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Social Complexity and Nesting Habits Are Factors in the
Evolution of Antimicrobial Defences in Wasps
Stephen J. Hoggard*, Peter D. Wilson, Andrew J. Beattie, Adam J. Stow
Department of Biological Sciences, Macquarie University, North Ryde, Australia
Abstract
Microbial diseases are important selective agents in social insects and one major defense mechanism is the secretion of
cuticular antimicrobial compounds. We hypothesized that given differences in group size, social complexity, and nest type
the secretions of these antimicrobials will be under different selective pressures. To test this we extracted secretions from
nine wasp species of varying social complexity and nesting habits and assayed their antimicrobial compounds against
cultures of Staphylococcus aureus. These data were then combined with phylogenetic data to provide an evolutionary
context. Social species showed significantly higher (18x) antimicrobial activity than solitary species and species with paper
nests showed significantly higher (11x) antimicrobial activity than those which excavated burrows. Mud-nest species
showed no antimicrobial activity. Solitary, burrow-provisioning wasps diverged at more basal nodes of the phylogenetic
trees, while social wasps diverged from the most recent nodes. These data suggest that antimicrobial defences may have
evolved in response to ground-dwelling pathogens but the most important variable leading to increased antimicrobial
strength was increase in group size and social complexity.
Citation: Hoggard SJ, Wilson PD, Beattie AJ, Stow AJ (2011) Social Complexity and Nesting Habits Are Factors in the Evolution of Antimicrobial Defences in
Wasps. PLoS ONE 6(7): e21763. doi:10.1371/journal.pone.0021763
Editor: Art F. Y. Poon, British Columbia Centre for Excellence in HIV/AIDS, Canada
Received April 3, 2011; Accepted June 6, 2011; Published July 6, 2011
Copyright: ß2011 Hoggard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work was funded by Australian Research Council (ARC) Discovery Grant (#DP0879229). The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: stephen.hoggard@mq.edu.au
Introduction
Disease risk in social insects can be influenced by a variety of
intrinsic and extrinsic factors. Large group sizes and limited
genetic diversity commonly associated with social species are
known to be factors [1] and different nest substrates and
climates present arrays of pathogens which may compromise a
colony. Despite this, social Hymenoptera have successfully
established on every continent (except Antarctica) suggesting
that they have found one or more strategies to successfully
combat disease. Indeed, multiple disease resistance strategies
have been observed in various hymenopteran species including
behavioural (e.g. allogrooming, nest cleaning), genetic (e.g.
increased diversity) or biochemical (e.g. immune response,
antibiotic secretions) [2,3].
Cuticular antimicrobial compounds [1,4,5] are important
because they target pathogens before they can infect an
individual. Previous studies have shown that antimicrobial
activity scales positively with group size in both bees and thrips
[1,4] supporting the theory that larger group sizes lead to
greater risk of disease. However, in order to establish the
generality of these findings, more insect lineages should be
examined.
Within the Hymenoptera, wasps are of particular interest as
both bees and ants arose from wasp lineages (Apoidea and
Vespoidea superfamilies respectively [6]) and examples of all
major nesting habits and levels social complexity are found within
extant wasp taxa [7]. Additionally, antibacterial peptides have
been isolated from the cuticle and venom of social wasp species
[5]. Therefore, wasps provide the opportunity to examine the
factors affecting disease resistance from some of the most primitive
states up until the most recent and complex derivatives.
Given the diversity of wasp taxa we predicted that we would
observe a large amount of variation in the relative strength of
antimicrobial defences among species, based on life history traits.
For this study, we extracted putative antimicrobial compounds
from the cuticle of a variety of wasp species which span a range of
nesting habits and social complexity. Using an established bioassay
[8], the activity of these compounds was measured and compared.
Based on previous studies we expected those species with the
greatest group sizes (i.e. the social species) to possess the strongest
antimicrobial compounds.
According to published phylogenies [6,9] solitary wasp species
are ancestral to social wasp lineages. Individuals of solitary species
do not cooperate with one another and aggregations, if any, result
from the availability of limited nesting sites [10,11]. While not
subjected to the evolutionary pressures of disease arising from
sociality, as the ancestral lineage of social species, we predicted
that solitary wasps would possess weak cuticular antimicrobial
compounds. In contrast, for social species we predicted they would
possess strong cuticular antimicrobial compounds used to protect
themselves from disease risks associated with large, high-density
group sizes. To test this hypothesis, we performed a phylogenetic
reconstruction of the examined species as there are no compre-
hensive phylogenies for Australian vespid or apoid wasps. These
data provided our results with an evolutionary context, allowing
for inferences to be made regarding the order in which traits
evolved or co-evolved.
PLoS ONE | www.plosone.org 1 July 2011 | Volume 6 | Issue 7 | e21763
Materials and Methods
Ethics Statement
No animal ethics approval was required for this study however
wasps were incapacitated with carbon dioxide during sampling
and prior to extraction of cuticular compounds and internal tissue
to minimise stress to the animals.
Sampling locations and species identification
1268 individual wasps comprising members of nine different
species were collected from public land across Sydney (New South
Wales) and Alice Springs (Northern Territory), Australia. Species
were identified by morphology and sequencing of 28S nrDNA and
COI mtDNA fragments (see below). In four wasp species,
identification could only be resolved to genus-level due to lack of
identification keys for many Australian wasp genera. In one
instance, identification could only be resolved to the level of sub-
family (designated as ‘Pepsinae Sp1’). Species were then
categorised by social complexity (social, communal aggregator or
solitary) and by nest type (paper nest, mud nest or burrow;
Table 1), representing broad categories of group size and
environmental exposure. Paper nests were defined as clusters of
cells constructed from pulp and attached to the substrate by a
petiole [7]. Mud nests were defined as sealed and provisioned cells
constructed from mud and above-ground [12,13]. Burrows were
defined as either provisioned terrestrial burrows [10,14], or as
those species which burrowed for prey which was subsequently
paralysed and ectoparasitised [15].
Bioassay
Putative antimicrobial compounds were assayed by established
methods to assay antimicrobial compounds obtained from thrips
and bees [1,4] and were removed from the cuticle of live wasps by
washing whole animals with 70% ethanol for 10 minutes,
followed by two rinses to maximise extraction. Solvents were
removed by vacuum evaporated at 25uC and the recovered
residue was resuspended in LB broth. Extracts were assayed
against Staphylococcus aureus using opposing gradients of extract
concentration and cell numbers across rows of 12 wells in 96-
well microtitre plates [8]. As S. aureus is exclusively found in birds
and mammals [16], it is unlikely that any species of wasp
has developed pathogen-specific defensive compounds that
would bias our study of broad-scale antimicrobial compounds.
Concentration-growth curves were generated for each species
with a minimum of five replicates per species, and a minimum of
three replicates per colony for social species, when sample size
permitted. Three control rows were used in each assay: LB broth,
resuspended extract with LB broth and a gradient of S. aureus cell
numbers with LB broth. Initially, the maximum concentration of
extract used was equivalent to a single wasp, however,
preliminary assays indicated this was too low to detect activity
in many species. Where additional samples were available the
maximum concentration of extract was increased to 2.0
(Cryptocheilus sp.,Sceliphron laetum and Austroscolia sp.)or4.0(Bembix
sp.). Where only a single individual was collected (Delta sp.,Abispa
ephippium and Pepsinae Sp1) the highest concentration of extract
was equivalent to 0.5 wasps. For social and communal aggregator
species washes from between three and eight individuals were
pooled and then diluted to the required wasp equivalent
concentrations. All assays used a one-half serial dilution for both
the S. aureus and extract gradients. Following incubation at 37uC
for 19 h, growth in treatment and control wells was measured as
an increase in optical density (OD) at 590 nm. These data were
expressed as [increase in OD of treatment well]/[increase in OD
of control well] and then used to determine the concentration of
extract required to kill or inhibit 50% of S. aureus growth (herein
referred to as IC50). A total of 50 assays were performed across
the nine species (Table 1).
Calculating Relative Antimicrobial Strength
Following Smith et al. (2008), a modified Gompertz function was
fitted to the data using R (version 2.5.1 [17]) to calculate the IC50
value for each assay. Mean IC50 (695% CI) was calculated for
each species and each category of social complexity and nest type.
Two-sample t-tests were performed between pairs of social
complexity and nest type categories. To standardise measurements
across different species, concentrations of wasp equivalents were
converted to concentrations of equivalent surface area. Adapting
methods previously applied to bees [1], mean surface area for each
species was estimated by generating elliptical cylinders using
measurements from up to ten individuals per species. Although
regular cylinders would have been appropriate for most species,
some of the larger species exhibited up to ,1.5x difference
between height and width, rendering regular cylinders an
imprecise measure.
Table 1. Characterisation of wasp species.
Species (Family)
n
Sociality Nest type IC50 (±95% CI)
n
r
Polistes humilis (Vespidae) 1077 (10) Soc. Paper 6.03 (62.26) 28
Ropalidia plebeiana (Vespidae) 49 (2) Soc. Paper 7.58 (65.91) 5
Bembix sp. (Crabronidae) 83 Com Burrow 31.97 (627.62) 6
Austroscolia sp. (Scoliidae) 47 Sol. Burrow 158.27 (6152.82) 5 (3)*
Cryptocheilus sp. (Pompilidae) 4 Sol. Burrow 14.47 1
Pepsinae Sp1 (Pompilidae) 1 Sol. Burrow 90.26 1
Abispa ephippium (Vespidae) 1 Sol. Mud No Inhibition 1
Sceliphron laetum (Sphecidae) 5 Sol. Mud No Inhibition 2
Delta sp. (Vespidae) 1 Sol. Mud No Inhibition 1
n: number of individuals (number of colonies for social species); Sociality: social (Soc.), communal aggregator (Com.), solitary (Sol.); IC50: mean equivalent surface area
(mm
2
) of wasp cuticle required to kill or inhibit 50% of S. aureus growth; n
r
: number of replicates per species.
*Only three replicates for Austroscolia sp. showed activity over the assayed concentration gradient and the IC50 value given was calculated using only these data.
doi:10.1371/journal.pone.0021763.t001
Evolution of Antimicrobial Defences in Wasps
PLoS ONE | www.plosone.org 2 July 2011 | Volume 6 | Issue 7 | e21763
DNA Extraction and Amplification
Using one member of each sampled species, DNA was extracted
from internal tissues in the thoracic region of wasps using
proteinase-K and ‘salting-out’ [18]. Phylogenetic reconstruction
was performed using two gene fragments; 28S nuclear rDNA and
COI mitochondrial DNA. Amplification of the 28S gene
fragments was performed using primers previously used in the
construction of microgastrid wasp phylogenies [19] and COI gene
fragments amplifications were performed using generic inverte-
brate primers for that region [20]. PCRs for both gene fragment
were carried out in 10 mL volumes containing 0.5U of GoTaq
Flexi DNA polymerase (Promega), 1 mM forward primer, 1 mM
reverse primer, 0.8 mM DNTPs, 1x GoTaq Buffer (Promega) and
2.0 mM MgCl
2
. PCR amplifications had an initial denaturation at
94uC for 3 min followed by six ‘touch down’ cycles of 94uC
denaturation for 30 s, annealing temperatures (60uC, 58uC, 56uC,
54uC, 52uC, 50uC) for 30 s and an extension step of 72uC for 45 s.
On the completion of the last touchdown cycle, another 35 cycles
were carried out at 50uC annealing temperature and a final
extension of 10 min at 72uC. Following PCR amplicons were
purified using ExoSap-IT (USB) according to the manufacturer
instructions and purified products were sequenced using their
corresponding forward primers with dye terminator reactions on a
3130x1 Genetic Analyser (Applied Biosystems).
Sequence Alignment
Phylogenetic reconstruction of nine wasp species (plus Apis
mellifera as an outgroup) was performed using a 941 bp sequence
generated by concatenating the two gene fragments: 28S nrDNA
(484 bp) and COI mtDNA (457 bp). Generated sequences
(Table 2) plus corresponding sequences from A. mellifera acquired
from GenBank (28S: AJ302936.1; COI: FJ582092.1) were aligned
using the ClustalW option with default parameters in MEGA v4.0
[21]. Length polymorphisms in sequence data were removed
following alignment and prior to concatenation of gene fragments.
Concatenation of the two sequences was deemed appropriate as a
partition homogeneity test performed in PAUP* v4.0b10 [22]
revealed no significant incongruencies between the two data sets
(p = 0.124). Phylogenies were created using both distance-based
(neighbour-joining) and Bayesian methods (maximum clade
credibility).
Distance-based phylogenetic analysis
Neighbour-joining phylogenetic reconstruction was performed
in MEGA v4.0 [21] using the maximum composite likelihood
model. Both transition and transversion substitutions were
included, assuming homogeneous patterns among lineages and
uniform rates among sites. Gaps were treated as complete
deletions. Bootstrap values were obtained using 10000 replicates.
Bayesian phylogenetic analysis
Bayesian phylogenetic analysis was performed using BEAST
v1.5.4 [23]. The input file for BEAST was generated using
BEAUti v1.5.4. Trees were generated using a single MCMC chain
of 20 million steps sampling every 5000 steps. Generalised time-
reversible plus gamma (GTR+G) was selected as the model for
nucleotide substitution using jModelTest v0.1.1 [24]. The
molecular clock rate was fixed to 1.0 and the Yule process was
selected as the tree prior. All other parameters were left in their
default state as generated by BEAUti. Integrity of generated data
was checked using Tracer v1.5 [25]. TreeAnnotator v1.5.4
(distributed with BEAST v.1.5.4) was used to generate a maximum
clade credibility tree (MCC) using a burn-in period of 400 trees
and a posterior probability limit of 0.5. The MCC tree was
visualised using FigTree v1.3.1 [26].
Results
Antimicrobial Activity
IC50 values could be calculated for 44 of the 50 assays
performed (Table 1). No antimicrobial activity was observed from
species belonging to the ‘mud nest’ category. Two of the five assays
performed using extract obtained from Austroscolia sp. also showed
no activity hence IC50 values were calculated using data from the
three assays for which IC50 values could be calculated. When
grouped by social complexity, mean IC50 values for social species
were significantly lower than those of solitary species (Two-sample
t-test: social = 6.26, solitary = 115.93; p = 0.038). When grouped
by nest type, mean IC50 values for paper nest species were
significantly lower than those of burrow species (Two-sample t-test:
paper nest = 6.26, burrow = 70.13; p = 0.015). As no species
belonging to the ‘mud nest’ category showed any antimicrobial
activity it was not possible to compare mean IC50 values with the
other nest types. Similarly these species were not included when
calculating differences in mean IC50 by social complexity.
Phylogenetic Reconstruction
Following the removal of length polymorphisms and concate-
nation of 28S and COI gene fragments, of the 941 bp sequence,
651 bp were found to be variable of which 346 bp were parsimony
informative. Both the neighbour-joining and maximum clade
credibility trees were highly congruent, except when placing the
clades containing the two pompilid species and Austroscolia sp.
which were switched between the two trees. In both trees the
placement of Austroscolia sp. was the least supported branch
(bootstrap support of 0.52; credibility support of 0.7853).
Additionally, our phylogenetic reconstruction showed that solitary
burrowing wasps diverged at basal nodes in both trees and that
social lineages arose following a single divergence event (bootstrap
support of 0.82; credibility support of 1.0; Figure 1).
Discussion
It is clear from the data that levels of antimicrobial defences vary
among different wasp taxa and that these differences are strongly
linked to levels of social complexity. When examined together, the
Table 2. GenBank accession numbers by species.
Species name 28S COI
Polistes humilis JF510015 JF510006
Ropalidia plebeiana JF510016 JF510007
Bembix sp. JF510020 JF510011
Austroscolia sp. JF510021 JF510012
Cryptocheilus sp. JF510022 JF510013
Pepsinae Sp1 JF510023 JF510014
Abispa ephippium JF510017 JF510008
Sceliphron laetum JF510019 JF510010
Delta sp. JF510018 JF510009
28S: GenBank accession number for the amplified 28S nrDNA fragment
sequence; COI: GenBank accession number for the amplified COI mtDNA
fragment sequence.
doi:10.1371/journal.pone.0021763.t002
Evolution of Antimicrobial Defences in Wasps
PLoS ONE | www.plosone.org 3 July 2011 | Volume 6 | Issue 7 | e21763
phylogenetic and antimicrobial data suggest that the production of
antimicrobial compounds may have first arisen in solitary wasps as a
response to environmental, probably soil-borne, pathogens.
Social complexity
We observed an eighteen-fold difference in the mean strength of
cuticular compounds when comparing social and solitary wasp
species. This difference did not take into account those species for
which a mean value could not be calculated, thus the actual
difference between the two groups could be much higher. This
finding is consistent with previous studies linking increased
antimicrobial defences with group size and sociality [1,4]. As
wasps are an ancestral lineage in Hymenoptera [6,9] and this
pattern has already been demonstrated among bees [1] the
relationship may hold throughout the social Hymenoptera.
Nest type
Burrowing wasps, exposed to soil-based pathogens, may have
developed broad-scale antimicrobial defences in response. These
may have evolved into stronger compounds in the social lineages.
Perhaps the lack of antimicrobial activity in solitary species that
construct mud nests is because they are constructed above-ground
where there is less risk of disease. Alternatively, individual species
may have developed specific compounds to combat niche
pathogens which are ineffective against S. aureus. We acknowledge
that there may be confounding of results when examining
comparisons of sociality and nest types as both ‘social’ species
were also ‘paper nest’ species. With a sufficiently large sample size
it may be possible to separate the effects of each of these traits,
however time required to perform a single assay prohibits this.
Phylogenetic considerations
Traditionally, wasp lineages have been placed in one of three
distinct superfamilies (Vespoidea, Apoidea and Chrysidoidea) [6]
however evidence from recent molecular-based phylogenies [9]
has cast doubt on traditional taxonomies [9] which may explain
some of the incongruencies between the phylogenetic trees
presented in this paper and pre-existing, morphology-based
phylogenies [6]. Our placement of species belonging to Vespidae
is supported by both traditional taxonomic and more recent
molecular phylogenies [6,27], however our placement of Sceliphron
and Bembix (which belong to the Apoidea superfamily) within
Vespoidea is more congruent with molecular phylogenies
published by Pilgrim et al. [9]. This is unsurprising as we did not
use morphological data in our phylogenetic reconstruction,
however to ensure that this result was not due to outgroup choice,
we replicated our phylogenetic analysis replacing Apis mellifera
(Apoidea) with Chrysis cembricola (Chrysididae; 28S: GQ374718.1;
COI: GQ374633.1) as an outgroup (Chrysidoidea is a sister taxa
to both Apoidea and Vespoidea). This substitution did not change
the placement of Apoidea species or branch support for their
nodes. We similarly removed Apis mellifera without replacement
and again, this did not alter the placement of Apoidea species
within the trees relative to the other taxa. The only difference
observed between any of these trees (excepting minor changes to
branch support values) was the placement of Austroscolia and
Pompilidae taxa, which as previously stated, are the least
supported branches in the analysis.
This study provides evidence for the origin of antimicrobial
defences in wasps and Hymenoptera as a whole, and increases our
understanding of trends in disease resistance strategies in all social
insects. Assaying against pathogens for which wasps have no
evolutionary relationship revealed those species which have
potentially evolved to cope with wide-ranging or rapidly evolving
pathogenic threats. The absence of such a response in mud-nest
constructing species may be indicate that they are not subject to
the same pathogenic evolutionary constraints as social and
ground-dwelling wasp species. Further investigation is required
to determine whether these species have lost their antimicrobial
defences or evolved specific compounds to cope with a much
narrower range of pathogenic threats.
Acknowledgments
We would like to thank Michael Elliott (Australian Museum) for his
assistance with morphological identification of wasp species and Dr
Christine Turnbull for her assistance with sample collection.
Author Contributions
Conceived and designed the experiments: SJH AS AB. Performed the
experiments: SJH. Analyzed the data: SJH PDW. Contributed reagents/
materials/analysis tools: SJH AS. Wrote the paper: SJH AS AB. Designed
the software module used in the antimicrobial analysis: PDW.
Figure 1. Distance-based neighbour-joining tree. Neighbour-joining phylogenetic reconstruction of nine wasp species using 941 bp sequence
generated by concatenating the two gene fragments: 28S nrDNA (484 bp) and COI mtDNA (457 bp). Social complexity and nest type are indicated
after the species names; social (Sol), communal aggregator (Com), solitary (Soc), paper nest (P), mud nest (M) and burrower (B). Bootstrap values were
obtained using 10000 replicates.
doi:10.1371/journal.pone.0021763.g001
Evolution of Antimicrobial Defences in Wasps
PLoS ONE | www.plosone.org 4 July 2011 | Volume 6 | Issue 7 | e21763
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