Functional Ecology
Appendix S1: Additional methodological details
Details of the inoculation protocol
Stock tissue was generated via mechanical inoculation of a single inoculum source into several
C. pepo host plants, from which pooled tissue was harvested and frozen in aliquots at -80°C.
This stock was used to generate plants for the trapping and observation field experiments and the
squash bug growth and choice experiments. A second batch of stock tissue was created in the
same way and used to inoculate plants for the nutrient analysis and visual-cue measurements. For
all inoculations frozen tissue was ground on a cold surface then combined with 15mL of chilled
0.1M potassium phosphate buffer and a small amount of carborundum powder. The solution was
then applied to cotyledon surfaces using cotton swabs. Plants designated for virus-free treatments
were inoculated in the same manner, but with a solution containing tissue from a healthy squash
plant.
Aphid and squash bug rearing
Aphid colonies (Aphis gossypii Glover, collected in State College, PA) were reared on 2-3-weekold Cucurbita pepo (cv. Dixie) plants in cages exposed to ambient seasonal photoperiod
(daylight) at ~24⁰C. Squash bugs (Anasa tristis) were collected as eggs laid on field-grown C.
pepo in and around State College, PA, during the late summer of 2008 (~200 eggs) and again in
2011 (~200 eggs). The resulting A. tristis colony was maintained on C. pepo under artificial
lighting (16:8 photoperiod) at ~24⁰C.
Insect functional groups delineated in the trapping experiment
Herbivores: piercing sucking (Aphididae), piercing sucking (Cicadellidae/Cercopidae), piercing
sucking (Miridae and other macerate-and-flush feeding style hemiptera), rasping sucking
(Thripidae), and chewing (beetles, lepidopterans - grouped together as “other herbivores”).
Predators: Syrphidae, Coccinellidae, predatory Diptera (Cecidomyiidae, genus Aphidoletes and
Chamaemyiidae, genus Leucopis, mostly larval forms on whole plants), parasitoids, spiders, and
other assorted generalist predators that occurred in low numbers (Sphecidae, Vespidae,
Carabidae, Staphylinidae, Chrysopidae, and Formicidae). Syrphids, Coccinellids, and predatory
Dipterans are key predators of aphids in many systems.
Other insects trapped were recorded but not classified as predators or herbivores (e.g.
Hymenopteran and Coleopteran pollinators, Dipterans that could not be identified in sufficient
detail to determine feeding guild, Collembolans, Acarines, and eggs that could not be attributed
to a particular guild).
Insect functional groups delineated in the observation experiment
Herbivore guilds were delineated as piercing-sucking (Cicadellidae/Cercopidae), macerate-andflush (Miridae), and other (includes Thripidae, chewing Coleopterans, clutches of Pentatomidae
Virus infection influences host plant interactions with non-vector herbivores and predators
Kerry E. Mauck, Erica Smyers, Consuelo M. De Moraes, Mark C. Mescher
Functional Ecology
eggs [each clutch = one count], and aphids—each of which occurred in low numbers). Predator
groups were similarly delineated and included the following: Chrysopidae, Syrphidae,
Coccinellidae, parasitoids, spiders, and other predators (Reduviidae, Nabidae, and Formicidae—
each of which occurred in very low numbers). Other insects observed included Hymenopteran
and Coleopteran pollinators and Dipterans for which a feeding guild could not be assigned.
Camera settings for leaf area and RGB measurements
Camera make and model
Exposure time
FNumber
ISOSpeedRatings
Focal length
Quality
White Balance
Canon EOS 5D Mark III
1/60 seconds
13
800
73mm
RAW
Sunny
Gas Chromatography-Mass Spectrometry protocol
Chromatography employed an Agilent 6890 gas chromatograph fitted with a DB-5MS column
(60m long, 0.25mm internal diameter, 0.25μm film) and an Agilent 5973 Network Mass
Selective Detector. Injection volume was 1μL with an inlet temperature of 230 °C in splitless
mode and a constant helium carrier gas flow set to 1 mL/ min. The temperature program began
with an isothermal run at 70°C for 5 minutes, followed by a ramp of 5°C/min. up to 325°C and a
final heating at 315°C for 5 minutes. The instrument was then cooled to 70°C as fast as possible
(~4 minutes), and equilibrated at this temperature for one minute prior to analysis of the next
sample. The transfer line to the mass spectrometer was set to 250°C and the mass spectrometer
source was operated at 200°C in electron ionization mode (tuned to the manufacturer’s
specifications). Scanning was set to 2 scans per second with a range of m/z 50-600. The solvent
delay was 13 minutes. Syringe washes were performed using acetone and hexanes. Chemstation
(2003) software was used to analyze the output. Areas of all peaks were calculated using the
software’s integration program. Identification of compounds was accomplished using the NIST
2008 mass spectral library (National Institute of Standards and Technology) and verified by
comparison to known standards in an amino acid standards mixture purchased from
Phenomenex, and to standards of glucose, fructose, and sucrose purchased from Sigma Aldrich.
Additional growth experiment with Anasa tristis
We performed an additional round of the growth experiment described in the methods of the
main manuscript. 37 newly hatched nymphs were distributed across two, 3-week old infected
plants and 32 nymphs across two, 3-week old healthy plants (one plant and 13-19 bugs to a cage
– starting numbers were initially equal but 5 nymphs died due to handling in the healthy plant
treatment). Bugs were allowed to feed on the plants for 3 weeks then counted, weighed, and
assessed for instar progression. Proportion surviving and proportions progressed into each instar
were compared for bugs grown on infected vs. healthy plants. Weights within each instar were
Virus infection influences host plant interactions with non-vector herbivores and predators
Kerry E. Mauck, Erica Smyers, Consuelo M. De Moraes, Mark C. Mescher
Functional Ecology
compared using the Mann-Whitney test (only the 3rd, 4th, and 5th instars had sufficient numbers
for statistical tests).
Virus infection influences host plant interactions with non-vector herbivores and predators
Kerry E. Mauck, Erica Smyers, Consuelo M. De Moraes, Mark C. Mescher