore the intensity of the wing polyphenic response of pea aphids exposed to cues from ladybird predators and crowding, and their response was compared to pea aphids that were not exposed to any cues (control).
3. The study used cues from two different ladybird species: Coccinella septempunctata L. (Coleoptera: Coccinellidae) and Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae) to investigate whether the wing polyphenic response of pea aphids to predator cues can be generalized
4. The intensity of the wing polyphenic response of pea aphids to crowding was found to be much stronger than their response to predator cues. There was no response to H. convergens cues and the response to C. septempunctata cues was mixed

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University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Faculty Publications in the Biological Sciences Papers in the Biological Sciences 2014 Comparison of the wing polyphenic response of pea aphids (Acyrthosiphon pisum) to crowding and predator cues Swapna R. Purandare University of Nebraska-Lincoln, swapna.purandare@huskers.unl.edu Brigitte Tenhumberg University of Nebraska - Lincoln, btenhumberg2@unl.edu Jennifer A. Brisson University of Nebraska-Lincoln, jennifer.brisson@rochester.edu Follow this and additional works at: https://digitalcommons.unl.edu/bioscifacpub  Part of the Biology Commons Purandare, Swapna R.; Tenhumberg, Brigitte; and Brisson, Jennifer A., "Comparison of the wing polyphenic response of pea aphids (Acyrthosiphon pisum) to crowding and predator cues" (2014). Faculty Publications in the Biological Sciences. 455. https://digitalcommons.unl.edu/bioscifacpub/455 This Article is brought to you for free and open access by the Papers in the Biological Sciences at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications in the Biological Sciences by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Comparison of the wing polyphenic response of pea aphids(Acyrthosiphon pisum) to crowding and predator cuesSwapna R. Purandare1,*, Brigitte Tenhumberg1,2, and Jennifer A. Brisson11School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68588-0118, U.S.A.2Department of Mathematics, University of Nebraska-Lincoln, Lincoln, NE 68588-0118, U.S.A.Abstract1. Pea aphids (Acyrthosiphon pisum Harris; Hemiptera: Aphididae) exhibit transgenerational wingpolyphenism, in which unwinged females produce genetically identical winged offspring inresponse to environmental cues such as overcrowding and predation risk that indicate poor habitatquality.2. Laboratory experiments were carried out to explore the intensity of the wing polyphenicresponse of pea aphids exposed to cues from ladybird predators and crowding, and their responsewas compared to pea aphids that were not exposed to any cues (control).3. The study used cues from two different ladybird species: Coccinella septempunctata L.(Coleoptera: Coccinellidae) and Hippodamia convergens Guérin-Méneville (Coleoptera:Coccinellidae) to investigate whether the wing polyphenic response of pea aphids to predator cuescan be generalized4. The intensity of the wing polyphenic response of pea aphids to crowding was found to be muchstronger than their response to predator cues. There was no response to H. convergens cues andthe response to C. septempunctata cues was mixed.KeywordsPolyphenism; wing induction; predation risk; dispersal; crowdingIntroductionPolyphenism is an extreme form of phenotypic plasticity in which alternate, discretephenotypes are produced from the same genotype as a response to environmental variation(Nijhout 1999). The pea aphid (Acyrthosiphon pisum) exhibits a transgenerational wingpolyphenism, in which unwinged asexual females produce genetically identical wingedoffspring. Winged morphs engage in long-range dispersal and thus can escape declininghabitat quality, but long-range dispersal is risky because it mainly occurs passively throughwind (Dieckmann et al. 1999, Compton 2002) and consequently a large proportion of*Corresponding author: Swapna R. Purandare, School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE68588-0118, U.S.A., swapna.purandare@huskers.unl.edu, Telephone: +1-402-472-2720, Fax: +1-402-472-2083.NIH Public AccessAuthor ManuscriptEcol Entomol. Author manuscript; available in PMC 2015 April 01.Published in final edited form as:Ecol Entomol. 2014 April 1; 39(2): 263–266. doi:10.1111/een.12080.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptdispersers die before reaching a new host plant (Ward et al. 1998). Furthermore, the costsfor producing winged morphs include an extended development time and reduced fecundity(Dixon 1998).Crowding conditions induce the production of winged offspring in aphids because host plantquality deteriorates when the number of feeding aphids is high (Sutherland 1969a);Sutherland 1969b). Aphids also experience elevated mortality risk if the density of naturalenemies and/or pathogens is high and hence, transgenerational wing polyphenism in aphidscan be induced by the presence of parasitoids (Sloggett and Weisser 2002), pathogens(Hatano et al. 2012) and aphid predators (Weisser et al. 1999, Kunert & Weisser 2003). Forwing induction via predation, it is sufficient for aphids to encounter cues associated withhigh predation risk like predator tracks (Dixon and Agarwala 1999, Mondor et al. 2005).Aphid predators are mobile and may leave an area soon after depositing cues. Thus, thepresence of predatory cues may not predict poor habitat quality with the same certainty ascrowding in which case we would expect a weaker intensity of the wing polyphenicresponse. In order to understand the costs and benefits of dispersal it is important to evaluatethe intensity of wing polyphenism associated with different habitat quality indicators. Wecompared the intensity of the wing polyphenic response of pea aphids to crowding and cuesof two different ladybird species C. septempunctata and H. convergens. Our results provideinsight into understanding the interactions between two fundamental ecological processes,predation and dispersal.Materials and MethodsInsect collection and rearingPredators—Adult C. septempunctata were collected from an alfalfa field in Lincoln, NE,in July 2011, while adult H. convergens were purchased from commercial suppliers (Hirt'sGardens) in May 2011. Both coccinellid species were reared in chiffon netted aluminumcages (44×51×61 cm) in growth chambers at approximately 25°C on a 16:8 (L: D)photoperiod on Vicia faba L. plants infested with pea aphids.Aphids—Three pea aphid clones were used to increase the generalization of our resultsbecause different aphid clones vary in their sensitivity to environmental cues and in theirwing polyphenic response (Sutherland 1969a); Weisser and Braendle 2001). To preventmixing of clones, all aphids were caged by enclosing the host plant V. faba using Plexiglasstubes (21.5 cm high and 6.5 cm diameter) with a mesh on top. The aphids were maintainedin the laboratory at 17±1.5 °C, 35-45% relative humidity on a 16:8 (L: D) photoperiod.Experimental procedureThe experiments were carried out in growth chambers at 17±1.5 °C, 35-45% relativehumidity and a 16:8 (L: D) photoperiod. Pea aphids were maintained on V. faba plants atlow density (six individuals per plant) until they started reproducing. Aphids reared on thesame plant were divided randomly between the treatments to avoid differences in thematernal environment that could influence their offspring phenotype. Pea aphids areviviparous and therefore adult asexual females have embryos in their ovaries. Since thePurandare et al. Page 2Ecol Entomol. Author manuscript; available in PMC 2015 April 01.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptwinged/unwinged morph determination in pea aphids is prenatal (Sutherland 1969a);Sutherland 1969b), all experimental treatments were applied to adult asexual females withinthe first three days of the beginning of their reproductive period.The experimental arena consisted of a plastic Petri plate (60mm×15 mm, Fisher) with twoMedicago arborea leaves inserted in 3 ml of 2 % bactoagar mixed with MiracleGro (plate).Leaves, rather than plants, were used in order to restrict the predator cues to a small area,which increased the probability that aphids encountered the cues during the experiment.Adult unwinged asexual female pea aphids reared on the same plant were divided randomlybetween the following treatments.• Predator cues: One well-fed adult of either H. convergens or C. septempunctatawas released in the experimental arena. The predators were allowed to search anddeposit cues (eggs, feces and tracks) at 22 °C on a 16:8 (L: D) photoperiod underfluorescent light for 24 hours. Since the response of aphids to ladybird tracks candepend on the predator sex (Ninkovic et al. 2013), we included both sexes in ourexperiments and randomly distributed them between the treatments. After removalof the predator, one adult, unwinged aphid female was placed in each plate.• Crowding: Ten adult unwinged aphid females were placed in a small empty plasticPetri dish (32.5mm×15 mm) for 24 hours and then each individual was transferredto its own plate. In our statistical analysis we included only one randomly chosenaphid per Petri dish.• Control: A single adult, unwinged aphid female was placed in a plate; predatorcues were absent.The response of aphids to predator tracks is known to decrease with the age of the tracks(Ninkovic et al. 2013). Therefore, female aphids were removed from all plates after 24hours, and the offspring produced during that duration were reared until they reachedadulthood (14±2 days), and the phenotype of the offspring after reaching maturity wasrecorded. We recorded the proportion of pea aphid females producing at least one wingedoffspring (“induced aphids”) and the proportion of winged offspring produced by inducedpea aphid females. The experiments were replicated for each of the three aphid clones (Hf-alf-07: 7 replicates, Roc-1: 6 replicates, HF-74: 4 replicates).All analyses were performed using R (v. 2.15.0, R Development Core Team 2012).Backwards model selection was performed and likelihood ratio tests were used to decidewhich model fit the data best. A generalized linear mixed model (GLMM) with a binomialerror distribution was used to analyze the proportion of induced aphids, treating clone andtreatment as fixed effects and starting date as a random effect. The same statistical modelwas used to analyze the proportion of winged offspring produced by each induced aphid.ResultsAphid clone had no significant effect on the proportion of aphids induced (Hf-alf-07 p=0.17;Roc-1 p= 0.33, Table S1) and proportion of winged offspring produced by the inducedaphids (Hf-alf-07 p=0.30; Roc-1 p= 0.92, Table S2). Crowding produced a strong wingPurandare et al. Page 3Ecol Entomol. Author manuscript; available in PMC 2015 April 01.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptpolyphenic response. In comparison to the control treatment (no cues), the proportion ofinduced aphids (p=0.001, Fig.1) and the proportion of winged offspring produced byinduced aphids (p=0.0001, Fig. 2,) were significantly higher than in the control. Exposure toC. septempunctata cues elicited a weak but significant response. Compared to the control theproportion of induced aphids increased significantly (p=0.003), but the proportion of wingedoffspring produced by induced aphids decreased (p=0.03). In contrast, exposure to H.convergens cues had no effect on the proportion of induced aphids (p=0.14) and wingedoffspring (p=0.87).DiscussionWe found a strong wing polyphenic response of pea aphids to crowding (roughly twice ashigh as the control, Fig 1), which is consistent with previous studies (Sutherland 1969a,Sutherland 1969b). We found no response of pea aphids to H. convergens cues and a mixedresponse to C. septempunctata cues. Even though the proportion of induced aphids (Pinduced)was significantly higher in aphids exposed to C. septempunctata cues, the proportion ofwinged offspring (Poffspring) was lower than the control aphids suggesting that the totaldispersal response to predator cues is negligible (PinducedPoffspring= 12% (C), 13% (HC), and14% (C7)). We suggest two potential reasons for the weak response to predator cues. Firstly,the wing polyphenic response is transgenerational in pea aphids and the delay between thetime the females are induced and the offspring dispersal response is substantial. Thus, thepredatory ladybird that induces the production of winged offspring in an aphid colony isunlikely to be present when the winged offspring mature (Minerotti & Weisser 2000). Theevolution of delayed predator induced dispersal is only adaptive under specialcircumstances. According to a model byPoethke et al. (2010), a substantial proportion (morethan 80%) of the population should disperse only if predation risk is high (≥ 0.8), predatorsrevisit patches (return probability ≥ 0.8), and predator-induced mortality increases after thefirst visit of a predator. These conditions may not apply to ladybirds foraging for aphids.Secondly, it is possible that our predator treatment did not provide a sufficient number ofcues to induce a strong wing polyphenic response. In our predator treatment, we usedisolated aphids to separate the effect of predator cues on wing induction from cues such astactile cues associated with crowding (Sutherland 1969a). Only a weak response by isolatedpea aphids to predator tracks has been observed in a different aphid-coccinellid system (peaaphids-Adalia bipunctata, Dixon and Agarwala 1999).Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.ReferencesCompton, SG. Sailing with the wind: dispersal by small flying insects. In: Bullock, JM.; Kenward,RE.; Hails, RS., editors. Dispersal Ecology. Oxford: Blackwell Science Ltd; 2002. p. 113-133.Dieckmann U, O'Hara B, Weisser WW. The evolutionary ecology of dispersal. Trends in Ecology &Evolution. 1999; 14:88–90.Dixon, AFG. Resource tracking in time. In: Dixon, AFG., editor. Aphid Ecology: An OptimizationApproach. London, U.K.: Chapman & Hall; 1998. p. 128-170.Purandare et al. Page 4Ecol Entomol. Author manuscript; available in PMC 2015 April 01.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptDixon AFG, Agarwala BK. Ladybird-induced life history changes in aphids. Proceedings of the RoyalSociety of London Series B: Biological Sciences. 1999; 266:1549–1553.Hatano E, Baverstock J, Kunert G, Pell JK, Weisser WW. Entomopathogenic fungi stimulatetransgenerational wing induction in pea aphids, Acyrthosiphon pisum (Hemiptera: Aphididae).Ecological Entomology. 2012; 37(1):75–82.Kunert G, Weisser WW. The interplay between density-and trait-mediate effects in predator-preyinteractions: a case study in aphid wing polymorphism. Oecologia. 2003; 135:304–312. [PubMed:12698353]Mondor EB, Rosenheim JA, Addicott JF. Predator-induced transgenerational phenotypic plasticity inthe cotton aphid. Oecologia. 2005; 142:104–108. [PubMed: 15372226]Minoretti N, Weisser WW. The impact of individual ladybirds (Coccinella septempunctata,Coleoptera: Coccinellidae) on individual aphid colonies. European Journal of Entomology. 2000;97:475–479.Nijhout HF. Control mechanisms of polyphenic development. Bioscience. 1999; 49:181–192.Ninkovic V, Feng Y, Olsson U, Pettersson J. Ladybird footprints induce aphid avoidance behavior.Biological Control. 2013; 65:63–71.Poethke HJ, Weisser WW, Hovestadt T. Predator induced dispersal and the evolution of conditionaldispersal in correlated environments. American Naturalist. 2010; 175:577–586.R Development Core Team. R: A Language and Environment for Statistical Computing. Vienna,Austria: R Foundation for Statistical Computing; 2012. [WWW document]. URL http://www.R-project.org [accessed on 26 December 2012]Sloggett JJ, Weisser WW. Parasitoids induce production of the dispersal morph of the pea aphid,Acyrthosiphon pisum. Oikos. 2002; 98:323–333.Sutherland ORW. The role of crowding in the production of winged forms by two strains of the peaaphid, Acyrthosiphon pisum . Journal of Insect Physiology. 1969a; 15:1385–1410.Sutherland ORW. The role of the host plant in the production of winged forms by two strains of thepea aphid Acyrthosiphon pisum . Journal of Insect Physiology. 1969b; 15:2179–2201.Ward SA, Leather SR, Pickup J, Harrington R. Mortality during dispersal and the cost of host-specificity in parasites: how many aphids find hosts? Journal of Animal Ecology. 1998; 67:763–773.Weisser WW, Braendle C, Minoretti N. Predator-induced morphological shift in the pea aphid.Proceedings of the Royal Society B: Biological Sciences. 1999; 266:1175–1181.Weisser WW, Braendle C. Body colour and genetic variation in winged morph production in the peaaphid. Entomologia Experimentalis et Applicata. 2001; 99:217–223.Purandare et al. Page 5Ecol Entomol. Author manuscript; available in PMC 2015 April 01.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1.Proportion of induced pea aphids for each treatment. The bars indicate the mean values, and the lines show the 95% confidenceinterval. Control = No cues, H. conv. = exposure to H. convergens cues, C. sept = exposure to C. septempunctata cues,Crowding = exposure to crowding. All comparisons are made with respect to the control.Purandare et al. Page 6Ecol Entomol. Author manuscript; available in PMC 2015 April 01.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 2.Proportion of winged offspring produced by induced pea aphids for each treatment. The bars indicate the means, and the linesshow the 95% confidence interval. Control = No cues, H. conv. = exposure to H. convergens cues, C. sept = exposure to C.septempunctata cues, Crowding = exposure to crowding. All comparisons are made with respect to the control.Purandare et al. Page 7Ecol Entomol. Author manuscript; available in PMC 2015 April 01.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

Comparison of the wing polyphenic response of pea aphids
(\u3ci\u3eAcyrthosiphon pisum\u3c/i\u3e) to crowding and predator cues

University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
Faculty Publications in the Biological Sciences Papers in the Biological Sciences 
2014 
Comparison of the wing polyphenic response of pea aphids 
(Acyrthosiphon pisum) to crowding and predator cues 
Swapna R. Purandare 
University of Nebraska-Lincoln, swapna.purandare@huskers.unl.edu 
Brigitte Tenhumberg 
University of Nebraska - Lincoln, btenhumberg2@unl.edu 
Jennifer A. Brisson 
University of Nebraska-Lincoln, jennifer.brisson@rochester.edu 
Follow this and additional works at: https://digitalcommons.unl.edu/bioscifacpub 
 Part of the Biology Commons 
Purandare, Swapna R.; Tenhumberg, Brigitte; and Brisson, Jennifer A., "Comparison of the wing polyphenic 
response of pea aphids (Acyrthosiphon pisum) to crowding and predator cues" (2014). Faculty 
Publications in the Biological Sciences. 455. 
https://digitalcommons.unl.edu/bioscifacpub/455 
This Article is brought to you for free and open access by the Papers in the Biological Sciences at 
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Comparison of the wing polyphenic response of pea aphids
(Acyrthosiphon pisum) to crowding and predator cues
Swapna R. Purandare1,*, Brigitte Tenhumberg1,2, and Jennifer A. Brisson1
1School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68588-0118, U.S.A.
2Department of Mathematics, University of Nebraska-Lincoln, Lincoln, NE 68588-0118, U.S.A.
Abstract
1. Pea aphids (Acyrthosiphon pisum Harris; Hemiptera: Aphididae) exhibit transgenerational wing
polyphenism, in which unwinged females produce genetically identical winged offspring in
response to environmental cues such as overcrowding and predation risk that indicate poor habitat
quality.
2. Laboratory experiments were carried out to explore the intensity of the wing polyphenic
response of pea aphids exposed to cues from ladybird predators and crowding, and their response
was compared to pea aphids that were not exposed to any cues (control).
3. The study used cues from two different ladybird species: Coccinella septempunctata L.
(Coleoptera: Coccinellidae) and Hippodamia convergens Guérin-Méneville (Coleoptera:
Coccinellidae) to investigate whether the wing polyphenic response of pea aphids to predator cues
can be generalized
4. The intensity of the wing polyphenic response of pea aphids to crowding was found to be much
stronger than their response to predator cues. There was no response to H. convergens cues and
the response to C. septempunctata cues was mixed.
Keywords
Polyphenism; wing induction; predation risk; dispersal; crowding
Introduction
Polyphenism is an extreme form of phenotypic plasticity in which alternate, discrete
phenotypes are produced from the same genotype as a response to environmental variation
(Nijhout 1999). The pea aphid (Acyrthosiphon pisum) exhibits a transgenerational wing
polyphenism, in which unwinged asexual females produce genetically identical winged
offspring. Winged morphs engage in long-range dispersal and thus can escape declining
habitat quality, but long-range dispersal is risky because it mainly occurs passively through
wind (Dieckmann et al. 1999, Compton 2002) and consequently a large proportion of
*Corresponding author: Swapna R. Purandare, School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE
68588-0118, U.S.A., swapna.purandare@huskers.unl.edu, Telephone: +1-402-472-2720, Fax: +1-402-472-2083.
NIH Public Access
Author Manuscript
Ecol Entomol. Author manuscript; available in PMC 2015 April 01.
Published in final edited form as:
Ecol Entomol. 2014 April 1; 39(2): 263–266. doi:10.1111/een.12080.
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dispersers die before reaching a new host plant (Ward et al. 1998). Furthermore, the costs
for producing winged morphs include an extended development time and reduced fecundity
(Dixon 1998).
Crowding conditions induce the production of winged offspring in aphids because host plant
quality deteriorates when the number of feeding aphids is high (Sutherland 1969a);
Sutherland 1969b). Aphids also experience elevated mortality risk if the density of natural
enemies and/or pathogens is high and hence, transgenerational wing polyphenism in aphids
can be induced by the presence of parasitoids (Sloggett and Weisser 2002), pathogens
(Hatano et al. 2012) and aphid predators (Weisser et al. 1999, Kunert & Weisser 2003). For
wing induction via predation, it is sufficient for aphids to encounter cues associated with
high predation risk like predator tracks (Dixon and Agarwala 1999, Mondor et al. 2005).
Aphid predators are mobile and may leave an area soon after depositing cues. Thus, the
presence of predatory cues may not predict poor habitat quality with the same certainty as
crowding in which case we would expect a weaker intensity of the wing polyphenic
response. In order to understand the costs and benefits of dispersal it is important to evaluate
the intensity of wing polyphenism associated with different habitat quality indicators. We
compared the intensity of the wing polyphenic response of pea aphids to crowding and cues
of two different ladybird species C. septempunctata and H. convergens. Our results provide
insight into understanding the interactions between two fundamental ecological processes,
predation and dispersal.
Materials and Methods
Insect collection and rearing
Predators—Adult C. septempunctata were collected from an alfalfa field in Lincoln, NE,
in July 2011, while adult H. convergens were purchased from commercial suppliers (Hirt's
Gardens) in May 2011. Both coccinellid species were reared in chiffon netted aluminum
cages (44×51×61 cm) in growth chambers at approximately 25°C on a 16:8 (L: D)
photoperiod on Vicia faba L. plants infested with pea aphids.
Aphids—Three pea aphid clones were used to increase the generalization of our results
because different aphid clones vary in their sensitivity to environmental cues and in their
wing polyphenic response (Sutherland 1969a); Weisser and Braendle 2001). To prevent
mixing of clones, all aphids were caged by enclosing the host plant V. faba using Plexiglass
tubes (21.5 cm high and 6.5 cm diameter) with a mesh on top. The aphids were maintained
in the laboratory at 17±1.5 °C, 35-45% relative humidity on a 16:8 (L: D) photoperiod.
Experimental procedure
The experiments were carried out in growth chambers at 17±1.5 °C, 35-45% relative
humidity and a 16:8 (L: D) photoperiod. Pea aphids were maintained on V. faba plants at
low density (six individuals per plant) until they started reproducing. Aphids reared on the
same plant were divided randomly between the treatments to avoid differences in the
maternal environment that could influence their offspring phenotype. Pea aphids are
viviparous and therefore adult asexual females have embryos in their ovaries. Since the
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winged/unwinged morph determination in pea aphids is prenatal (Sutherland 1969a);
Sutherland 1969b), all experimental treatments were applied to adult asexual females within
the first three days of the beginning of their reproductive period.
The experimental arena consisted of a plastic Petri plate (60mm×15 mm, Fisher) with two
Medicago arborea leaves inserted in 3 ml of 2 % bactoagar mixed with MiracleGro (plate).
Leaves, rather than plants, were used in order to restrict the predator cues to a small area,
which increased the probability that aphids encountered the cues during the experiment.
Adult unwinged asexual female pea aphids reared on the same plant were divided randomly
between the following treatments.
• Predator cues: One well-fed adult of either H. convergens or C. septempunctata
was released in the experimental arena. The predators were allowed to search and
deposit cues (eggs, feces and tracks) at 22 °C on a 16:8 (L: D) photoperiod under
fluorescent light for 24 hours. Since the response of aphids to ladybird tracks can
depend on the predator sex (Ninkovic et al. 2013), we included both sexes in our
experiments and randomly distributed them between the treatments. After removal
of the predator, one adult, unwinged aphid female was placed in each plate.
• Crowding: Ten adult unwinged aphid females were placed in a small empty plastic
Petri dish (32.5mm×15 mm) for 24 hours and then each individual was transferred
to its own plate. In our statistical analysis we included only one randomly chosen
aphid per Petri dish.
• Control: A single adult, unwinged aphid female was placed in a plate; predator
cues were absent.
The response of aphids to predator tracks is known to decrease with the age of the tracks
(Ninkovic et al. 2013). Therefore, female aphids were removed from all plates after 24
hours, and the offspring produced during that duration were reared until they reached
adulthood (14±2 days), and the phenotype of the offspring after reaching maturity was
recorded. We recorded the proportion of pea aphid females producing at least one winged
offspring (“induced aphids”) and the proportion of winged offspring produced by induced
pea aphid females. The experiments were replicated for each of the three aphid clones (Hf-
alf-07: 7 replicates, Roc-1: 6 replicates, HF-74: 4 replicates).
All analyses were performed using R (v. 2.15.0, R Development Core Team 2012).
Backwards model selection was performed and likelihood ratio tests were used to decide
which model fit the data best. A generalized linear mixed model (GLMM) with a binomial
error distribution was used to analyze the proportion of induced aphids, treating clone and
treatment as fixed effects and starting date as a random effect. The same statistical model
was used to analyze the proportion of winged offspring produced by each induced aphid.
Results
Aphid clone had no significant effect on the proportion of aphids induced (Hf-alf-07 p=0.17;
Roc-1 p= 0.33, Table S1) and proportion of winged offspring produced by the induced
aphids (Hf-alf-07 p=0.30; Roc-1 p= 0.92, Table S2). Crowding produced a strong wing
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polyphenic response. In comparison to the control treatment (no cues), the proportion of
induced aphids (p=0.001, Fig.1) and the proportion of winged offspring produced by
induced aphids (p=0.0001, Fig. 2,) were significantly higher than in the control. Exposure to
C. septempunctata cues elicited a weak but significant response. Compared to the control the
proportion of induced aphids increased significantly (p=0.003), but the proportion of winged
offspring produced by induced aphids decreased (p=0.03). In contrast, exposure to H.
convergens cues had no effect on the proportion of induced aphids (p=0.14) and winged
offspring (p=0.87).
Discussion
We found a strong wing polyphenic response of pea aphids to crowding (roughly twice as
high as the control, Fig 1), which is consistent with previous studies (Sutherland 1969a,
Sutherland 1969b). We found no response of pea aphids to H. convergens cues and a mixed
response to C. septempunctata cues. Even though the proportion of induced aphids (Pinduced)
was significantly higher in aphids exposed to C. septempunctata cues, the proportion of
winged offspring (Poffspring) was lower than the control aphids suggesting that the total
dispersal response to predator cues is negligible (PinducedPoffspring= 12% (C), 13% (HC), and
14% (C7)). We suggest two potential reasons for the weak response to predator cues. Firstly,
the wing polyphenic response is transgenerational in pea aphids and the delay between the
time the females are induced and the offspring dispersal response is substantial. Thus, the
predatory ladybird that induces the production of winged offspring in an aphid colony is
unlikely to be present when the winged offspring mature (Minerotti & Weisser 2000). The
evolution of delayed predator induced dispersal is only adaptive under special
circumstances. According to a model byPoethke et al. (2010), a substantial proportion (more
than 80%) of the population should disperse only if predation risk is high (≥ 0.8), predators
revisit patches (return probability ≥ 0.8), and predator-induced mortality increases after the
first visit of a predator. These conditions may not apply to ladybirds foraging for aphids.
Secondly, it is possible that our predator treatment did not provide a sufficient number of
cues to induce a strong wing polyphenic response. In our predator treatment, we used
isolated aphids to separate the effect of predator cues on wing induction from cues such as
tactile cues associated with crowding (Sutherland 1969a). Only a weak response by isolated
pea aphids to predator tracks has been observed in a different aphid-coccinellid system (pea
aphids-Adalia bipunctata, Dixon and Agarwala 1999).
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
References
Compton, SG. Sailing with the wind: dispersal by small flying insects. In: Bullock, JM.; Kenward,
RE.; Hails, RS., editors. Dispersal Ecology. Oxford: Blackwell Science Ltd; 2002. p. 113-133.
Dieckmann U, O'Hara B, Weisser WW. The evolutionary ecology of dispersal. Trends in Ecology &
Evolution. 1999; 14:88–90.
Dixon, AFG. Resource tracking in time. In: Dixon, AFG., editor. Aphid Ecology: An Optimization
Approach. London, U.K.: Chapman & Hall; 1998. p. 128-170.
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Dixon AFG, Agarwala BK. Ladybird-induced life history changes in aphids. Proceedings of the Royal
Society of London Series B: Biological Sciences. 1999; 266:1549–1553.
Hatano E, Baverstock J, Kunert G, Pell JK, Weisser WW. Entomopathogenic fungi stimulate
transgenerational wing induction in pea aphids, Acyrthosiphon pisum (Hemiptera: Aphididae).
Ecological Entomology. 2012; 37(1):75–82.
Kunert G, Weisser WW. The interplay between density-and trait-mediate effects in predator-prey
interactions: a case study in aphid wing polymorphism. Oecologia. 2003; 135:304–312. [PubMed:
12698353]
Mondor EB, Rosenheim JA, Addicott JF. Predator-induced transgenerational phenotypic plasticity in
the cotton aphid. Oecologia. 2005; 142:104–108. [PubMed: 15372226]
Minoretti N, Weisser WW. The impact of individual ladybirds (Coccinella septempunctata,
Coleoptera: Coccinellidae) on individual aphid colonies. European Journal of Entomology. 2000;
97:475–479.
Nijhout HF. Control mechanisms of polyphenic development. Bioscience. 1999; 49:181–192.
Ninkovic V, Feng Y, Olsson U, Pettersson J. Ladybird footprints induce aphid avoidance behavior.
Biological Control. 2013; 65:63–71.
Poethke HJ, Weisser WW, Hovestadt T. Predator induced dispersal and the evolution of conditional
dispersal in correlated environments. American Naturalist. 2010; 175:577–586.
R Development Core Team. R: A Language and Environment for Statistical Computing. Vienna,
Austria: R Foundation for Statistical Computing; 2012. [WWW document]. URL http://www.R-
project.org [accessed on 26 December 2012]
Sloggett JJ, Weisser WW. Parasitoids induce production of the dispersal morph of the pea aphid,
Acyrthosiphon pisum. Oikos. 2002; 98:323–333.
Sutherland ORW. The role of crowding in the production of winged forms by two strains of the pea
aphid, Acyrthosiphon pisum . Journal of Insect Physiology. 1969a; 15:1385–1410.
Sutherland ORW. The role of the host plant in the production of winged forms by two strains of the
pea aphid Acyrthosiphon pisum . Journal of Insect Physiology. 1969b; 15:2179–2201.
Ward SA, Leather SR, Pickup J, Harrington R. Mortality during dispersal and the cost of host-
specificity in parasites: how many aphids find hosts? Journal of Animal Ecology. 1998; 67:763–
773.
Weisser WW, Braendle C, Minoretti N. Predator-induced morphological shift in the pea aphid.
Proceedings of the Royal Society B: Biological Sciences. 1999; 266:1175–1181.
Weisser WW, Braendle C. Body colour and genetic variation in winged morph production in the pea
aphid. Entomologia Experimentalis et Applicata. 2001; 99:217–223.
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Figure 1.
Proportion of induced pea aphids for each treatment. The bars indicate the mean values, and the lines show the 95% confidence
interval. Control = No cues, H. conv. = exposure to H. convergens cues, C. sept = exposure to C. septempunctata cues,
Crowding = exposure to crowding. All comparisons are made with respect to the control.
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Figure 2.
Proportion of winged offspring produced by induced pea aphids for each treatment. The bars indicate the means, and the lines
show the 95% confidence interval. Control = No cues, H. conv. = exposure to H. convergens cues, C. sept = exposure to C.
septempunctata cues, Crowding = exposure to crowding. All comparisons are made with respect to the control.
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University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Brigitte Tenhumberg Papers Papers in the Biological Sciences 2014 Comparison of the wing polyphenic response of pea aphids (Acyrthosiphon pisum) to crowding and predator cues Swapna R. Purandare swapna.purandare@huskers.unl.edu Brigitte Tenhumberg University of Nebraska - Lincoln, btenhumberg2@unl.edu Jennifer A. Brisson University of Nebraska - Lincoln, jennifer.brisson@rochester.edu Follow this and additional works at: https://digitalcommons.unl.edu/bioscitenhumberg Purandare, Swapna R.; Tenhumberg, Brigitte; and Brisson, Jennifer A., "Comparison of the wing polyphenic response of pea aphids (Acyrthosiphon pisum) to crowding and predator cues" (2014). Brigitte Tenhumberg Papers. 1. https://digitalcommons.unl.edu/bioscitenhumberg/1 This Article is brought to you for free and open access by the Papers in the Biological Sciences at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Brigitte Tenhumberg Papers by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. IntroductionPolyphenism is an extreme form of phenotypic plasticity in which alternate, discrete phenotypes are produced from the same genotype as a response to environmental varia-tion (Nijhout, 1999). The pea aphid (Acyrthosiphon pisum) exhibits a transgenerational wing polyphenism, in which unwinged asexual females produce genetically identical winged offspring. Winged morphs engage in long-range dispersal and thus can escape declining habitat quality, but long-range dispersal is risky because it mainly occurs passively through wind (Dieckmann et al., 1999; Comp-ton, 2002), and consequently a large proportion of dispers-ers die before reaching a new host plant (Ward et al., 1998). Furthermore, the costs for producing winged morphs in-clude an extended development time and reduced fecun-dity (Dixon, 1998).Crowding conditions induce the production of winged offspring in aphids because host plant quality deteriorates when the number of feeding aphids is high (Sutherland, 1969a,b). Aphids also experience elevated mortality risk if the density of natural enemies and/or pathogens is high, and hence transgenerational wing polyphenism in aphids can be induced by the presence of parasitoids (Sloggett & Weisser, 2002), pathogens (Hatano et al., 2012), and aphid predators (Weisser et al., 1999; Kunert & Weisser, 2003). For wing induction via predation, it is sufficient for aphids to encounter cues associated with high predation risk such as predator tracks (Dixon & Agarwala, 1999; Mondor et al., 2005).Aphid predators are mobile and may leave an area soon after depositing cues. Thus, the presence of predatory cues may not predict poor habitat quality with the same certainty as crowding, in which case we would expect a weaker intensity of the wing polyphenic response. In order to understand the costs and benefits of dispersal, it is im-portant to evaluate the intensity of wing polyphenism as-sociated with different habitat quality indicators. We com-pared the intensity of the wing polyphenic response of pea aphids to crowding and cues of two different ladybird spe-cies, Coccinella septempunctata and Hippodamia convergens. Our results provide an insight into understanding the in-teractions between two fundamental ecological processes, predation and dispersal.Published in Ecological Entomology 39 (2014), pp. 263–266; doi 10.1111/een.12080Copyright © 2013 Royal Entomological Society. Used by permission.Accepted September 12, 2013; published online November 18, 2013.Supporting information for this article follows the References.Comparison of the wing polyphenic response of  pea aphids (Acyrthosiphon pisum) to crowding  and predator cuesSwapna R. Purandare,1 Brigitte Tenhumberg,1,2 and Jennifer A. Brisson11. School of Biological Sciences, University of Nebraska–Lincoln, Lincoln, Nebraska, USA2. Department of Mathematics, University of Nebraska–Lincoln, Lincoln, Nebraska, USACorresponding author – Swapna R. Purandare, School of Biological Sciences, University of Nebraska–Lincoln,  Lincoln, NE 68588-0118, USA, email swapna.purandare@huskers.unl.eduAbstract1. Pea aphids (Acyrthosiphon pisum Harris; Hemiptera: Aphididae) exhibit transgenerational wing polyphenism, in which un-winged females produce genetically identical winged offspring in response to environmental cues such as overcrowding and predation risk that indicate poor habitat quality.2. Laboratory experiments were carried out to explore the intensity of the wing polyphenic response of pea aphids exposed to cues from ladybird predators and crowding, and their response was compared with pea aphids that were not exposed to any cues (control).3. The study used cues from two different ladybird species—Coccinella septempunctata L. (Coleoptera: Coccinellidae) and Hippo-damia convergens Guérin-Méneville (Coleoptera: Coccinellidae)—to investigate whether the wing polyphenic response of pea aphids to predator cues can be generalized.4. The intensity of the wing polyphenic response of pea aphids to crowding was found to be much stronger than their response to predator cues. There was no response to H. convergens cues and the response to C. septempunctata cues was mixed.Keywords: Crowding, dispersal, polyphenism, predation risk, wing induction263digitalcommons.unl.edu264 Pu r a n d a r e,  Te n h u m b e r g,  a n d br i s s o n,  Ec o l o g i c a l En t o m o l o g y  39  (2014) Materials and methodsInsect collection and rearingPredators. Adult C. septempunctata were collected from an alfalfa field in Lincoln, Nebraska, in July 2011, while adult H. convergens were purchased from commercial suppliers (Hirt’s Gardens, Wadsworth, Ohio) in May 2011. Both coc-cinellid species were reared in chiffon-netted aluminum cages (44×51×61 cm) in growth chambers at approximately 25°C and LD 16:8 h on Vicia faba L. plants infested with pea aphids.Aphids. Three pea aphid clones were used to increase the generalization of our results because different aphid clones vary in their sensitivity to environmental cues and in their wing polyphenic response (Sutherland, 1969a; Weisser & Braendle, 2001). To prevent mixing of clones, all aphids were caged by enclosing the host plant V. faba using Plexi-glass tubes (21.5 cm high and 6.5 cm diameter) with a mesh on top. The aphids were maintained in the laboratory at 17 ± 1.5°C, RH 35–45%, and LD 16:8 h.Experimental procedureThe experiments were carried out in growth chambers at 17 ± 1.5°C, RH 35–45%, and LD 16:8 h. Pea aphids were maintained on V. faba plants at low density (six individuals per plant) until they started reproducing. Aphids reared on the same plant were divided randomly between the treat-ments to avoid differences in the maternal environment that could influence their offspring phenotype. Pea aphids are viviparous, and therefore adult asexual females have embryos in their ovaries. Since the winged/unwinged morph determination in pea aphids is prenatal (Suther-land, 1969a,b), all experimental treatments were applied to adult asexual females within the first 3 days of the begin-ning of their reproductive period.The experimental arena consisted of a plastic Petri plate (60mm×15 mm, Fisher) with two Medicago arborea leaves in-serted in 3ml of 2% bactoagar mixed with Miracle-Gro (The Scotts Miracle-Gro Company, Marysville, Ohio) (plate). Leaves, rather than plants, were used in order to restrict the predator cues to a small area, which increased the prob-ability that aphids encountered the cues during the experi-ment. Adult unwinged asexual female pea aphids were di-vided randomly between the following treatments:• Predator cues. One well-fed adult of either H. convergens or C. septempunctata was released in the experimental arena. The predators were allowed to search and de-posit cues (eggs, feces, and tracks) at 22°C and LD 16:8 h under fluorescent light for 24 h. As the response of aphids to ladybird tracks can depend on the predator sex (Ninkovic et al., 2013), we included both sexes in our experiments and randomly distributed them be-tween the treatments. After removal of the predator, one adult, unwinged aphid female was placed in each plate.• Crowding. Ten adult unwinged aphid females were placed in a small empty plastic Petri dish (32.5mm×15 mm) for 24 h, and then each individual was transferred to its own plate. In our statistical analysis, we included only one randomly chosen aphid per Petri dish.• Control. A single adult, unwinged aphid female was placed in a plate; predator cues were absent.The response of aphids to predator tracks is known to decrease with the age of the tracks (Ninkovic et al., 2013). Therefore, female aphids were removed from all plates after 24 h, the offspring produced during that du-ration were reared until they reached adulthood (14 ± 2 days), and the phenotype of the offspring after reach-ing maturity was recorded. We recorded the proportion of pea aphid females producing at least one winged off-spring (“induced aphids”) and the proportion of winged offspring produced by induced pea aphid females. The experiments were replicated for each of the three aphid clones (Hf-alf-07, seven replicates; Roc-1, six replicates; HF-74, four replicates).All analyses were performed using r (v. 2.15.0; R De-velopment Core Team, 2012). Backward model selection was performed and likelihood ratio tests were used to de-cide which model fitted the data best. A generalized lin-ear mixed model (GLMM) with a binomial error distribu-tion was used to analyze the proportion of induced aphids, treating clone and treatment as fixed effects and starting date as a random effect. The same statistical model was used to analyze the proportion of winged offspring pro-duced by each induced aphid.ResultsAphid clone had no significant effect on the proportion of aphids induced (Hf-alf-07, P = 0.17; Roc-1, P = 0.33; ta-ble S1 in supporting information) and the proportion of winged offspring produced by the induced aphids (Hf-alf-07, P = 0.30; Roc-1, P = 0.92; table S2 in supporting information). Crowding produced a strong wing poly-phenic response. In comparison to the control treatment (no cues), the proportion of induced aphids (P = 0.001, fig. 1) and the proportion of winged offspring produced by induced aphids (P = 0.0001, fig. 2) were significantly higher than in the control. Exposure to C. septempunc-tata cues elicited a weak but significant response. Com-pared with the control, the proportion of induced aphids increased significantly (P = 0.003), but the proportion of winged offspring produced by induced aphids decreased (P = 0.03). By contrast, exposure to H. convergens cues had no effect on the proportion of induced aphids (P = 0.14) and winged offspring (P = 0.87).DiscussionWe found a strong wing polyphenic response of pea aphids to crowding (roughly twice as high as the control; fig. 1), Co m P a r i s o n o f T h e w i n g P o l y P h e n i C r e s P o n s e o f P e a a P h i d s   265                                                   which is consistent with previous studies (Sutherland, 1969a,b). We found no response of pea aphids to H. con-vergens cues and a mixed response to C. septempunc-tata cues. Even though the proportion of induced aphids (Pinduced) was significantly higher in aphids exposed to C. septempunctata cues, the proportion of winged offspring (Poffspring) was lower than the control aphids, suggest-ing that the total dispersal response to predator cues is negligible [PinducedPoffspring = 12% (Control), 13% (H. conver-gens), and 14% (C. septempunctata)]. We suggest two poten-tial reasons for the weak response to predator cues. First, the wing polyphenic response is transgenerational in pea aphids, and the delay between the time the females are in-duced and the offspring dispersal response is substantial. Thus, the predatory ladybird that induces the production of winged offspring in an aphid colony is unlikely to be present when the winged offspring mature (Minoretti & Weisser, 2000). The evolution of delayed predator-induced dispersal is only adaptive under special circumstances. Ac-cording to a model by Poethke et al. (2010), a substantial proportion (more than 80%) of the population should dis-perse only if the predation risk is high (≥ 0.8), predators revisit patches (return probability ≥ 0.8), and predator-in-duced mortality increases after the first visit of a preda-tor. These conditions may not apply to ladybirds foraging for aphids. Secondly, it is possible that our predator treat-ment did not provide a sufficient number of cues to in-duce a strong wing polyphenic response. In our predator treatment, we used isolated aphids to separate the effect of predator cues on wing induction from cues (such as tactile cues) associated with crowding (Sutherland, 1969a). Only a weak response by isolated pea aphids to predator tracks has been observed in a different aphid-coccinellid system (pea aphids–Adalia bipunctata; Dixon & Agarwala, 1999).Acknowledgments – We would like to thank two anon-ymous reviewers for their helpful comments, Emily Zahn for her help with aphid rearing and conducting the experi-ments, and Travis Hinkelman for the useful discussions. This work was supported by the University of Nebraska–Lincoln School of Biological Sciences Startup Funds and NIEHS award 4R00ES017367 (JAB) and University of Nebraska–Lincoln School of Biological Sciences Special Funds (SRP).ReferencesCompton, S. G. (2002) Sailing with the wind: Dispersal by small flying insects. Dispersal Ecology (ed. by J. M. Bullock, R. E. Kenward, and R. S. Hails), pp. 113–133. Blackwell Sci-ence Ltd., Oxford, UK.Dieckmann, U., O’Hara, B., & Weisser, W. W. (1999) The evo-lutionary ecology of dispersal. Trends in Ecology & Evolu-tion, 14, 88–90.Dixon, A. F. G. (1998) Resource tracking in time. Aphid Ecology: An Optimization Approach (ed. by A. F. G. Dixon), pp. 128–170. Chapman & Hall, London, UK.Dixon, A. F. G., & Agarwala, B. K. (1999) Ladybird-induced life history changes in aphids. Proceedings of the Royal So-ciety of London Series B: Biological Sciences, 266, 1549–1553.Hatano, E., Baverstock, J., Kunert, G., Pell, J. K. & Weisser, W. W. (2012) Entomopathogenic fungi stimulate transgenera-tional wing induction in pea aphids, Acyrthosiphon pisum (Hemiptera: Aphididae). Ecological Entomology, 37, 75–82.Kunert, G., & Weisser, W. W. (2003) The interplay be-tween density and trait-mediate effects in predator-prey Figure 2. Proportion of winged offspring produced by in-duced pea aphids for each treatment. The bars indicate the means, and the lines show the 95% confidence interval. Con-trol, no cues; H. conv., exposure to Hippodamia convergens cues; C. sept, exposure to Coccinella septempunctata cues; crowding, exposure to crowding. All comparisons are made with respect to the control.Figure 1. Proportion of induced pea aphids for each treat-ment. The bars indicate the mean values, and the lines show the 95% confidence interval. Control, no cues; H. conv., expo-sure to Hippodamia convergens cues; C. sept, exposure to Cocci-nella septempunctata cues; crowding, exposure to crowding. All comparisons are made with respect to the control.266 Pu r a n d a r e,  Te n h u m b e r g,  a n d br i s s o n,  Ec o l o g i c a l En t o m o l o g y  39  (2014) interactions a case study in aphid wing polymorphism. Oecologia, 135, 304–312.Minoretti, N., & Weisser, W. W. (2000) The impact of individ-ual ladybirds (Coccinella septempunctata, Coleoptera: Cocci-nellidae) on individual aphid colonies. European Journal of Entomology, 97, 475–479.Mondor, E. B., Rosenheim, J. A. & Addicott, J. F. (2005) Preda-tor induced transgenerational phenotypic plasticity in the cotton aphid. Oecologia, 142, 104–108.Nijhout, H. F. (1999) Control mechanisms of polyphenic devel-opment. Bioscience, 49, 181–192.Ninkovic, V., Feng, Y., Olsson, U., & Pettersson, J. (2013) Lady-bird footprints induce aphid avoidance behavior. Biological Control, 65, 63–71.Poethke, H. J., Weisser, W. W. & Hovestadt, T. (2010) Preda-tor induced dispersal and the evolution of conditional dis-persal in correlated environments. American Naturalist, 175, 577–586.R Development Core Team. (2012) R: A Language and Environ-ment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria http://www.R-project.org [accessed on 26 December 2012].Sloggett, J. J. & Weisser, W. W. (2002) Parasitoids induce pro-duction of the dispersal morph of the pea aphid, Acyrthosi-phon pisum. Oikos, 98, 323–333.Sutherland, O. R. W. (1969a) The role of crowding in the production of winged forms by two strains of the pea aphid, Acyrthosiphon pisum. Journal of Insect Physiology, 15, 1385–1410.Sutherland, O.R.W. (1969b) The role of the host plant in the production of winged forms by two strains of the pea aphid Acyrthosiphon pisum. Journal of Insect Physiology, 15, 2179–2201.Ward, S. A., Leather, S. R., Pickup, J., & Harrington, R. (1998) Mortality during dispersal and the cost of host-specificity in parasites: How many aphids find hosts? Journal of Ani-mal Ecology, 67, 763–773.Weisser, W. W. & Braendle, C. (2001) Body colour and genetic variation in winged morph production in the pea aphid. Entomologia Experimentalis et Applicata, 99, 217–223.Weisser, W. W., Braendle, C., & Minoretti, N. (1999) Predator-induced morphological shift in the pea aphid. Proceedings of the Royal Society B: Biological Sciences, 266, 1175–1181.Supplementary Table S1: Proportion of pea aphids induced.  The predicted proportion of induced aphids for each of the treatment is depicted in Figure 1.  SE = standard error.  Fixed Effects Estimate SE P Value Control and Clone HF-74 (Intercept) –0.72 0.38 0.054 Predator Treatment-C7 0.66 0.21 0.0017** Predator Treatment-HC 0.31 0.21 0.14 Crowding Treatment-CR 1.81 0.56 0.001** Clone HF-alf-07 –0.61 0.45 0.17 Clone Roc-1 0.4 0.44 0.33         Supplementary Table S2: Proportion of winged offspring produced by induced pea 1 aphids. The predicted proportion of induced aphids for each of the treatment is 2 depicted in Figure 2. SE = standard error. 3  4 Fixed Effects Estimate SE P Value Control and Clone HF-74 (Intercept) –0.55 0.31 0.0795 Predator Treatment-C7 –0.26 0.12 0.03 * Predator Treatment-HC –0.02 0.13 0.87 Crowding Treatment-CR 0.78 0.19 4.97e-05 *** Clone HF-alf-07 0.39 0.38 0.30 Clone Roc-1 –0.04 0.37 0.92  5  6 

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Comparison of the wing polyphenic response of pea aphids (\u3ci\u3eAcyrthosiphon pisum\u3c/i\u3e) to crowding and predator cues

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