Colony Breeding Structure of the Invasive Termite Reticulitermes
urbis (Isoptera: Rhinotermitidae)
AND ANNE-GENEVIEVE BAGNE
J. Econ. Entomol. 106(5): 2216Ð2224 (2013); DOI: http://dx.doi.org/10.1603/EC13157
ABSTRACT Invasive species cause severe environmental and economic problems. The invasive
success of social insects often appears to be related to their ability to adjust their social organization
to new environments. To gain a better understanding of the biology of invasive termites, this study
investigated the social organization of the subterranean termite, Reticulitermes urbis, analyzing the
breeding structure and the number of reproductives within colonies from three introduced popula-
tions. By using eight microsatellite loci to determine the genetic structure, it was found that all the
colonies from the three populations were headed by both primary reproductives (kings and queens)
and secondary reproductives (neotenics) to form extended-family colonies. R. urbis appears to be the
only Reticulitermes species with a social organization based solely on extended-families in both native
and introduced populations, suggesting that there is no change in their social organization on
introduction. F-statistics indicated that there were few neotenics within the colonies from urban areas,
which did not agree with results from previous studies and Þeld observations. This suggests that
although several neotenics may be produced, only few become active reproductives. The results also
imply that the invasive success of R. urbis may be based on different reproductive strategies in urban
and semiurbanized areas. The factors inßuencing an individual to differentiate into a neotenic in
Reticulitermes species are discussed.
KEY WORDS termite, invasive species, breeding system, neoteny
Anthropic activities and global climatic changes have
led to a signiÞcant increase in the number of species
accidentally or deliberately introduced into new areas
(Vitousek et al. 1997, Sala et al. 2000). Some intro-
duced species have become well established in their
new ranges, causing severe environmental and eco-
nomic problems (Wilcove et al. 1998, Mooney and
Cleland 2001, Pimentel et al. 2005).
The invasive success of social insects is thought to
be at least partially attributable to changes in social
organization (Moller 1996, Chapman and Bourke
2001, Holway et al. 2002). Social organization, in par-
ticular the number and relatedness of reproductives
within social groups, provides ecological ßexibility,
allowing rapid adaptation to new environmental con-
ditions. Shifts in the social organization are well illus-
trated in Hymenoptera. For example, colonies of in-
troduced populations of the wasp Vespula germanica
have several reproductive queens, whereas native
populations have only one reproductive queen per
colony (Donovan et al. 1992, Kasper et al. 2008). In-
troduced populations of the bumblebee, Bombus ter-
restris, have large colonies with two reproducing gen-
erations per year, whereas native populations have
small colonies with only one reproducing generation
per year (Buttermore 1997, Nagamitsu and Yamagishi
2009). Introduced populations of several taxa of ants
have a unicolonial social organization, including the
most destructive invasive species such as Anoplolepis
gracilipes, Linepithema humile, Pheidole megacephala,
and Wasmannia auropunctata (Morel et al. 1990, Van-
loon et al. 1990, Holway et al. 2002, Tsutsui and Suarez
2003, Le Breton et al. 2004, Fournier et al. 2009, Blight
et al. 2012). Unicolonial populations are characterized
by the absence of colony boundaries between nests
that contain many queens and interchange individuals
and brood (Ho¨lldobler and Wilson 1990). Although
Isoptera have not been studied to the same extent as
Hymenoptera, they also show potential changes in
social organization of introduced populations (Dron-
net et al. 2005; Husseneder et al. 2005, 2012; Perdereau
et al. 2010).
Invasive Isoptera are mainly represented by sub-
terranean termites (Evans et al. 2013). The cryptic
lifestyle of these termites makes particularly difÞcult
to study their biological and social traits. Their social
organization is complex and variable, both between
Institut de Recherche sur la Biologie de lÕInsecte UMR CNRS
7261, Universite´Franc¸ois Rabelais, Faculte´des Sciences, Parc de
Grandmont, 37200 Tours, France.
Corresponding authors, e-mail: firstname.lastname@example.org.
Dipartimento di Scienze Biologiche, Geologiche e Ambientali Ð
Universita`di Bologna, via Selmi 3, 40126 Bologna, Italy.
Laboratoire dÕEthologie Expe´rimentale et Compare´e UMR CNRS
7153, Universite´Paris 13, 99 Ave. J.-B. Cle´ment, 93430 Villetaneuse,
0022-0493/13/2216Ð2224$04.00/0 䉷2013 Entomological Society of America
and within species (Cle´ment 1981, Reilly 1987). Most
colonies are founded by a monogamous pair of pri-
mary reproductives (alates) and have a simple-family
structure, that is, one queen, one king, and their prog-
eny. Colonies with an extended-family structure occur
when secondary reproductives (neotenics), differen-
tiating from nymphs or workers, can supplement or
replace the primary reproductives (Buchli 1958).
Neotenics are the offspring of primary pairs. However,
in three speciesÐReticulitermes speratus from Japan,
Reticulitermes virginicus from North America, and Re-
ticulitermes lucifugus from ItalyÐneotenics are pro-
duced by thelytokous parthenogenesis with terminal
fusion (Matsuura and Nishida 2001; Matsuura et al.
2004, 2009; Vargo et al. 2012; Luchetti et al. 2013).
Colonies may occasionally fuse into genetically com-
plex groups, resulting in a mixed-family structure (De-
Heer and Vargo 2008, Perdereau et al. 2010, Luchetti
et al. 2013). The type of family structure and the
number of neotenics within colonies may differ in
introduced and native populations. This has been well
illustrated in the genera Coptotermes and Reticu-
litermes, which commonly infest wood in buildings
and are the termites that have the greatest economic
impact in the United States and Europe (Vargo and
Husseneder 2009). In Reticulitermes ﬂavipes (previ-
ously named Reticulitermes santonensis) (Dronnet et
al. 2005, Perdereau et al. 2010) and three Coptotermes
species (Coptotermes lacteus, Coptotermes acinacifor-
mis, and Coptotermes frenchi) (Lenz and Barrett
1982), there are more neotenics in introduced colo-
nies than in native populations. Colonies headed by
multiple neotenics can grow and expand, sometimes
forming large networks of interconnected reproduc-
tive centers. It appears, therefore, that the presence of
neotenics within a colony is an advantage, allowing
colony sustainability and increasing the capacity to
colonize new areas, despite increased inbreeding (De-
Heer and Vargo 2006). Moreover, it has been sug-
gested that the presence of many neotenics, by allow-
ing the growth of larger colonies, is responsible for the
formation of mixed-families in invasive populations of
R. ﬂavipes (Perdereau et al. 2010). Knowledge of
breeding systems is essential to give an in-depth un-
derstanding of the biology of invasive subterranean
termites and to develop effective pest control pro-
grams (Eger et al. 2012, Vargo and Parman 2012).
R. urbis (Rhinotermitidae) is a Reticulitermes spe-
cies recently described in Europe (Bagne`res et al.
2003). Phylogenetic studies showed that R. urbis was
related to a complex of species living in the Balkans
(Uva et al. 2004, Luchetti et al. 2007, Leniaud et al.
2010). This species is widely distributed in the
Peloponnese, northÐwestern Greece, Croatia, and
Bosnia Herzegovina, and it shows a certain degree of
differentiation between northern and southern pop-
ulations (Luchetti et al. 2007, Velona`et al. 2010, Ku-
lijer et al. 2013). Invasive populations of R. urbis have
been found in southern France and in northÐeastern
and southÐeastern Italy, mainly in urban areas, with
the possible exception of southÐeast Italy, where col-
onies have also been found in natural sites, such as
natural forests. These distribution and genetic data
suggest that R. urbis has been introduced by trade,
although the source populations of these invasive col-
onies have not been identiÞed (Marini and Mantovani
2002; Uva et al. 2004; Luchetti et al. 2007, 2013; Leniaud
et al. 2009, 2010).
R. urbis seems to have similar characteristics to
other introduced termites. It has a supercolony struc-
ture in an introduced population in Dome`ne (RhoˆneÐ
Alpes region, France), suggesting the presence of nu-
merous neotenics (Leniaud et al. 2009). All
populations studied in France, Italy, Croatia, and
Greece contain extended-family colonies only (Le-
niaud et al. 2009, 2010; Luchetti et al. 2013), which is
a rare case in Reticulitermes species. However, the
estimation of the number of active secondary repro-
ductives was not determined in previous studies on R.
urbis. To gain further insight into the breeding system
of this invasive termite, this study investigated the
colony genetic structure of three introduced popula-
tions from different urban areas in France and Italy.
The breeding structure and the number of reproduc-
tives were inferred by genotyping at eight microsat-
Materials and Methods
Sampling. Samples were collected from three in-
troduced populations of R. urbis, two in France and
one in Italy (Fig. 1). Samples were collected in France
from nine collection points in Saint Cyr sur Mer (St)
(30 km east of Marseille; Fig. 1A) and 10 collection
points near Sophia Antipolis (SA) (20 km west of Nice;
Fig. 1B) in 2008 and 2009. Samples were collected in
Italy from seven collection points in Bagnacavallo
(Ba) (55 km east of Bologna; Fig. 1C) in 2001 and 2002.
Termites were collected from wood fragments or dam-
aged wooden constructions. At least 20 workers were
collected from each collection point and stored in 96%
ethanol at 4⬚C until DNA extraction. The species was
determined by chemotaxonomy and DNA analysis by
using an ITS2 marker.
Genotyping. Five hundred twenty workers (20
workers from each of the 26 collection points) were
genotyped at eight microsatellite loci, that is, Rf6–1,
Rf21–1, originally isolated from R. ﬂavipes (Vargo
2000), and RS2, RS10, RS15, RS16, RS43, and RS62,
isolated from R. ﬂavipes (previously named R. san-
tonensis) in France (Dronnet et al. 2004). The
genomic DNA was extracted by using a Wizard
Genomic DNA PuriÞcation Kit (Promega, Madison,
WI). Polymerase chain reaction (PCR) ampliÞcation
was performed by using a Multiplex PCR Kit
(QIAGEN, Venlo, Netherlands), as described by the
manufacturer by using a Biometra 96 T1 or a Strat-
agene thermal cycler with an initial denaturation step
at 95⬚C (15 min), followed by 40 cycles at 94⬚C (30 s),
57⬚C (1 min 30 s), and 72⬚C (1 min), and Þnally an
extension step at 60⬚C (30 min). Multiplex reactions
were carried out with the following primer pairs and
Þnal concentrations: Rs85 and Rf6 –1 at 50 and 100 nM,
Rs76 and Rf11–1 at 150 nM, Rs10 and Rf15–2 at 50 nM,
October 2013 PERDEREAU ET AL.: BREEDING STRUCTURE OF Reticulitermes urbis 2217
and Rs15 and Rf1–3 at 50 nM. The PCR products were
analyzed as described by Dronnet et al. (2004). The
PCR products were separated by electrophoresis on a
6% polyacrylamide gel in a LI-COR 4000L sequencer.
The alleles were scored by using GENE PROFILER
4.03 (Scanalytics, Inc., Fairfax, Va).
Colony Boundaries. Microsatellite analyses were
carried out to determine whether different collection
points belonged to the same colony. Genotypic fre-
quencies were compared for all pairs of collection
points by using a log-likelihood (G)-based differen-
tiation test from GENEPOP on the Web (Raymond
and Rousset 1995). The overall signiÞcance was de-
termined by using Fisher combined probability test,
with a Bonferroni correction for multiple compari-
sons. Samples from two collection points were con-
sidered to belong to different colonies if genotypic
differentiation was statistically signiÞcant (Vargo
2003b, DeHeer and Vargo 2004, Dronnet et al. 2005).
G-tests have proven useful and are widely used to
delineate colonies of social insects (Vargo and Huss-
Breeding Structure of Colonies. After deÞning the
colony boundaries, GENEPOP on the Web option one
was used to test, for each population, the deviation
from HardyÐWeinberg equilibrium. The genotypic
disequilibrium was also estimated by using Fstat 126.96.36.199
(Goudet 1995) to avoid any problems that might occur
from nonindependent genotypes within colonies.
Then, the breeding system of each colony was then
determined by using GENEPOP on the Web (Ray-
mond and Rousset 1995). Colonies were classiÞed into
three types based on their family structure, by com-
paring the number and frequency of alleles and ge-
notypes observed in the colonies with expected ge-
notypes according to standard criteria for the
respective termite families (Vargo 2000, Bulmer et al.
2001, DeHeer and Vargo 2004, Vargo and Carlson
2006). Colonies were classiÞed as simple-families
when worker genotypes were consistent with direct
offspring of a single pair of reproductives and when
the observed frequencies did not differ signiÞcantly
from those expected under Mendelian segregation of
alleles from two parents. SigniÞcance was determined
by a G-test (P⬍0.05) combined across all loci. Col-
onies were considered as extended-families when
there were no more than four alleles at any one locus
and when worker genotypes were not consistent with
a single pair of reproductives (e.g., more than four
genotypes at a locus or three or more homozygote
genotypes) or when genotype frequencies deviated
signiÞcantly from those expected in simple-family col-
Fig. 1. Locations of the three populations studied in Europe and map of collection points in St-Cyr-sur-Mer (A, collection
points St), Sophia Antipolis (B, collection points SA), and Bagnacavallo (C, collection points Ba). For each population,
collection points belonging to the same colony (Table 1) are indicated by the same color and symbol.
2218 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 106, no. 5
onies. Colonies were considered as mixed-families
when more than four alleles were found at a locus, a
pattern that is consistent with offspring produced by
more than two unrelated reproductives.
F-statistics and Relatedness Estimations. The colony
level F-statistics (Weir and Cockerham 1984) and co-
efÞcient of relatedness (r) (Queller and Goodnight
1989) were estimated by using Fstat 188.8.131.52 (Goudet
1995). Results were compared with the simulated ter-
mite breeding structure models proposed by Thorne
et al. (1999), where the various components of vari-
ation are classiÞed as individual (I), colony (C), and
total (T). In these models, F
is the coefÞcient of
inbreeding for individuals relative to the total popu-
is the estimated genetic differentiation
between colonies; F
is the coefÞcient of inbreeding
for individuals within colonies and provides informa-
tion on the number of reproductives and relatedness
among them. The number of neotenic within colonies
increases the value of F
, which approaches zero. It
can become positive if genotyped individuals come
from genetically differentiated colonies either be-
cause of colonies fused or a sharing of foraging tunnels
(Thorne et al. 1999). The signiÞcance of the F-statis-
tics was assessed from 95% CIs by bootstrapping over
loci, with 1,000 replications, with a probability
that their conÞdence limits did not overlap zero. The
same software was used to determine the estimated
gene diversity (Nei 1987) within each population.
Isolation by Distance. Isolation by distance was cal-
culated for all collection points for each population
and for two colonies that had a sufÞcient number of
collection points (a colony from St comprising six
collection points and a colony from SA comprising
Þve collection points). The F
points within each colony was calculated. The correla-
tion coefÞcient between F
) and Ln of geo-
graphic distances (Slatkin 1993, Rousset 1997) was ob-
tained by using the Mantel Test in GENEPOP on the
Colony Boundaries. None of the loci showed con-
sistent patterns of genotypic disequilibrium. Geno-
typic differentiation tests for the French populations
grouped the nine collection points from St into three
colonies and the 10 collection points from SA into four
colonies (signiÞcant G-tests between pairs of collec-
tion points P⬍0.0003). For the Italian population
from Bagnacavallo, genotypic differentiation tests
identiÞed six colonies among the seven collection
points (signiÞcant G-test between pairs of collection
points P⬍0.0009) (Fig. 1; Table 1).
Breeding Structure of Colonies. Based on the num-
ber of genotypes per colony at the eight microsatellite
loci, all the 13 colonies identiÞed within the three
introduced populations of R. urbis were classiÞed as
extended-family (Table 1). Seven colonies (St A,
SA B-D, Ba D-F) had more than four genotypes for
at least one locus. In the remaining six colonies
(St B-C, SA A, Ba A-C), the number of geno-
types was consistent with simple-family colonies, but
the distribution of genotypes within the colonies dif-
fered signiÞcantly from that expected if the workers
had been the offspring of a single pair of reproductives
(G-test across all loci; P⬍0.05). These results indi-
cated that all workers were the offspring of more than
two related breeders within the 13 colonies.
F-statistics and Relatedness Estimations. The ex-
tended-family structure of the 13 R. urbis colonies
was conÞrmed by the values of relatedness and
F-statistics, in particular by the F
value that is
sensitive to the number and origin of reproductives
(Table 2). The F-statistics for the extended-family
colonies from the Bagnacavallo and St populations
were consistent with those expected for colonies
with two neotenic reproductives that had interbred
for one generation (B(i), Table 2); F
negative and signiÞcantly different from zero in
both populations. The F-statistics and relatedness es-
timated for the extended-family colonies from the SA
population gave a different picture, consistent with
that expected for colonies with breeding between 10
and 300 neotenic reproductives over three genera-
tions (B(iv), Table 2). In this case, F
negative but not signiÞcantly different from zero.
Isolation by Distance. SigniÞcant isolation by dis-
tance was scored for collection points from the SA
population (Mantel test: n⫽10, r⫽0.45, P⫽0.001,
range of F
:⫺0.016 to 0.469), indicating high popu-
lation viscosity and nonrandom mating in colonies
from SA. For the St and Bagnacavallo populations, no
signiÞcant isolation by distance was detected between
the collection points for each population, indicating
that collection points within each population were no
more likely to be related to their neighbors than to
more distant collection points (Mantel test: n⫽9, r⫽
0.013, P⫽0.39, range of F
:⫺0.001 to 0.587; Mantel
test: n⫽7, r⫽0.13, P⫽0.97, range of F
: 0.071Ð 0.536).
Table 1. R. urbis colony collection points from the three pop-
ulations with the colony ID, the family structure, the number of
collection points (N
), and the number of genotyped individuals
Colony collection points Colony ID Family
St1, St2, St3, St4, St5, St7 St_A Extended 6 120
St6 St_B Extended 1 20
St8, St9 St_C Extended 2 40
SA1 SA_A Extended 1 20
SA2, SA3, SA4 SA_B Extended 3 60
SA5 SA_C Extended 2 20
SA6, SA7, SA8, SA9, SA10 SA_D Extended 5 100
Ba1 Ba_A Extended 1 20
Ba2, Ba4 Ba_B Extended 2 40
Ba3 Ba_C Extended 1 20
Ba5 Ba_D Extended 1 20
Ba6 Ba_E Extended 1 20
Ba7 Ba_F Extended 1 20
Collection points are shown in Fig. 1.
October 2013 PERDEREAU ET AL.: BREEDING STRUCTURE OF Reticulitermes urbis 2219
Colony-level analyses did not show signiÞcant isola-
tion by distance between collection points for the two
colonies studied (the colony of St St A and the col-
ony from SA SA D) (Mantel test: n⫽6, r⫽0.015, P⫽
0.39; Mantel test: n⫽5, r⫽0.30, P⫽0.092).
Genetic Diversity of the Populations. A signiÞcant
deviation from HardyÐWeinberg equilibrium was de-
tected when all loci were pooled, and for at least one
locus when each locus was analyzed separately for the
three populations studied. The allelic richness (Rs)of
the eight microsatellite loci was 1.61 in St, 2.22 in SA,
and 2.49 in Bagnacavallo (range: 1Ð3.40), with an av-
erage gene diversity (H
) of 0.163 in St, 0.318 in SA,
and 0.289 in Bagnacavallo (range: 0Ð0.512) (Table 3).
The allelic richness was signiÞcantly higher in the
Bagnacavallo population (2.49) than in the St popu-
lation (1.61) (MannÐWhitney Test; W ⫽46; P⬍0.05)
but was not signiÞcantly different from the SA pop-
ulation. Of the 32 alleles identiÞed from the three
populations, 14 alleles (44%) were found exclusively
in the Bagnacavallo population, suggesting that it was
introduced independently of the French population.
To compare the genetic diversity in introduced pop-
ulations and native populations, the gene diversity and
allele numbers were calculated from the values pre-
viously determined in Leniaud et al. (2009) for the
Balkans population (native area). The gene diversity
) and number of alleles for the Balkan population
based on the eight loci used in this study were esti-
mated as 0.216 and 3.75, respectively. None of the
differences in gene diversity between introduced and
native populations were considered signiÞcant
(MannÐWhitney tests H
Balkans vs. H
Mer, W ⫽76, P⬎0.05; H
Balkans vs. H
Table 2. F-statistics and relatedness coefﬁcients (r) for genotyped nestmates from the three populations
Extended families of R. urbis (n⫽6) 0.061 (⫺0.231/0.416) 0.326 (0.163/0.570) ⫺0.393 (⫺0.583/⫺0.202) 0.614 (0.373/0.825)
Extended families of R. urbis (n⫽3) 0.015 (⫺0.451/0.695) 0.327 (0.054/0.712) ⫺0.464 (⫺0.801/⫺0.008) 0.644 (0.195Ð0.905)
(iii) FranceÑSophia Antipolis
Extended families of R. urbis (n⫽4) 0.194 (⫺0.088/0.405) 0.266 (0.058/0.466) ⫺0.098 (⫺0.480/0.200) 0.446 (0.102Ð0.733)
Simulated breeding systems
(A) Colonies headed by monogamous
0 0.25 ⫺0.33 0.50
(B) Colonies with breeding among
⫽1. X⫽10.33 0.42 ⫺0.14 0.62
⫽1. X⫽30.57 0.65 ⫺0.22 0.82
⫽10. X⫽30.37 0.38 ⫺0.02 0.56
⫽100. X⫽30.33 0.34 0 0.50
(C) Nest budding with interconnected
(i) X⫽0. P⫽0.5 0.33 0.37 ⫺0.06 0.56
⫽1. X⫽3. P⫽0.5 0.66 0.56 0.22 0.68
(D) Workers from unrelated nests mix
at collection sites
⫽1. X⫽1. P⫽0.5 0.33 0.20 0.17 0.29
⫽1. X⫽3. P⫽0.8 0.57 0.43 0.25 0.55
Values expected for the possible breeding systems of termites, derived from computer simulations by Thorne et al. (1999) are also given.
CIs of 95% are shown in parentheses.
Xis the number of generations of replacement reproductives within a colony, N
is the number of replacement females, N
is the number
of replacement males produced per generation and Pis the proportion of workers coming from one of the two nests.
Table 3. Variability at eight microsatellite markers in the three populations
French populations Italian population
St-Cyr-sur-Mer Sophia Antipolis Bagnacavallo
Rf6-1 4 2 0.502 2 0.431 2 0.332
Rf21-1 3 1 0 2 0.315 2 0
RS2 4 1 0 2 0.435 2 0.399
RS10 4 1.98 0.108 2 0.122 1.72 0.043
RS15 5 2.87 0.362 3 0.386 3.40 0.282
RS16 4 1 0 2.99 0.482 2.97 0.512
RS43 3 1 0 1 0 2.88 0.329
RS62 5 2 0.331 2.8 0.374 2.97 0.414
Mean (⫾SD) 1.61 ⫾0.71 2.22 ⫾0.68 2.49 ⫾0.63
Overall 32 0.163 0.318 0.289
The number of alleles (Na), the allelic richness (Rs), and the gene diversity (H
) were calculated from the whole sample.
2220 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 106, no. 5
Antipolis, W ⫽54, P⬎0.05; H
Balkans vs. H
nacavallo, W ⫽56, P⬎0.05). The number of alleles in
Balkans population was appeared signiÞcantly higher
only for the St population (MannÐWhitney tests Na
Balkans vs. Na St, W ⫽93, P⬍0.01; Na Balkans vs. Na
SA, W ⫽84, P⬎0.05; Na Balkans vs. Na Bagnacavallo,
To understand the possible social structure changes
in invasive termite populations, the social organization
of the subterranean termite R. urbis was determined
by analyzing the breeding system and the number of
reproductives in colonies of three introduced popu-
lations. No supercolony structure was identiÞed, as
previously described in the Dome`ne population (Le-
niaud et al. 2009), but three colonies were found in the
population of St (France), four in the population of SA
(France), and six in the population of Bagnacavallo
(Italy). The SA population located in a semiurbanized
habitat had an extensive colony covering 650 m com-
pared with the St and Bagnacavallo populations, which
had a relatively dense urban habitat (the largest col-
ony in the urban area covered 220 m in the St popu-
lation, St A). Interestingly, isolation by distance was
only found in the SA population, indicating dispersion
by budding with independent reproductive centers in
this semiurbanized forest in addition to dispersion by
swarming. However, in urban habitats (St and Bag-
nacavallo), human activities may play an important
role in the dispersal and/or fragmentation of colonies.
The human contribution to the colony expansion in
urban habitat was also observed in the invasive pop-
ulation of R. ﬂavipes in Paris (Dronnet et al. 2005).
The results also showed that all the 13 colonies from
the three introduced populations of R. urbis had re-
lated and inbred secondary reproductives (thus form-
ing extended-families), as previously observed in
other populations of these species. All the 36 R. urbis
colonies analyzed so far from several populations in
Italy, France, Croatia, and Greece had an extended-
family structure (Leniaud et al. 2009, 2010, Luchetti et
al. 2013, this article) (Table 4). The presence of neo-
tenics in every colony of all the populations of R. urbis
studied is surprising, as populations of other Reticu-
litermes species have been shown to have a variable
proportion of simple- and extended-families with at
time mixed-families (Table 4). Moreover, the family
structure of colonies from the introduced range
(France and Italy) seems to be the same as that ob-
served within colonies from native populations (Cro-
atia and Greece). In introduced populations of several
termite species, the family structure and/or the num-
ber of secondary reproductives within colonies have
been found to be different from that in the native
populations (Lenz and Barrett 1982, Dronnet et al.
2005, Vargo and Husseneder 2009, Perdereau et al.
2010, Husseneder et al. 2012). The systematic produc-
tion of neotenics within colonies of R. urbis seems,
therefore, to be usual phenomenon in this species, in
both introduced and native populations.
The role of secondary reproductives (neotenics)
within a colony is to supplement or replace the pri-
mary reproductives (Buchli 1958). In the Þrst case,
neotenics allow 1) rapid growth of the colony and 2)
the organization of widespread networks of intercon-
nected reproductive centers (Dronnet et al. 2005, Per-
dereau et al. 2010). In the second case, the presence
of secondary reproductives may allow colony frag-
mentation into autonomous reproductive centers that
will constitute new nests (Thorne et al. 1999). Know-
ing the number of neotenics in the colony is, therefore,
essential to understand the colony dispersal strategy of
a termite species.
Table 4. Colony breeding structures in Reticulitermes spp. inferred from microsatellite markers and number of neotenics estimated
Species No. colonies Simple family Extended family No. neotenics, Estimated
R. urbis 36 0% 100% 25%, ⱕ20%
R. lucifugus 7 57% 14% NR 29%
R. ﬂavipes (introduced populations) 39 0% 90% 100%, 10Ð300 10%
R. grassei 189 36% 62% 57%, ⬍10 2%
R. ﬂavipes (native populations) 504 72% 25% 86%, ⬍10 3%
R. hageni 36 91% 9% ⬍10 0%
R. malletei 13 54% 46% ⬍10 0%
R. virginicus 12 88% 13% ⬍10 0%
R. hesperus 30 73% 27% ⬍10 0%
R. speratus 15 27% 27% NR 46%
NR ⫽not reported.
Bulmer et al. 2001; Vargo 2003a,b, 2006, 2013; Copren 2004; DeHeer and Vargo 2004; DeHeer et al. 2005; Dronnet et al. 2005; Hayashi et
al. 2005; Vargo and Carlson 2006; DeHeer and Kamble 2008; Nobre et al. 2008; Parman and Vargo 2008; Leniaud et al. 2009, 2010; Perdereau
et al. 2010, 2011; Luchetti et al. 2013.
October 2013 PERDEREAU ET AL.: BREEDING STRUCTURE OF Reticulitermes urbis 2221
For the Þrst time, the number of active secondary
reproductives within colonies of R. urbis has been
estimated. There seemed to be differences in the num-
ber of neotenics between introduced populations in
urban and semiurbanized. F-statistics and relatedness
coefÞcients indicated that between 10 and 300 neo-
tenics had interbred for several generations within the
semiurbanized colonies in the SA population, whereas
in urban areas (Bagnacavallo and St populations), only
two neotenics had interbred for one generation.
Ghesini and Marini (2009) found that 3 yr after setting
up laboratory colonies, the majority of R. urbis colo-
nies from the Italian population of Bagnacavallo con-
tained two secondary reproductives, together with a
few workers. However, the presence of a few neoten-
ics within R. urbis colonies in urban areas was not in
agreement with previous studies; Leniaud et al. (2009)
suggested that the Dome`ne urban supercolony of R.
urbis had characteristics similar to an invasive species,
including a high number of active secondary repro-
ductives. In the Þeld, it was also observed that termites
collected from wood could have ⬇10Ð40 neotenics
per colony in urban, semiurbanized, and natural areas
of Italy, including Bagnacavallo (Luchetti et al. 2013;
A. Luchetti, personal communication). The small
number of neotenics indicated by the F-statistics
within the urban colonies and the observations in the
Þeld would suggest that, in some instances, even if
several secondary reproductives differentiate, only
two are reproductively active within a colony. Many
studies have been undertaken into the nature of ge-
netic and environmental factors inßuencing an indi-
vidual to differentiate into a neotenic in the Reticu-
litermes species. In three species, R. speratus, R.
virginicus, and R. lucifugus, neotenics are genetically
determined, being produced by parthenogenesis
(Matsuura et al. 2009, Vargo et al. 2012, Luchetti et al.
2013). It was suggested that parthenogenesis may be
widespread among Reticulitermes species; however, it
has not been found in R. urbis (Luchetti et al. 2013).
It was recently demonstrated that environmental
and climatic conditions had a strong effect on the
numbers of reproductives within colonies in two sub-
terranean termites species (R. ﬂavipes and Reticu-
litermes grassei): it was found that there were more
neotenics within a colony in cool, moist environments
(Vargo et al. 2013). Our data are not consistent with
this Þnding, as the populations of St (neotenics ⱕ2)
and SA (10 Ð300 neotenics) are located along the Med-
iterranean coast with a hot, dry climate and Bagna-
cavallo (neotenics ⱕ2), in the northÐeast Italy, is
moist and cool in winter but hot in summer.
Furthermore, R. speratus female neotenics and eggs
produce a volatile inhibitory pheromone preventing
the differentiation of new female neotenics (Matsuura
et al. 2010, Yamamoto and Matsuura 2011). It would be
interesting to test whether the difference between the
numbers of reproductively active neotenics within R.
urbis colonies may be because of an unknown pher-
omone control. Further studies are needed to gain a
better understanding of the mechanisms inßuencing
the differentiation into secondary reproductives in R.
urbis as well as in other subterranean termites.
This study did not reveal any differences in the
breeding structure and the genetic diversity of the
introduced populations of R. urbis, compared with
native populations. Nevertheless, it is important to
determine the source population to gain further un-
derstanding of the biology and dispersal strategy of
this termite. It has been suggested several times that
the capacity of a colony to produce a large number of
reproductives may be a common characteristic of in-
vasive Reticulitermes termites (Dronnet et al. 2005,
Leniaud et al. 2010, Perdereau et al. 2010). In R. urbis,
the invasive success seems to be based on different
reproductive strategies in urban and semiurbanized
areas. To help the population to expand in different
habitats (urban and semiurbanized), colonies may ad-
just the number of active neotenics within colonies to
promote either colony growth or colony fragmenta-
We thank Mario Marini for sampling the Bagnacavallo
population. We also thank Tony Tebby for his help to im-
prove the English of the manuscript. This work was sup-
ported by the PACA (Provence-Alpes-Coˆte dÕAzur) region
funds to A.G.B. and by Canziani funds to B.M.A.V. performed
most of the analyses on the R. urbis Bagnacavallo population
during an Erasmus stay in the French laboratory (IRBI) in
Bagne`res, A. -G., P. Uva, and J. -L. Cle´ment. 2003. Descrip-
tion dÕune nouvelle espe`ce de termite: Reticulitermes ur-
bis n.sp. (Isopt., Rhinotermitidae). Bull. Soc. Entomol. Fr.
Blight, O., L. Berville, V. Vogel, A. Hefetz, M. Renucci, J.
Orgeas, E. Provost, and L. Keller. 2012. Variation in the
level of aggression, chemical and genetic distance among
three supercolonies of the Argentine ant in Europe. Mol.
Ecol. 21: 4106Ð4121.
Buchli, H. 1958. LÕorigine des castes et les potentialite´s on-
toge´nique des Termites europe´ens du genre Reticu-
litermes. Ann. Sci. Nat. Zool. Biol. Anim. 11: 263Ð429.
Bulmer, M. S., E. S. Adams, and J.F.A. Traniello. 2001. Vari-
ation in colony structure in the subterranean termite
Reticulitermes ﬂavipes. Behav. Ecol. Sociobiol. 49: 236Ð
Buttermore, R. E. 1997. Observations of successful Bombus
terrestris (L.) (Hymenoptera: Apidae) colonies in south-
ern Tasmania. Aust. J. Entomol. 36: 251Ð254.
Chapman, R. E., and A.F.G. Bourke. 2001. The inßuence of
sociality on the conservation biology of social insects.
Ecol. Lett. 4: 650Ð662.
Cle´ment, J. -L. 1981. Enzymatic polymorphism in the Eu-
ropean populations of various Reticulitermes species
(Isoptera), pp. 49Ð62. In P. E. Howse and J. -L. Cle´ment
(eds.), Biosystematics of Social InsectsAcademic Press,
London, New York, NY.
Copren, K. A. 2004. Variation in nestmate recognition be-
havior in the western subterranean termite, Reticu-
litermes hesperus (Isoptera: Rhinotermitidae). UC Davis,
2222 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 106, no. 5
DeHeer, C. J., and S. T. Kamble. 2008. Colony genetic or-
ganization, fusion and inbreeding in Reticulitermes ﬂavi-
pes from the Midwestern U.S. Sociobiology 51: 307Ð325.
DeHeer, C. J., and E. L. Vargo. 2004. Colony genetic orga-
nization and colony fusion in the termite Reticulitermes
ﬂavipes as revealed by foraging patterns over time and
space. Mol. Ecol. 13: 431Ð441.
DeHeer, C. J., and E. L. Vargo. 2006. An indirect test of
inbreeding depression in the termites Reticulitermes ﬂa-
vipes and Reticulitermes virginicus. Behav. Ecol. Sociobiol.
DeHeer, C. J., and E. L. Vargo. 2008. Strong mitochondrial
DNA similarity but low relatedness at microsatellite loci
among families within fused colonies of the termite Re-
ticulitermes ﬂavipes. Insectes Soc. 55: 190Ð199.
DeHeer, C. J., M. Kutnik, E. L. Vargo, and A. G. Bagneres.
2005. The breeding system and population structure of
the termite Reticulitermes grassei in Southwestern
France. Heredity 95: 408Ð415.
Donovan, B. J., A.M.E. Howie, N. C. Schroeder, A. R. Wallace,
and P.E.C. Read. 1992. Comparative characteristics of
nests of Vespula-Germanica (F) and Vespula-Vulgaris (L)
(Hymenoptera, Vespinae) from Christchurch-City, New
Zealand.NZJ.Zool. 19: 61Ð71.
Dronnet, S., A. -G. Bagne`res, T. R. Juba, and E. L. Vargo.
2004. Polymorphic microsatellite loci in the European
subterranean termite, Reticulitermes santonensis Feytaud.
Mol. Ecol. Notes 4: 127Ð129.
Dronnet, S., M. Chapuisat, E. L. Vargo, C. Lohou, and A. G.
Bagne`res. 2005. Genetic analysis of the breeding system
of an invasive subterranean termite, Reticulitermes san-
tonensis, in urban and natural habitats. Mol. Ecol. 14:
Eger, J. E., M. D. Lees, P. A. Nees, T. H. Atkinson, E. M.
Thoms, M. T. Messenger, J. J. DeMark, L. -C. Lee, E. L.
Vargo, and M. P. Tolley. 2012. Elimination of subterra-
nean termite (Isoptera: Rhinotermitidae) colonies using
a reÞned cellulose bait matrix containing novißumuron
when monitored and replenished quarterly. J. Econ. En-
tomol. 105: 533Ð539.
Evans, T. A., B. T. Forschler, and J. K. Grace. 2013. Biology
of invasive termites: a worldwide review. Ann. Rev. En-
tomol. 58: 455Ð474.
Fournier, D., J. C. Biseau, and S. Aron. 2009. Genetics, be-
haviour and chemical recognition of the invading ant
Pheidole megacephala. Mol. Ecol. 18: 186Ð199.
Ghesini, S., and M. Marini. 2009. Caste differentiation and
growth of laboratory colonies of Reticulitermes urbis
(Isoptera, Rhinotermitidae). Insectes Soc. 56: 309Ð318.
Goudet, J. 1995. FSTAT (vers 1.2): a computer program to
calculate F-statistics. J. Heredity 86: 485Ð486.
Hayashi, Y., O. Kitade, M. Gonda, T. Kondo, H. Miyata, and
K. Urayama. 2005. Diverse colony genetic structures in
the Japanese subterranean termite Reticulitermes speratus
(Isoptera: Rhinotermitidae). Sociobiology 46: 175Ð184.
Ho¨lldobler, B., and E. O. Wilson. 1990. The ants. The
Belknap Press of Harvard University Press, Cambridge,
Holway, D. A., L. Lach, A. V. Suarez, N. D. Tsutsui, and T. J.
Case. 2002. The causes and consequences of ant inva-
sions. Ann. Rev. Ecol. Syst. 33: 181Ð233.
Husseneder, C., M. T. Messenger, N. Y. Su, J. K. Grace, and
E. L. Vargo. 2005. Colony social organization and pop-
ulation genetic structure of an introduced population of
Formosan subterranean termite from New Orleans, Lou-
isiana. J. Econ. Entomol. 98: 1421Ð1434.
Husseneder, C., D. M. Simms, J. R. Delatte, C. L. Wang, J. K.
Grace, and E. L. Vargo. 2012. Genetic diversity and col-
ony breeding structure in native and introduced ranges of
the Formosan subterranean termite, Coptotermes formo-
sanus. Biol. Invasions 14: 419Ð437.
Kasper, M. L., A. F. Reeson, and A. D. Austin. 2008. Colony
characteristics of Vespula germanica (F.) (Hymenoptera,
Vespidae) in a Mediterranean climate (southern Austra-
lia). Aust. J. Entomol. 47: 265Ð274.
Kulijer, D., S. Dupont, and A. -G. Bagne`res. 2013. Distribu-
tion and Natural Habitat of the Invasive Termite species
Reticulitermes urbis in the Balkans (Isoptera: Rhinoter-
mitidae). Entomol. Gen. 34: 189Ð196.
Le Breton, J., J.H.C. Delabie, J. Chazeau, A. Dejean, and H.
Jourdan. 2004. Experimental evidence of large-scale
unicoloniality in the tramp ant Wasmannia auropunctata
(Roger). J. Insect Behav. 17: 263Ð271.
Leniaud, L., F. Dedeine, A. Pichon, S. Dupont, and A. -G.
Bagne`res. 2010. Geographical distribution, genetic di-
versity and social organization of a new European ter-
mite, Reticulitermes urbis (Isoptera: Rhinotermitidae)
Biol. Invasions 12: 1389Ð1402.
Leniaud, L., A. Pichon, P. Uva, and A. G. Bagne`res. 2009.
Unicoloniality in Reticulitermes urbis: a novel feature in a
potentially invasive termite species Bull. Entomol. Res.
Lenz, M., and R. A. Barrett. 1982. Neotenic formation in
Þeld colonies of Coptotermes lacteus (Froggatt) in Aus-
tralia, with comments on the roles of neotenics in the
genus Coptotermes (Isoptera: Rhinotermitidae). Sociobi-
ology 7: 47Ð59.
Luchetti, A., M. Marini, and B. Mantovani. 2007. Filling the
European gap: biosystematics of the eusocial system Re-
ticulitermes (Isoptera, Rhinotermitidae) in the Balkanic
Peninsula and Aegean area. Mol. Phylogenet. Evol. 45:
Luchetti, A., A. Velona`, M. Mueller, and B. Mantovani. 2013.
Breeding systems and reproductive strategies in Italian
Reticulitermes colonies (Isoptera: Rhinotermitidae). In-
sectes Soc. 60: 203Ð211.
Marini, M., and B. Mantovani. 2002. Molecular relation-
ships among European samples of Reticulitermes
(Isoptera, Rhinotermitidae). Mol. Phylogenet. Evol. 22:
Matsuura, K., and T. Nishida. 2001. Comparison of colony
foundation success between sexual pairs and female asex-
ual units in the termite Reticulitermes speratus (Isoptera:
Rhinotermitidae). Popul. Ecol. 43: 119Ð124.
Matsuura, K., M. Fujimoto, and K. Goka. 2004. Sexual and
asexual colony foundation and the mechanism of facul-
tative parthenogenesis in the termite Reticulitermes spe-
ratus (Isoptera, Rhinotermitidae). Insectes Soc. 51: 325Ð
Matsuura, K., E. L. Vargo, K. Kawatsu, P. E. Labadie, H.
Nakano, T. Yashiro, and K. Tsuji. 2009. Queen succes-
sion through asexual reproduction in termites. Science
Matsuura, K., C. Himuro, T. Yokoi, Y. Yamamoto, E. L. Vargo,
and L. Keller. 2010. IdentiÞcation of a pheromone reg-
ulating caste differentiation in termites. Proc. Natl. Acad.
Sci. USA 107: 12963Ð12968.
Moller, H. 1996. Lessons for invasion theory from social
insects. Biol. Conserv. 78: 125Ð142.
Mooney, H. A., and E. E. Cleland. 2001. The evolutionary
impact of invasive species. The evolutionary impact of
invasive species. Proc. Natl Acad. Sci. USA 98: 5446 Ð5451.
Morel, L., R. K. Vandermeer, and C. S. Lofgren. 1990. Com-
parison of nestmate recognition between monogyne and
polygyne populations of Solenopsis invicta (Hymenop-
tera, Formicidae). Ann. Entomol. Soc. Am. 83: 642Ð647.
October 2013 PERDEREAU ET AL.: BREEDING STRUCTURE OF Reticulitermes urbis 2223
Nagamitsu, T., and H. Yamagishi. 2009. Nest density, ge-
netic structure, and triploid workers in exotic Bombus
terrestris populations colonized Japan. Apidologie 40:
Nei, M. 1987. Molecular evolutionary genetics. Columbia
University Press, New York, NY.
Nobre, T., L. Nunes, and D. E. Bignell. 2008. Colony inter-
actions in Reticulitermes grassei population assessed by
molecular genetic methods. Insectes Soc. 55: 66Ð73.
Parman, V., and E. L. Vargo. 2008. Population density, spe-
cies abundance, and breeding structure of subterranean
termite colonies in and around infested houses in central
North Carolina. J. Econ. Entomol. 101: 1349Ð1359.
Perdereau, E., A. -G. Bagne`res, S. Dupont, and F. Dedeine.
2010. High occurrence of colony fusion in a European
population of the American termite Reticulitermes ﬂavi-
pes. Insectes Soc. 57: 393Ð402.
Perdereau, E., F. Dedeine, J. -P. Christide`s, S. Dupont, and
A. -G. Bagne`res. 2011. Competition between invasive
and indigenous species: an insular case study of subter-
ranean termites. Biol. Invasions 13: 1457Ð1470.
Pimentel, D., R. Zuniga, and D. Morrison. 2005. Update on
the environmental and economic costs associated with
alien-invasive species in the United States. Ecol. Econ. 52:
Queller, D. C., and K. F. Goodnight. 1989. Estimating re-
latedness using genetic markers. Evolution 43: 258Ð275.
Raymond, M., and F. Rousset. 1995. An exact test for pop-
ulation differentiation. Evolution 49: 1280Ð1283.
Reilly, L. M. 1987. Measurements of inbreeding and average
relatedness in a termite population. Am. Nat. 130: 339Ð
Rousset, F. 1997. Genetic differentiation and estimation of
gene ßow from F-statistics under isolation by distance.
Genetics 145: 1219Ð1228.
Sala, O. E., F. S. Chapin, J. J. Armesto, E. Berlow, J. Bloom-
ﬁeld, R. Dirzo, E. Huber–Sanwald, L. F. Huenneke, R. B.
Jackson, A. Kinzig, et al. 2000. Biodiversity: global bio-
diversity scenarios for the year 2100. Science 287: 1770Ð
Slatkin, M. 1993. Isolation by distance in equilibrium and
non-equilibrium populations. Evolution 47: 264Ð279.
Thorne, B. L., J.F.A. Traniello, E. S. Adams, and M. Bulmer.
1999. Reproductive dynamics and colony structure of
subterranean termites of the genus Reticulitermes
(Isoptera Rhinotermitidae): a review of the evidence
from behavioral, ecological and genetic studies. Ethol.
Ecol. Evol. 11: 149Ð169.
Tsutsui, N. D., and A. V. Suarez. 2003. The colony structure
and population biology of invasive ants. Conserv. Biol. 17:
Uva, P., J. L. Clement, J. W. Austin, J. Aubert, V. Zaffagnini,
A. Quintana, and A. G. Bagneres. 2004. Origin of a new
Reticulitermes termite (Isoptera, Rhinotermitidae) in-
ferred from mitochondrial and nuclear DNA data. Mol.
Phylogenet. Evol. 30: 344Ð353.
Vanloon, A. J., J. J. Boomsma, and A. Andrasfalvy. 1990. A
new polygynous Lasius species (Hymenoptera, Formi-
cidae) from Central-Europe. 1. Description and general
biology. Insectes Soc. 37: 348Ð362.
Vargo, E. L. 2000. Polymorphism at trinucleotide microsat-
ellite loci in the subterranean termite Reticulitermes ﬂa-
vipes. Mol. Ecol. 9: 817Ð829.
Vargo, E. L. 2003a. Genetic structure of Reticulitermes ﬂa-
vipes and R. virginicus (Isoptera: Rhinotermitidae) col-
onies in an urban habitat and tracking of colonies fol-
lowing treatment with hexaßumuron bait. Environ.
Entomol. 32: 1271Ð1282.
Vargo, E. L. 2003b. Hierarchical analysis of colony and pop-
ulation genetic structure of the eastern subterranean ter-
mite, Reticulitermes ﬂavipes, using two classes of molec-
ular markers. Evolution 57: 2805Ð2818.
Vargo, E. L., and J. R. Carlson. 2006. Comparative study of
breeding systems of sympatric subterranean termites
(Reticulitermes ﬂavipes and R. hageni) in Central North
Carolina using two classes of molecular genetic markers.
Environ. Entomol. 35: 173Ð187.
Vargo, E. L., and C. Husseneder. 2009. Biology of subter-
ranean termites: insights from molecular studies of Re-
ticulitermes and Coptotermes. Ann. Rev. Entomol. 54: 379 Ð
Vargo, E. L., and C. Husseneder. 2011. Genetic structure of
termite colonies and populations, pp. 321Ð347 In D. Big-
nell, Y. Roisin, and N. Lo (eds.), Biology of Termites: A
Modern Synthesis. Springer, Heidelberg, Germany.
Vargo, E. L., and V. Parman. 2012. Effect of Þpronil on
subterranean termite colonies (Isoptera: Rhinotermiti-
dae) in the Þeld. J. Econ. Entomol. 105: 523Ð532.
Vargo, E. L., T. R. Juba, and C. J. DeHeer. 2006. Relative
abundance and comparative breeding structure of sub-
terranean termite colonies (Reticulitermes ﬂavipes, Re-
ticulitermes hageni, Reticulitermes virginicus, and Cop-
totermes formosanus) in a South Carolina lowcountry site
as revealed by molecular markers. Ann. Entomol. Soc.
Am. 99: 1101Ð1109.
Vargo, E. L., P. E. Labadie, and K. Matsuura. 2012. Asexual
queen succession in the subterranean termite Reticu-
litermes virginicus. Proc. R. Soc. B Biol. Sci. 279: 813Ð819.
Vargo, E. L., L. Leniaud, L. E. Swoboda, S. E. Diamond, M. D.
Weiser, D. M. Miller, and A. G. Bagne`res. 2013. Clinal
variation in colony breeding structure and level of in-
breeding in the subterranean termites Reticulitermes ﬂa-
vipes and R. grassei. Mol. Ecol. 22: 1447Ð1462.
Velona`, A., S. Ghesini, A. Luchetti, M. Marini, and B. Man-
tovani. 2010. Starting from Crete, a phylogenetic re-
analysis of the genus Reticulitermes in the Mediterranean
area. Mol. Phylogenet. Evol. 56: 1051Ð1058.
Vitousek, P. M., C. M. Dantonio, L. L. Loope, M. Rejmanek,
and R. Westbrooks. 1997. Introduced species: a signiÞ-
cant component of human-caused global change.NZJ.
Ecol. 21: 1Ð16.
Weir, B. S., and C. C. Cockerham. 1984. Estimating F-sta-
tistics for the analysis of population structure. Evolution
Wilcove, D. S., D. Rothstein, J. Dubow, A. Phillips, and E.
Losos. 1998. Quantifying threats to imperiled species in
the United States. Bioscience 48: 607Ð615.
Yamamoto, Y., and K. Matsuura. 2011. Queen pheromone
regulates egg production in a termite. Biol. Lett. 7: 727Ð
Received 2 April 2013; accepted 6 June 2013.
2224 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 106, no. 5