Actinomycetes with Antimicrobial Activity Isolated from Paper Wasp (Hymenoptera: Vespidae: Polistinae) Nests

INSECT-SYMBIONT INTERACTIONS Actinomycetes with Antimicrobial Activity Isolated from Paper Wasp (Hymenoptera: Vespidae: Polistinae) Nests

ANNE A. MADDEN,1,2 ANDREW GRASSETTI,1,3 JONATHAN-ANDREW N. SORIANO,1,4 1 AND PHILIP T. STARKS

Environ. Entomol. 42(4): 703Ð710 (2013); DOI: http://dx.doi.org/10.1603/EN12159 ABSTRACT ActinomycetesÑa group of antimicrobial producing bacteriaÑhave been successfully cultured and characterized from the nest material of diverse arthropods. Some are symbionts that produce antimicrobial chemicals found to protect nest brood and resources from pathogenic microbes. Others have no known Þtness relationship with their associated insects, but have been found to produce antimicrobials in vitro. Consequently, insect nest material is being investigated as a new source of novel antimicrobial producing actinomycetes, which could be harnessed for therapeutic potential. To extend studies of actinomycete-insect associations beyond soil-substrate dwelling insects and wood boring excavators, we conducted a preliminary assessment of the actinomycetes within the nests of the paper wasp, Polistes dominulus (Christ). We found that actinomycetes were readily cultured from nest material across multiple invasive P. dominulus populationsÑincluding members of the genera Streptomyces, Micromonospora,andActinoplanes.Thirtyoftheseisolateswereassayedfor antimicrobial activity against the challenge bacteria Pseudomonas aeruginosa, Escherichia coli, Staph- ylococcus aureus, Serratia marcescens,andBacillus subtilis.Sixtypercentofisolatesinhibitedthegrowth of at least one challenge strain. This study provides the Þrst assessment of bacteria associated with nests of P. dominulus,andtheÞrst record of antimicrobial producing actinomycetes isolated from social wasps. We provide a new system to explore nest associated actinomycetes from a ubiquitous and cosmopolitan group of insects.

KEY WORDS antibiotic, natural products, actinobacteria, Polistes dominula

Antimicrobial producing bacteria have been identi- Grubbs et al. 2011), termites (Visser et al. 2012), bees Þed in nest material of divergent arthropod lineages. (Promnuan et al. 2009), and solitary wasps (e.g., The best studied of these bacteria are actinomycetes Poulsen et al. 2011; Kaltenpoth et al. 2006, 2010). Even (actinobacteria)Ña bacterial phylum that produces a anovelactinomycetespecies,Actinomadura apis, was wide variety of antimicrobial chemicals (reviewed in isolated from a honey bee hive (Promnuan et al. 2011). Lam 2006). Some of these arthropod bacteria associ- Many constituents of these associated microbial ations include derived symbioses, where the actino- communities produce antibiotics in vitro, some of mycetes assist in nest or colony hygiene by producing which are novel (Poulsen et al. 2011, Mueller et al. antimicrobial chemicals (reviewed in Seipke et al. 2008). For example, Poulsen et al. (2011) studied sol- 2011a and Kaltenpoth 2009). In addition, recent evi- itary mud dauber wasps of the species Chalybion cali- dence indicates that arthropod habitats frequently fornicum and Sceliphron caementarium, and isolated at contain a veritable bouquet of diverse, nonsymbiont least 15 actinomycete strains of the genus Streptomy- actinomycete species (e.g., Poulsen et al. 2011, Muel- ces. Extensive chemical investigations showed that ler et al. 2008, Kost et al. 2007, Sen et al. 2009, Prom- these symbionts produce eight unique antimicrobials, nuan et al. 2009, Hulcr et al. 2011). Such actinomycete including the novel antimicrobial sceliphrolactam associates include both ubiquitous and rare species in (Poulsen et al. 2011, Oh et al. 2011). the nest material of ants (e.g., Barke et al. 2010, Sen et The ecological function of many actinomycete anal. 2009), beetles (Scott et al. 2008. Hulcr et al. 2011, timicrobials remains nebulous. It is clear, however, that arthropod nest material is a valuable source of novel actinomycetes. This is particularly relevant to The sequences reported in this article have been deposited in the human health, as the recent increase in antibiotic GenBank database (accession nos. HQ336264ÐHQ336293). 1 Department of Biology, Tufts University, 120 Dana Laboratory, resistant pathogens necessitates the discovery of novel 163 Packard Ave., Medford, MA 02155. drugs (Clardy et al. 2006). Because most clinically 2 Corresponding author, e-mail: [email protected]. available antibiotics originate from actinomycetes and 3 Current Location: Department of Molecular and Cellular Biology, fungi (Newman et al. 2003 in Pelaez 2006, reviewed in Dartmouth College, 7560 Remsen Bldg., Hanover, NH 03755-384. 4 Current Location: Plastic Surgery Research Laboratory, Massa- Lam 2006), the current epidemic of antibiotic resistant chusetts General Hospital, Boston, MA 02114. pathogens has spawned a renewed interest in identi-

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Fig. 1. Polistes dominulus nest in Massachusetts. fying uncharacterized habitats from which to isolate dominulus nests in Massachusetts, we recently iden- unique actinomycetes that produce novel antibiotics tiÞed a previously uncharacterized fungal species (Pelaez 2006). Insect nest associates are consequently (Madden et al. 2012), suggesting this may be a habitat being investigated as a likely source of such novelty that contains novel bacteria as well. (e.g., Poulsen et al. 2011, Bode 2009). Despite the Therefore, we investigated P. dominulus nests to potential for microbial or chemical novelty, investi- provide an initial bacterial characterization of this gations of nest material have been conÞned to rela- insect, and to determine if nests of social wasps contain tively few wood excavating or soil-nest/constructing actinomycetes with antimicrobial activity. Eventual insects. novel chemical isolation and structure elucidation tra- Paper wasps of the genus Polistes are social, pro- ditionally requires culturable isolates. We conse- gressively provisioning hymenoptera, which construct quently conducted a preliminary investigation of the open-faced nests out of macerated plant pulp and actinomycetes associated with the nests of P. dominu- saliva (Evans and WestÐEberhard 1970). One species lus using culture dependent methods and assessing in in particular, Polistes dominulus (Fig. 1), is a globally vitro microbial growth inhibition capability. This distributed paper wasp that annually constructs nests broadens the studies of actinomycete insect associates often associated with anthropogenic structures (re- to encompass the previously undescribed realm of a viewed in Liebert et al. 2006). Their proclivity for this globaly distributed, and well-studied insect lineage accessible habitat, and their status as a successful in- whose microbiota has previously remained unex- vader, has contributed to their use as a model system plored. in the Þelds of behavior, evolution, and invasion ecology (Starks and Turillazzi 2006, reviewed in Liebert et Materials and Methods al. 2006). Despite the resultant wealth of information that exists on this species, to date no studies have Nest Collection. Mature, active P. dominulus nests investigated their associated microbial communities. were aseptically removed at the top of their nest pedi- Indeed, few studies have been conducted on the bac- cel and stored at 4ЊC. Nests were identiÞed as P. teria of any Polistes species that have not solely ad- dominulus and determined to be active if at the time dressed the Wolbachia endosymbiont (Stahlhut et al. of collection (August) they were found to have live P. 2006, but see Morel and Fouillaud 1992). As part of a dominulus residents on them at night. In the labora- preliminary characterization of the fungi within P. tory, all active individuals, larvae, pupae, and eggs August 2013 MADDEN ET AL.: ACTINOMYCETE NEST ASSOCIATES OF PAPER WASPS 705 were aseptically removed from the nest. Nests were ware, Germany). Resulting sequences were compared visually inspected and any found to be parasitized by with those in the GenBank sequence repository using brood parasitoids (Madden et al. 2010) were removed the BLAST algorithm, and highest matching identity from the study. Nests were collected from three geo- was recorded for probable identiÞcation. A phyloge- graphically distinct regions in Eastern Massachusetts netic tree was constructed using the parameters de- in the towns of Waltham (n ϭ 4), Easton (n ϭ 3), and scribed by Liu et al. (2009). Brießy, MEGA v4.0 (Ta- Medford (n ϭ 5). These sites were determined to be mura et al. 2007) was used for both the construction geographically isolated because they were separated by of the multiple alignment (using ClustalW and the IUB adistanceexceedingtheforagingrangeofthisspecies DNA weight matrix) and the phylogenetic trees using (Ugolini and Cannicci 1996). They were chosen to re- the neighbor-joining tree-making algorithms (Saitou duce the probability that one insect had visited, and and Nei 1987). Pairwise and evolutionary distances for thereby contaminated, all nests collected because of po- the neighbor-joining algorithms were estimated using tential low nest Þdelity (Sumner et al. 2007). the Kimura two-parameter model (with both transi- Microorganism Isolation. For each nest, nest ma- tions and transversions included) (Kimura 1980), with terial (including paper, pedicel, meconium, and honey positions containing gaps or missing data excluded if present) was aseptically mechanically homogenized (complete deletion option). with forceps, and placed in a conical tube with a 1ϫ Strain sequences from Haeder et al. (2009) and phosphate buffered saline solution diluted to contain other high homology strains, as determined by BLAST 5mMphosphate.Tubeswerevortexedandthere- analysis, were included in the multiple alignments for sulting homogenate was diluted in the same solvent comparison. Sequences were trimmed to Ϸ1,300 bp and plated in 1% LuriaÐBertani (Miller) medium (15% before alignment to reduce false distance analyses. bacto agar) (BD [Becton, Dickinson and Company], The bacterial 16S rRNA gene sequence of Biﬁdobac- Sparks Glencoe, MD), Actinomycete Isolation me- terium longum subsp. suis (AB437360), downloaded dium (BD), and a low nutrient medium: 1% Skim Milk from the GenBank database, was used as an outgroup. and 18% Gellan Gum (GELZAN CM, SigmaÐAldrich, The resultant tree was evaluated using bootstrap anal- Milwaukee, WI). Plates were incubated at ambient ysis (Felsenstein 1985) based on 1,000 replicates. room temperature (Ϸ22ЊC) and bacterial colonies Antimicrobial Production Assay. The ability of the were streaked for puriÞcation as they grew (contin- actinomycete isolates to inhibit bacterial growth was uously after Ϸ72 h) (Supp. Table 1 [online only]). assessed using a modiÞed agar cross-streak method Colonies exhibiting morphologies indicative of acti- (Waksman et al. 1965). This method involves two nomycetes were selected for further puriÞcation. challenge bacteria being streaked perpendicularly to Molecular Analysis. Genomic DNA was extracted one another. After incubation, antimicrobial activity is from cultured microorganisms using the UltraClean assessed as the presence of a clear, no growth zone microbial DNA extraction kit (MoBio, Carlsbad, CA) between the putative antimicrobial producer, and the per the manufacturerÕs instructions, including a 10 min challenge species. Isolated and control actinomycete heating step at 70ЊCtoenhancelysis.Alargefragment strains were challenged with a broad spectrum of of the 16S rRNA gene was ampliÞed, as there is enough bacterial species including Escherichia coli, the clini- variability in this fragment to allow for determinative cally relevant Pseudomonas aeruginosa, Serratia marc- identity to the genus level in this phylum (Stacke- escens, Staphylococcus aureus (methicillin sensitive), brandt et al. 1997). The fragment was ampliÞed using and Bacillus subtilis. Ahybridassaymediumcontain- the universal eubacterial primers 27F and 1492R ing 50% LuriaÐBertani (Miller) medium (BD) and (Heuer et al. 1997, Eden et al. 1991). The polymerase 17% bacto agar (BD) was used because the majority chain reaction (PCR) cocktail contained: 5 ␮lof5ϫ of isolated strains grew on this medium well, as did the Green GoTaq Reaction Buffer (Promega, Madison, more fastidious challenge bacteria. Assay media plates WI), 5 ␮ltemplateDNA,11.95␮lwater,1.5␮lof25 were prepared at a depth of 3 mm. Isolated actino- mM MgCl2 (Promega), 0.15 ␮l(100pM/␮l) of each mycetes were streaked to conßuency over half of the primer, 1 ␮lofdNTPMix(10mM)(Promega),and plate from axenic, mature cultures grown on the same 0.25 ␮lGoTaqPolymerase(50U/␮l) (Promega) for a medium. Plates containing the growing isolated acti- total reaction volume of 25 ␮l. PCR was conducted using nomycetes were incubated for three days at 30ЊCto athermocycler(model2700,GeneAmpPCRSystem, allow for robust growth. Applied Biosystems, San Francisco, CA) with the cycling Challenge bacteria were grown in 50% LuriaÐBertani parameters of 95ЊCfor5min,then30cyclesof95ЊCfor (Miller) broth (BD) at 37ЊCtoexponentialphase.They 1min,54ЊCfor1min,72ЊCfor1min,andaÞnal extension were subsequently diluted to the McFarland 0.5 turbid- at 72ЊCfor5min.AmpliÞed products of the estimated ity standard in the same broth to control for cell con- fragment length were conÞrmed by running them on a centration (McFarland 1907). A sterile cotton swab was 1.5% agarose electrophoresis gel and visualizing with dipped into the liquid culture for2s(toallowforsatu- ethidium bromide stain. ration) and then pressed against the side of the tube to Successfully ampliÞed samples were puriÞed and remove excess liquid. The challenge organism was cross- sequenced by MacrogenUSA (Rockville, MD) using streaked on the plates, perpendicularly to the actinomy- the ampliÞcation primers. Sequence ends were cete growth. Plates that had not been inoculated, were trimmed and a consensus contiguous sequence was streaked with the challenge organism in the same man- created using DNA Baser Software (Heracles Soft- ner to serve as negative controls. Streptomyces griseus, a 706 ENVIRONMENTAL ENTOMOLOGY Vol. 42, no. 4

Fig. 2. Neighbor-joining phylogenetic tree based on partial 16S rDNA gene sequences, showing the relationships among actinomycetes isolated from P. dominulus nests. Accession numbers are given in parentheses. Biﬁdobacterium longum was used as an outgroup. Neighbor-joining algorithms were applied using the software package MEGA v4.0 (Tamura et al. 2007). Bootstrap percentages (based on 1,000 replications) are shown at branch points. Bar represents 0.005 substitutions per nucleotide position. Symbols next to strains indicate the three populations sampled. Symbol shades indicate the three populations sampled (black, Medford; striped, Easton; white, Waltham), and symbol shapes indicate unique nests within each population. known producer of the antibiotic streptomycin, was used chosen for sequencing. BLAST searches of the se- as a positive control. Growth inhibition was measured quenced 16S rRNA gene fragments within GenBank after bacterial plates were incubated at 37ЊCfor18h. revealed high similarity with bacterial sequences from Assays were performed in triplicate and inhibition was three different actinomycete genera: Streptomyces, scored as a clear zone of growth inhibition (ϩ)orno Micromonospora, and Actinoplanes. clear zone of growth inhibition (Ϫ). Results were aver- Six bacterial strains with a high sequence similarity aged across replicates. (Ն98% 16S rRNA gene) to Micromonospora spp. were isolated from four nests at two Þeld site locations (Fig. 2). A strain with a nearest sequence homology to Results Actinoplanes deccanensis (99%) was isolated from one Actinomycete Isolation. Bacterial isolates with mac- nest, at one location (Fig. 2). roscopic colony morphologies consistent with actino- Most of the isolated strains sequenced (77%) were mycetes were isolated from 12 of the 15 collected P. Streptomyces. These strains were isolated from eight dominulus nests, and from all three sites sampled (Fig. nests and from all three Þeld sites (Fig. 2). Subsequent 2). Of the 39 strains isolated, 30 were haphazardly ampliÞcation and sequencing of the 16S rDNA re- August 2013 MADDEN ET AL.: ACTINOMYCETE NEST ASSOCIATES OF PAPER WASPS 707

Table 1. Antibiotic activity of actinomycetes isolated from the nests of Polistes dominulus

Gram negative Gram positive a Strain homology Strain Escherichia Pseudomonas Serratia Bacillus Staphylococcus coli aeruginosa marcescens subtilis aureus Streptomyces ﬂavovirens M23 ϪϪϪϪϪ species complex M24 ϪϪϪϪϪ M25 ϩϪϪϪϪ M01 ϪϪϪϪϪ M05 ϪϪϪϪϪ M41 ϪϪϪϪϪ M29 ϩϪϪϩϩ M06 ϪϪϪϪϪ M08 ϪϪϪϪϪ M20 ϪϪϪϪϪ M43 ϪϪϪϪϪ M26 ϪϪϪϩϪ M45 ϪϪϪϪϪ M22 ϪϪϪϪϪ M30 ϩϪϪϩϪ Streptomyces griseus M38 ϪϩϪϩϩ species complex M19 ϪϪϪϪϪ M02 ϪϪϪϩϩ M37 ϪϪϪϩϩ M28 ϪϪϪϩϪ S. griseus ϩϪϩϩϩ M03 ϪϪϪϩϩ M04 ϪϪϪϩϩ M15 ϪϩϪϩϪ Actinoplanes sp. M32 ϪϪϪϩϩ Micromonospora spp. M42 ϪϪϪϪϩ M34 ϪϪϪϩϩ M40 ϩϪϪϪϪ M11 ϪϪϪϩϩ M13 ϪϪϪϩϩ M12 ϪϪϪϩϩ

Antibiotic activity was determined using a modiÞed agar cross-streak assay on 50% LuriaÐBertani (Miller) agar plates (n ϭ 3). Results for aknownantibioticproducingstrainofStreptomyces griseus are included for comparison (n ϭ 8). Strains are arranged as they appear on the phylogenetic tree (Fig. 2). a There was a Ն98% 16S rRNA gene fragment sequence similarity. vealed that these actinomycete strains formed two though low sample sizes and the inability to detect well-supported clades. One cluster included strains unique, nonreplicate strains prohibited statistical test- exhibiting a high degree of 16S rRNA gene sequence ing of this hypothesis. Fifty-three percent of the similarity (98Ð100%) to a strain of S. griseus and iso- strains showed growth inhibition activity against at lates from other insect nest material (Fig. 2). This least one of the gram-positive strains in the panel includes Streptomyces sp. Ae32_2, which is an antifun- (Table 1). Notably, strains with a higher sequence gal producing actinomycete isolated from Panamanian similarity to S. griseus, Micromonospora spp., and A. leaf-cutter ant fungus garden nests of Acromyrmex deccanensis, inhibited the growth of the gram positives echinatior (Haeder et al. 2009) (Fig. 2). These strains in the panel (B. subtilis and S. aureus)(Table1). were isolated from four nests collected from all three Twenty percent of the conÞrmed actinomycete iso- locations sampled (Fig. 2). lates displayed antimicrobial activity against the gram Asecondclusterofactinomycetestrainswasiso- negatives included in the challenge panel (Table 1). lated from Þve P. dominulus nests, from all three lo- Six of the strains with a high degree of homology to cations sampled (Fig. 2). These strains had a high Streptomyces spp. and Micromonospora spp. inhibited degree of sequence similarity (Ն99%) to Streptomyces E. coli (Table 1). Only two strains (with a high ﬂavovirens, and to a second isolated ectosymbiont sequence similarity to S. griseus and a Micromono- cultured by Haeder et al. (2009)Ñ Streptomyces sp. spora sp.), out of the total tested, exhibited any Av25_5. Haeder et al. (2009) isolated this strain from growth inhibition of P. aeruginosa (Table 1). None the nest of the Panamanian leaf-cutter ant Acro- of the 30 isolates inhibited the growth of S. marc- myrmex volcanus (Fig. 2). escens (Table 1). Antimicrobial Activity. Of the 30 strains isolated and conÞrmed to be actinomycetes, 60% exhibited Discussion antimicrobial activity against one of the Þve challenge strains: E. coli, P. aeruginosa, S. marcescens, S. aureus, This is the Þrst assessment to reveal that diverse and B. subtilis. Antibiotic activity appeared to be pre- actinomycetes can be readily isolated from the nest dicted by phylogenetic relatedness (Table 1), al- material of a paper nest building vespid. The 30 se- 708 ENVIRONMENTAL ENTOMOLOGY Vol. 42, no. 4 quenced actinomycetes isolated from these nests be- The Streptomyces spp. portion of our isolates formed long to the two most bioactively-rich actinomycete two well-supported clusters in our phylogenetic tree, families: Streptomycetaceae and Micromonospo- with similar strains being isolated across nests and raceae (reviewed in Berdy 2005), including the three across P. dominulus populations (Fig. 2). Many of genera, Streptomyces, Micromonospora, and Actino- these strains produced antimicrobial activity in vitro planes (Fig. 2). While Streptomyces, and arguably Mi- (Table 1), and are similar to strains identiÞed in anal- cromonospora, are common soil microbes (Williams ogous screens of actinomycetes of various insectsÑ and Wellington 1982 in El-Tarabily and Sivasitham- such as the Streptomyces spp. identiÞed by Haeder et param 2006, but see review by Lazzarini et al. 2001), al. (2009) in association with leaf cutter ants (Fig. 2). Actinoplanes is a rare actinomycete genus (Yi-Quing et Therefore, it is tempting to speculate a Þtness rela- al. 2008). It is particularly surprising that we were able tionship between these microbes and the wasps, par- to isolate a strain of this genus, as we were not con- ticularly as paper wasps are expected to face a similar ducting a comprehensive study of the total culturable assortment of microbial pathogens to those insects in actinomycetes within this habitat. To the authorsÕ established symbioses (Jeanne 1996). However, it is knowledge, this is the Þrst strain of this genus isolated likely that at least some of the actinomycetes within from insect nest material. the P. dominulus nest are dormant spores. This ratio- Under our assay conditions, the antimicrobial ac- nale is based partially on the knowledge that nests of tivity among isolated strains was variable, even among P. dominulus are known to be collection points for closely related isolates (Table 1). This is consistent local heavy metal particles from combustion engines with other studies on intraspeciÞcdifferenceswithin (Urbini et al. 2006) and therefore may be traps for streptomycete species (AntonyÐBabu et al. 2008, aerially dispersed spores as well. Hulcr et al. 2011). Our isolates displayed predomi- While relatively little is known about how paper nately gram-positive antagonism; however, it is not wasps control nest hygiene (but see Turillazzi et al. clear if this activity arose from one, or multiple anti- 2004, Turillazzi 2006), Hoggard et al. (2011) found that microbial compounds. Streptomyces spp. are capable paper nest building wasps produce active cuticular of producing over 100,000 different antibiotics (Watve antimicrobial compounds. This may preclude the need et al. 2001), with some strains producing multiple for a symbiont to produce such compounds. Our data antimicrobials (Seipke et al. 2011b). This is true even appear to be consistent with other studies relating to under in vivo conditions, where a cocktail of antimi- actinomycetes of other insect nest substrates, suggest- crobials is expected to assist in nest hygiene (Kroiss et ing arthropod nest material is a habitat for culturable, al. 2010). Because of such complexity, further studies allochthonous actinomycetes that include Streptomy- relating to extracts from our isolates, produced under as well as other, more rare genera (e.g., Promnuan numerous culture conditions, would need to be con- ces ducted to understand the mechanism behind the bio- et al. 2009, Inglis et al. 1993, Haeder et al. 2009, Bignell activity displayed in our assays. et al. 1991), regardless of potential symbiotic associ- Most of the strains we isolated belong to the genus ations. Streptomyces, consistent with similar studies investi- Future studies characterizing the antimicrobial ac- gating nest-associated insect material. This includes tivity witnessed in our studies will be necessary to those studies relating to leaf-cutter ants (Haeder et al. determine if the actinomycetes isolated within this 2009, Barke et al. 2010, Zucchi et al. 2011, Kost et al. study produce novel chemistry. By further targeting 2007), wood boring beetles (Hulcr et al. 2011, Scott et the full diversity of the microbial community asso- al. 2008, Grubbs et al. 2011), honey and stingless bees ciated with these wasps, we will be able to better (Promnuan et al. 2009, Gilliam and Prest 1987), soli- understand how these wasps maintain nest hygiene, tary bees (Batra et al. 1973, Inglis et al. 1993), digger and what microbes may impact their Þtness. This wasps (Kaltenpoth et al. 2006), mud dauber wasps work is thus an important Þrst step in exploring a (Kumar et al. 2011a,b; Poulsen et al. 2011), and ter- new system for the study of arthropod associated mites (e.g., Bignell et al. 1991, Visser et al. 2012). actinomycetes. Furthermore, a study by Ruddick and Williams (1972), suggests that spores of Streptomyces spp. are associated with the cuticle of many arthropods. There- Acknowledgments fore, it is not surprising that strepomycetes are often found in nest material. The abundance of streptomycetes The authors would like to thank Eli Siegel of Tufts Uni- that we found may also be reßective of the fact that they versity for providing all challenge bacteria and the reference are abundant in many environmental habitatsÑinclud- strain of Streptomyces griseus.Thismanuscriptgreatlyben- ing plant material such as that used by P. dominulus to eÞted from the comments and suggestions of two anonymous reviewers. Funding was provided by a National Science construct nests (see references in Williams et al. 1989). Foundation Graduate Research Fellowship (NSF Division of It should be noted though, that the perceived unique Graduate Education 0806676), a Tufts Institute of the Envi- ubiquity of this genus in insect material when compared ronment Fellowship, and Tufts University Graduate Student with other actinomycetes may be a byproduct of stan- Research Awards to A.A.M, and an NSF Research Experience dard culturing practices, which select for their fast and for Undergraduates site award (NSF Division of Biological dominant growth (Manteca and Sanchez 2009). Infrastructure 0649190) to P.T.S. August 2013 MADDEN ET AL.: ACTINOMYCETE NEST ASSOCIATES OF PAPER WASPS 709

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