Ants defend aphids against lethal disease
SUPPLEMENTARY MATERIAL
Charlotte Nielsen, Anurag A. Agrawal & Ann E. Hajek
Field sites
Experiments were carried out at 3 field sites near Ithaca, New York, USA (N 42.44, W
76.50): in August 2005 on Niemi Road and at the Plant-Insect Interactions Field Lab on
Freese Road and in July 2007 at Brooktondale Meadow. At the Plant-Insect Interactions
Field Lab site, two naturally established milkweed plants (Asclepias syriaca) with Aphis
asclepiadis colonies (11-66 individuals per colony) that were actively tended by 1-10
Formica podzolica workers were selected. The plants were growing approximately 5 m
from the nearest F. podzolica nest. At the Niemi Road field site, 9 transplanted and 2
naturally established milkweed plants with A. asclepiadis colonies (8-151 individuals)
that were actively tended by 1-10 F. podzolica workers were selected. The 9 transplanted
plants were 1.5 m from the nearest F. podzolica nest and the remaining 2 plants were
growing approximately 5 m from the nearest nest. At the 2007 site, 26 naturally
established milkweed plants with A. asclepiadis colonies (13 - >400 individuals per
colony) actively tended by 1-11 F. podzolica workers, were selected. The plants were
growing < 5 m from the nearest F. podzolica nest.
Aphis asclepiadis
Aphids used as test insects originated either from naturally established colonies in the
field or laboratory colonies of A. asclepiadis originally collected from the field near
Ithaca. Aphids were maintained in the laboratory on potted milkweed plants or, for short
periods of time, on cut milkweed leaves. All aphids were kept at room temperature until
use for production of experimental aphids. Experimental aphids were produced every
day.
Production of experimental aphids
Sporulating cadavers: Aphids became infected after exposure to conidia from a
sporulating in vitro culture of P. neoaphidis (ARSEF 7214; USDA Agricultural Research
Service Collection of Entomopathogenic Fungal Cultures, Ithaca, NY) isolated from
Aphis glycines collected in NY, USA in 2003. The actively ejected conidia were obtained
from a fungal mat as described by Papierok & Hajek (1997). Healthy apterous adults and
4th instar nymphs were transferred to a milkweed leaf placed in 3% water agar in a 30-ml
plastic cup, and a sporulating fungal mat was placed over the aphids to shower conidia
onto them. After approximately 1 hr of incubation in light at 20oC, the sporulating mat
was removed and the cup was sealed. Inoculated aphids were incubated at 20oC, with a
16 hr photoperiod and cups were checked on a daily basis. Sporulating cadavers were
used in assays within eight hours after host death.
Uninfected aphid cadavers. Uninfected aphid cadavers were produced by freezing
healthy apterous adults and 4th instar nymphs at -20 oC for 10 min. Resulting cadavers
were always used in assays within eight hours.
Living aphids contaminated with conidia. Healthy apterous adults and 4th instar nymphs
were transferred to a milkweed leaf placed in 3% water agar in a 30-ml plastic cup, after
which a sporulating fungal mat was placed over the aphids. After 1-3 hr of incubation in
light at 20oC, the sporulating mat was removed and the cup was sealed. The conidialshowered aphids were always used within five hours after conidial showering.
For all treatments experimental aphids were placed in 29 ml plastic cups after production.
Cups contained 3% water agar to avoid dehydration and for living experimental aphids
cut milkweed leaves were provided as food. Cups containing experimental aphids were
transported to the field in a cooler.
Field methods
Experimental aphids were carefully introduced into colonies of the milkweed aphid using
a fine paintbrush. The first set of treatments (2005) had three types of experimental
aphids (sporulating cadaver, cadaver of an aphid killed by freezing, living aphid) and the
second set of treatments (2007) had two types (living conidia-contaminated aphid, living
uncontaminated aphid). For each replicate, a set of treatments was introduced to the same
colony on the same day. For each set of treatments, one type of experimental aphid was
introduced to a colony at a time, with at least 30 min before a different type of
experimental aphid was introduced. Timing was initiated as soon as an ant entered the
aphid colony. The experimental aphid was carefully observed continuously by one person
for five min while ant behaviour was recorded.
Removal of aphids or aphid cadavers by ants was recorded as follows: (i) the
experimental aphid was carried down the plant stem to the ground, (ii) the experimental
aphid was flung from the side of a leaf, or (iii) the experimental aphid was deposited
more than 1 cm away from the colony on the same food plant. Each experiment was
conducted for five minutes or until the experimental aphid was removed.
For studies with conidia-contaminated aphids, ant behaviour after each tending event was
categorized as (i) the ant removed the experimental aphid, (ii) the ant picked up and
groomed the experimental aphid, (iii) the ant groomed itself, or (iv) the ant did not take
any action and moved on to tending other aphids in the colony.
Data analysis
Removal of experimental aphids was analyzed using a Cox proportional-hazard
regression model, with treatment, aphid colony size and number of tending ants in the
aphid colony used as dependent variables. The removal distributions were analyzed using
Kaplan-Meier survival tests with the Breslow statistic (Allison, 1995). Data from studies
exploring responses to sporulating cadavers versus conidia-contaminated living aphids
were analyzed separately.
Multiple regressions were used to analyze the effect of types of experimental aphid on the
number of touches by ants, the number of times that ants picked up aphids, the number of
tending events and tending time, with treatment and number of tending ants in the aphid
colony used as dependent variables (Proc GLM; SAS Institute, 1999). The effect of types
of experimental aphids introduced to the colony on the proportion of experimental aphids
that ended on the ground was analyzed by logistic regression with treatment, aphid
colony size and number of tending ants in the aphid colony as dependent variables (Proc
GENMOD; SAS Institute, 1999). Logistic regression was used to analyze ant responses
after tending (N=210), with a multinomial distribution and cumulative logit as a link
function (four categories: self-grooming, aphid-grooming, removing experimental aphid
and no reaction) with treatment as a dependent variable (Proc GENMOD; SAS Institute
1999).
Allison, P. D. 1995. Survival Analysis using SAS. A practical Guide. SAS Institute Cary,
NC: SAS Publ.
Papierok, B. & Hajek, A. E. 1997 Entomophthorales. In Manual of Techniques in Insect
Pathology (ed. L. A. Lacey), pp. 187-212. London: Academic Press.
SAS Institute. 1999 SAS/STAT User’s Guide, Version 8. Cary, NC: SAS Publ.