Electronic Supplementary Material
Trophallaxis and prophylaxis: social immunity in the carpenter ant
Camponotus pennsylvanicus.
Casey Hamilton1,2*, Brian T. Lejeune1 and Rebeca B. Rosengaus1
1
Department of Biology, Northeastern University, Boston Massachusetts USA 02115
2
Current address: Department of Biological Sciences, Towson University, Towson
Maryland USA 21252
*Author for correspondence (chamil6@students.towson.edu)
Notes
Note 1. Droplets were initially “stolen” during trophallaxis with a pulled capillary tube by
placing treated ants with nestmates starved for 24 hours. However, in the interest of time
and sufficient sample size, we used “forced” regurgitation by applying slight pressure to
the gaster, forcing the contents of the crop out of the ant’s mouth. SDS-PAGE of stolen
and forced droplets from naïve ants was performed to determine if “forced” samples were
representative of natural trophallactic droplets. No differences in protein profiles were
detected between stolen and forced droplets (ESM Figure 1). Samples for antimicrobial
activity assays were taken 48 hours post treatment, which was the observed peak
antimicrobial response in Camponotus fellah and Tenebrio molitor under similar immune
challenges (de Souza et al. 2008; Haine et al. 2008). Behavioural observations were
conducted at 24 hours post-treatment, however, because 48 hours of isolation caused
increases in both naïve and immune challenged ants in C. fellah (de Souza et al. 2008).
Figures
Figure 1. SDS-PAGE of stolen and forced droplet samples from naïve ants. Lane 1 shows
a molecular weight standard (ProSieve® Protein Markers, Cambrex Bio Science
Rockland, Inc.); lanes 2-4 show “stolen” droplets of naïve ants; lanes 5-7 show “forced”
droplets of naïve ants. The similarity of the protein profiles of samples obtained from
both methods indicates that “forced” regurgitation did not result in the discharge of
liquids from additional sources besides the crop.
Figure 2. Median frequency of trophallaxis between pairs of workers of C.
pennsylvanicus after 24 hours of isolation for four colonies (a, b, c, and d). Three
replicates for each pairing were observed for each colony. Pairs consisted of naïve
workers (N), workers injected with Ringer solution (R), workers injected with heat-killed
S. marcescens (I), and workers injected with lipopolysaccharides (L).
Figure 3. Antimicrobial activity (median diameter of zone of inhibition) of droplets from
naïve workers (Naive), workers injected with Ringer solution (Ringer), workers injected
with heat-killed S. marcescens (Immunized), and workers injected with
lipopolysaccharides (LPS), for colonies a, b, c, and d. Numbers indicate sample size.
Figure 4. SDS-PAGE of forced droplet samples from two Naïve (2,3), two Ringerinjected (4,5), two immunized (6,7), and two lipopolysaccharide-injected (8,9)
individuals. Lane 1 shows a molecular weight standard (ProSieve® Protein Markers,
Cambrex Bio Science Rockland, Inc.). The arrow indicates the enhanced cathepsin D
protein bands (~35 kD) in immunized and lipopolysaccharide-injected ants.
Figure 5. Cathepsin D median percent of total protein in regurgitate samples based on
band densiometry analysis in ImageJ of four SDS-PAGE gels (2 samples from each
treatment per gel). Significant differences among treatments were observed (KruskalWallis: 2=8.276, df=3, p=0.041).
Figure 6. Survival curves from a Cox proportional regression. Ant survival across seven
colonies was tracked following a challenge with active S. marcescens. Curves represent
survival of Ringer-injected donor ants (), ants that received droplets from Ringerinjected donors (), immunized donor ants (), and ants that received droplets from
immunized donors (). After controlling for the effect of colony of origin, ants that
received droplets from immunized donors exhibited the highest survival. Relative to this
treatment, the hazard ratios of death were 2.1 (for the control donor treatment, Wald=9,
df=1, p=0.003), 1.8 (for the control recipient treatment, Wald=4.7, df=1, p=0.03) and 1.6
times (for the immunized donor treatment, Wald=3.3, df=1, p=0.08) higher.
References
de Souza, D. J., Vlaenderen, J. V., Moret, Y. & Lenoir, A. 2008 Immune response affects
ant trophallactic behavior. Journal of Insect Physiology 54, 828-832. (doi:
10.1016/j.jinsphys.2008.03.001)
Haine, E. R., Pollitt, L. C., Moret, Y., Siva-Jothy, M. T. & Rolff, J. 2008 Temporal
patterns in immune responses to a range of microbial insults (Tenebrio molitor).
Journal of Insect Physiology 54, 1090-1097. (doi:10.1016/j.jinsphys.2008.04.013)