Discovery and Description of the Bacterial Associates of a Gregarious Riparian with Explosive Defensive Chemistry

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Authors McManus, Reilly

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DISCOVERY AND DESCRIPTION OF THE BACTERIAL ASSOCIATES OF A GREGARIOUS RIPARIAN BEETLE WITH EXPLOSIVE DEFENSIVE CHEMISTRY

by

Reilly McManus

______Copyright © Reilly McManus 2017

A Thesis Submitted to the Faculty of the

THE GRADUATE INTERDISCIPLINARY PROGRAM IN ENTOMOLOGY & INSECT SCIENCES

In Partial Fulfillment of the Requirements

For the Degree of

MASTER OF SCIENCE

In the Graduate College

THE UNIVERSITY OF ARIZONA

2017

STATEMENT BY AUTHOR

The thesis titled Discovery and Description of the Bacterial Associates of a Gregarious Riparian Beetle with Explosive Defensive Chemistry prepared by Reilly McManus has been submitted in partial fulfillment of requirements for a master’s degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this thesis are allowable without special permission, provided that an accurate acknowledgement of the source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED: Reilly McManus

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

July 27, 2017 Dr. Wendy Moore Date Associate Professor, Department of Entomology

2

Table of Contents

Abstract 5

Introduction 6

Materials and Methods 8

Results 13

Discussion 20

Supplementary Data 24

References 28

3

List of Tables and Figures

Tables

Table 1: 16S amplicon MiSeq samples (Brachinus elongatulus) 9

Table 2: PCR survey of Brachinus elongatulus with specific 11 primers

Table 3: Brachinus elongatulus transcriptome survey for Spiroplasma 12

Table 4: Taxonomic identities of 10 most abundant bacterial OTUs 16

Table 5: Identities of six core OTUs present in all individuals 17

Table S1: Accession numbers used to infer molecular phylogeny of 24 Spiroplasma

Figures

Figure 1: Collection locations of Brachinus elongatulus 8

Figure 2: Most abundant 14

Figure 3: Frequencies of bacterial phylotypes 15

Figure 4: Spiroplasma OTU abundance per individual 18

Figure 5: Inferred molecular phylogeny of Spiroplasma 19

4 ABSTRACT

Bombardier (Carabidae: Brachininae), well known for their unique explosive defensive chemistry, are found in riparian corridors throughout the American Southwest where they commonly form large diurnal aggregations in moist areas under rocks, in crevices, and in leaf litter. Using high throughput 16S amplicon sequencing, we provide the first microbiome survey of a bombardier beetle, Brachinus elongatulus. Results include the surprising discovery that this species has a core set of six bacterial phylotypes found in all 13 specimens collected from two sites in Arizona separated from one another by over 80 kilometers. The core microbiome includes three species of Lactobacillales, including representatives of Enterococcus and Weissella. Members of these genera have been implicated in the production of volatile carboxylic acids that act as fecal aggregation pheromone components, which regulate aggregation in the German cockroach, and they may also play a role in Brachinus aggregation behavior. Our results also show that some B. elongatulus individuals collected from both sites are heavily infected with another bacterium, Spiroplasma. Various species of this facultative endosymbiont have been shown to have either a commensal, mutualistic or pathogenic relationship with their host. Most interestingly, from the perspective of B. elongatulus natural history, some Spiroplasma species have been shown to provide their dipteran hosts defensive protection against nematode infection, which is also a constant threat for Brachinus species. Our findings reveal intriguing possibilities that may influence bombardier beetle behavior and physiology, and this research lays the groundwork for future experiments to directly assess these beetle-bacterial relationships.

5 INTRODUCTION

Members of the genus Brachinus (Coleoptera: Brachininae) belong to a group of carabids known as bombardier beetles, which produce a noxious exothermic defensive spray to deter their predators (Arndt el al. 2015). Adults are found in moist areas under rocks, in crevices and in leaf litter along riparian corridors in the United States, Mexico, and Europe. Females oviposit in the moist mud or gravel along the waters edge. After hatching, the triungulan larva seeks out a pupa of a water beetle (Hydrophilidae, Dysticidae, Gyrinidae) which they consume as they undergo full larval development (Erwin 1967, Saska 2004). As adults, Brachinus forage at night scavenging on decaying organic matter and acting as opportunistic predators before forming a new aggregation by dawn. Similar to the behavior of some of their gyrinid hosts, Brachinus do not return to their aggregation of origin after dispersing at night (Heinrich 1980, WM pers. obs.). Due to living in close proximity to riparian corridors, adult Brachinus have often been found to be infected with parasitic nematophoran and nematodes (RM and WM pers. obs.).

Brachinus are gregarious and commonly form diurnal multispecies aggregations. These multi-species aggregations contain not only members of their own genus, but may include members of other carabid genera as well, such as Agonum, Chlaenius, Platynus and Galerita (Schaller et al. in prep). The cues involved in the formation of these aggregations are unknown, but are thought to be tactile and/or chemical in nature. Multispecies aggregations are extremely rare among arthropods with only a few cases being reported in harvestmen (Archnida: Opiliones), net-winged beetles (Coleoptera: Lycidae) and whirligig beetles (Coleoptera: Gyrinidae) (Machado & Vasconcelos 1998, Eisner et al. 1962, Heinrich 1980).

Other gregarious insects, such as German cockroaches (Ectobiidae) and firebrats (Lepismatidae), have been shown to aggregate in response to volatile fecal pheromones produced by their gut microbiota (Woodbury & Gries 2013, Wada- Katsumata et al. 2015). Digestive microbes in termites have also been implicated in providing cues for nest mate recognition (Minkley 2006). Gregarious and social insects can horizontally transmit bacteria via feeding and defecating in common areas, but the direct transfer of gut bacteria among conspecific nonsocial insect hosts has not been well studied. To our knowledge, the transfer of bacteria among conspecific and heterospecific hosts in multispecies aggregations has yet to be studied. Brachinus would be model organisms to investigate this phenomenon. To date the role of microbiota in influencing Brachinus aggregation behavior is unknown. Sociality has been shown to play a role in insects having specialized communities of gut bacteria.

Bumblebees, honeybees, and fungus-growing are known to socially transmit microbes that aid in protection against parasites or fungal and bacterial pathogens (Koch 2011). Social and termites have gut communities dominated by specialized bacteria not present in their non-social contemporaries (Martinson et al. 2011, Koch 2011, Sabree et al 2012). Evaluating insect populations for the presence

6 of a core microbiome is often undertaken to identify key host/microbe associations. Microorganisms that are detected in all host individuals in a particular habitat may play an important role in the function, and/or protection of the host (Poulsen 2012, Otani et al. 2014, Benjamino 2016). It is also possible these shared microorganisms are artifacts due to the microbes being present in the environment. Cockroaches have been shown to have extensive core microbiomes that remain persistent even when dietary shifts occur (Tinker 2016, Schauer 2012). The hindguts of termites harbor complex symbiotic communities composed of bacteria, protists, and archaeans. The hindgut of Reticulitermes flavipes, for example, houses a large set of core microbes. Species of the most dominant genus Treponema are implicated in having an important impact on the physiology of the termite’s hindgut (Benjamino 2016).

Insect bacterial associates are often divided into three categories: obligate, facultative, and environment-associated endosymbionts. Obligate endosymbionts are mutualists that are required for the host insect’s survival. These endosymbionts are often vertically transmitted and are not viable outside of the insect host, such as Buchnera aphidicola, which is needed to produce essential amino acids for its aphid hosts (Moran et al. 2008). Facultative endosymbionts are not necessary for host survival or reproduction; therefore they may not be present in every individual in a population. Instead they utilize their hosts to increase and support their own viability and transmission. Facultative endosymbionts can have a wide range of fitness effects on the host ranging from parasitism to mutualism. Examples of facultative endosymbiotic bacteria include , Rickettsia, and Spiroplasma, among others, whose effects range from manipulating host reproduction to increasing host survival (Liberti et al. 2015). Environment-associated bacteria often have no discernable impact on the insect host and the community members vary depending upon the environment and/or diet of the host insect. A large-scale study of gut microbiota across 21 orders of insects found environment and diet had an impact on microbial communities found in insects, with omnivorous insects having significantly higher bacterial diversity when compared to carnivorous and herbivorous insects (Yun et al. 2014).

The present study is the first exploration into the bacterial associates of a bombardier beetle and the first survey of a carabid beetle microbiome using high throughput sequencing methods. Two previous studies have examined the bacterial communities present in the digestive tracts of three species of carabid beetles— Harpalus pensylvanicus, Anisodactylus sanctaecrucis (both of which specialize on eating seeds) and Poecilus chalcites, a predator of living arthropods. Both of these studies used using terminal restriction fragment length polymorphism (tRFLP) in tandem with 16S rRNA gene cloning (Lundgren 2007, Lehman 2009). The objectives of our study were to (1) characterize the microbial communities of B. elongatulus using high-throughput 16S amplicon sequencing, (2) determine if there is a core set of OTUs common across 13 individuals from two localities, (3) determine if B. elongatulus hosts any potential obligate or facultative endosymbionts and (4) lay the

7 groundwork for future investigations into how the microbiome of B. elongatulus might shape their behavior and/or physiology.

MATERIALS AND METHODS

Insect collection and dissections Brachinus elongatulus were field-collected from riparian areas in the Rincon Mountains (Happy Valley, AZ) and in the Santa Rita Mountains (Madera Canyon, AZ) (Figure 1). All specimens were kept alive and transferred back to the lab for dissections. All dissections were performed within 24 hours of collection. The specimens were preserved in 100% ethanol just prior to dissection. Specimens were surfaced sterilized using an 8% bleach solution followed by a 1 minute wash in phosphate-buffered saline (PBS) and a 1 minute wash in 100% ethanol. For eleven specimens, internal organs, excluding the crop, were dissected out of the abdomen using flame-sterilized forceps and flame-sterilized glass petri dishes. For two specimens the whole bodies, minus the wings and elytra, were processed. Dissected tissues and whole bodies were placed in individual 1.5mL Eppendorf tubes containing lysozyme buffer (180ul for tissues and 360ul for whole bodies: 20mM Tris-HCl (pH 8.0), 2mM EDTA, 1.2% Triton X) containing 10mg ml-1 of lysozyme.

Figure 1. Collection locations of B. elongatulus. Locations from which B. elongatulus sequences were used. MiSeq collections from Happy Valley and Madera Canyon. Transcriptome sequences from Madera Canyon. Spiroplasma sequences from all three locations.

DNA extraction, PCR amplification, and high-throughput sequencing For DNA extraction, tissues in lysozyme buffer were ground using sterile

8 DNase/RNase free pestles. Samples were then incubated at 37˚C for 90 minutes. DNA was precipitated and purified following the Qiagen Blood and Tissue Kit protocol for gram-positive bacteria. The bacterial hypervariable V4 of 16S rDNA was PCR amplified in duplicate using the primers 515F 5’- GTGCCAGCMGCCGCGGTAA-3) and 806R (5’- GGACTACHVGGGTWTCTAAT- 3’) modified with adaptors and unique barcodes for Illumina MiSeq sequencing (Caporaso et al. 2012). The thermocycler protocol (adapted from the Earth Microbiome Project protocol) was denaturation at 94°C for 3 minutes followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 50°C for 60 seconds, and extension at 72°C for 90 seconds, with a final extension of 72°C for 10 minutes (Gilbert et al. 2014).

Duplicate PCR products for each sample were merged and quantified using a Qubit fluorometer (Qiagen) and gel electrophoresis. Equimolar amounts of each sample were pooled and then purified using QIAquick PCR Purification Kit (Qiagen). The bacterial library was sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA, USA) at New York Medical College Genomics Core Laboratory (Valhalla, NY) with 250 x 2 chemistry.

Illumina sequence data processing Reads were demultiplexed and quality filtered using mothur v3 (Schloss 2009). Sequences were merged and clustered at a 97% pair-wise similarity cutoff with UPARSE (Edgar 2013). Singletons were removed and chimera checking was performed using default settings during clustering. 422 operational taxonomic units (OTUs) were generated. Bacterial assignments were performed using the RDP classifier (Wang et al. 2007) with the Greengenes database (McDonald et al. 2012). Taxonomic assignments were checked and revised by blasting the representative set of bacterial sequences against the NCBI nucleotide database (Benson et al. 2012). The R package “phyloseq” (McMurdie & Holmes 2013) was used to organize, visualize and analyze data. Since preliminary results did not show significant differences among tissues types, individuals dissected by tissue type, data for 3 or 5 tissue types (depending on the individual) were merged together in phyloseq (Table 1) to form a representative individual.

Table 1: 16S Amplicon MiSeq Samples (Brachinus elongatulus). Tissue composition, unique ID, sex, and collecting information for each representative individual.

Taxon ID Unique ID Body Part Sex Collecting Information Individual_1 DNA4248 Fat body Female USA: AZ: Pima County: Madera Canyon. DNA4249 MGMT 31.74038, - DNA4250 Ileum 110.887765, 1340m, DNA4251 Defensive 1 Sept 2016. R.

9 system McManus A. Yanahan A. Hoover R. Keating DNA4252 Ovaries Individual_2 DNA4253 Ovaries Female USA: AZ: Pima County: Madera Canyon. DNA4254 Ileum 31.74038, - DNA4255 MGMT 110.887765, 1340m, Defensive 1 Sept 2016. R. DNA4256 system McManus A. Yanahan DNA4257 Fat body A. Hoover R. Keating Individual_3 DNA4258 Ileum Female USA: AZ: Pima County: Defensive Madera Canyon. DNA4259 system 31.74038, - 110.887765, 1340m, DNA4260 MGMT 1 Sept 2016. R. DNA4261 Fat body McManus A. Yanahan DNA4262 Ovaries A. Hoover R. Keating Individual_4 DNA4274 MGMT Female USA: AZ: Pima County: Madera Canyon. DNA4275 Ileum 31.712994, - Defensive 110.873208, 1664m, DNA4276 system 9 Sept 2016. R. DNA4277 Ovaries McManus A. Yanahan DNA4278 Fat body W. Moore Individual_5 DNA4279 MGMT Female USA: AZ: Pima County: Madera Canyon. DNA4280 Ileum 31.712994, - Defensive 110.873208, 1664m, DNA4281 system 9 Sept 2016. R. DNA4282 Ovaries McManus A. Yanahan DNA4283 Fat body W. Moore Individual_6 Female USA: AZ: Pima County: DNA4284 MGMT Madera Canyon. DNA4285 Ileum 31.712994, - Defensive 110.873208, 1664m, DNA4286 system 9 Sept 2016. R. DNA4287 Ovaries McManus A. Yanahan W. Moore DNA4288 Fat body Individual_7 Whole body Female Same as Individual_4- DNA4289 6 Individual_8 Whole body Female Same as Individual_4- DNA4241 6 Individual_9 Male USA: AZ: Pima Co., DNA4228 MGMT

10 DNA4229 Ileum Turkey Creek Trail, Happy Valley, 32.15607 -110.47414, 1270m, 8 July 2016, J. DNA4230 Organs Schaller Individual_10 Male USA: AZ: Pima Co., DNA4231 MGMT Turkey Creek Trail, DNA4232 Ileum Happy Valley, 32.15607 -110.47414, 1270m, 8 July 2016, J. DNA4233 Organs Schaller Individual_11 Male USA: AZ: Pima Co., DNA4234 MGMT Turkey Creek Trail, DNA4235 Ileum Happy Valley, 32.15607 -110.47414, 1270m, 8 July 2016, J. DNA4236 Organs Schaller Individual_12 Female USA: AZ: Pima Co., DNA4237 MGMT Turkey Creek Trail, DNA4238 Ileum Happy Valley, 32.15607 -110.47414, 1270m, 8 July 2016, J. DNA4239 Organs Schaller Individual_13 Female USA: AZ: Pima Co., DNA4297 MGMT Turkey Creek Trail, DNA4298 Ileum Happy Valley, 32.15607 -110.47414, 1270m, 8 July 2016, J. DNA4299 Organs Schaller

Spiroplasma survey using PCR Thirty-one B. elongatulus DNA extractions from various tissue types, including muscle tissue, digestive tract, and other internal organs, see Table 2) were screened for Spiroplasma using Spiroplasma-diagnostic primers 23f (5’- CTCAGGATGAACGCTGGCGGCAT-3’) and TKSSsp (5’-TAGCCGTGGCTTTCTGGTAA-3’) producing a 410 base pair fragment aligning to the V1-V2 region of 16S rDNA. This primer pair was selected for its ability to amplify almost all Spiroplasma, including male-killing and non-male killing strains (Watts et al. 2009). However, these primers do have the potential to amplify other types of bacteria as well. The thermocycler protocol was denaturation at 94°C for 2 minutes followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 90 seconds, with a final extension of 72°C for 5 minutes. PCR products were sequenced with an ABI 3730 at the University of Arizona’s Genomics Analysis & Technology Core (Tucson, AZ).

11 Table 2: PCR Survey of Brachinus elongatulus with Spiroplasma specific primers. Specimens used in Spiroplasma PCR survey that sequenced successfully.

DNA number Body part Collecting Sex Information DNA4230 Internal organs, USA: AZ: Pima Co., Male excluding ileum, Turkey Creek Trail, midgut, and Happy Valley, malpighian tubules 32.15607 - 110.47414, 1270m, 8 July 2016, J. Schaller DNA4233 Internal organs, USA: AZ: Pima Co., Male excluding ileum, Turkey Creek Trail, midgut, and Happy Valley, malpighian tubules 32.15607 - 110.47414, 1270m, 8 July 2016, J. Schaller DNA2210 Right middle leg USA: AZ, Cochise Unknown Co. Chiricahua Mts., upper Turkey Creek 2036m31.85077N, 109.32567W30-31 May 2010 J. Schaller DNA4372 Right middle leg USA: AZ: Pima Male County: Madera Canyon. 31.74038, -110.887765, 1340m, 13 March 2017 R. McManus A. Hoover C. Langford DNA4283 Fat body USA: AZ: Pima Female County: Madera Canyon. 31.712994, - 110.873208, 1664m, 9 Sept 2016. R. McManus A. Yanahan W. Moore

12 DNA4375 Right middle leg USA: AZ: Pima Female County: Madera Canyon. 31.712994, - 110.873208, 1664m, 9 Sept 2016. R. McManus A. Yanahan W. Moore DNA4288 Fat body USA: AZ: Pima Female County: Madera Canyon. 31.712994, - 110.873208, 1664m, 9 Sept 2016. R. McManus A. Yanahan W. Moore DNA4287 Ovaries USA: AZ: Pima Female County: Madera Canyon. 31.712994, - 110.873208, 1664m, 9 Sept 2016. R. McManus A. Yanahan W. Moore DNA2174 Right middle leg USA: Arizona, Pima Unknown Co., Santa Rita Mts., Box Canyon, 31.7989N, 110.77713W, 1432m, 25 June 2009 D. Maddison, W.Moore, K. Kanda, B. Allin DNA2172 Right middle leg USA: Arizona, Unknown Santa Cruz Co., Pajarito Mts., Sycamore Canyon, 31.43194N, 111.18859W, 1191m, 13 March 2010 W.Moore, J. Schaller, B. Smith

13 DNA2173 Right middle leg USA: Arizona, Unknown Santa Cruz Co., Pajarito Mts., Sycamore Canyon, 31.43194N, 111.18859W, 1191m, 13 March 2010 W.Moore, J. Schaller, B. Smith

Spiroplasma survey of transcriptome data Eleven B. elongatulus transcriptomes (Table 3) from an ongoing project were surveyed for the presence of Spiroplasma reads. The transcriptomes were blasted against a local microbial 16S rRNA database in CLC Genomic Workbench v8.5.3. Transcripts that hit to Spiroplasma were extracted in their entirety and blasted against the NCBI nucleotide database (Benson et al. 2012) to confirm their classification. Seventeen transcripts were identified as Spiroplasma and ranged in length from 230-612bp. Each transcript aligned to a different region across the entirety of the 16S gene.

Table 3: Brachinus elongatulus Transcriptome Survey for Spiroplasma. Transcriptome samples used to survey for Spiroplasma.

RNA number Body part Collecting Sex Information RNA007 20 defensive gland San Pedro River Unknown cells for reservoir chamber pooled from 10 beetles

RNA012 Whole body Pena Blanca Unknown RNA033 19 or 20 defensive Madera Canyon Unknown gland cells for near Bog Springs reservoir chamber April 12, 2015 pooled from 10 beetles

RNA035 20 reaction Madera Canyon Unknown chambers pooled near Bog Springs from 10 beetles April 12, 2015

RNA036 9 beetle bodies (- Madera Canyon Unknown defensive glands) near Bog Springs April 12, 2015

14

RNA037 20 defensive gland Madera Canyon Unknown cells for reservoir near Bog Springs chamber pooled April 12, 2015 from 10 beetles

RNA039 20 reaction Madera Canyon Unknown chambers pooled near Bog Springs from 10 beetles April 12, 2015

RNA040 10 beetle bodies (- Madera Canyon Unknown defensive glands) near Bog Springs April 12, 2015

RNA041 20 defensive gland Madera Canyon Unknown cells for reservoir near Bog Springs chamber pooled April 12, 2015 from 10 beetles

RNA043 20 reaction Madera Canyon Unknown chambers pooled near Bog Springs from 10 beetles April 12, 2015

RNA044 10 beetle bodies (- Madera Canyon Unknown defensive glands) near Bog Springs April 12, 2015

Molecular phylogenetic inference of Spiroplasma To infer the evolutionary relationships of Spiroplasma, we used 16S rDNA to construct a molecular phylogeny. Representative sequences from each OTU identified as Spiroplasma with more than 150 reads from the MiSeq survey were used. All of the Spiroplasma sequences from the PCR and transcriptome surveys were used, 9 and 17 sequences respectively. Additional sequences were downloaded from the NCBI nucleotide database (Benson et al. 2012; see Supplementary Table 1 for accession numbers). The sequences were aligned using MAFFT v 7.310 (Katoh & Standley 2013) with default settings in Mesquite (Maddison & Maddison 2017). A maximum likelihood phylogeny was inferred using RAxML v8.2.8 (Stamatakis 2014).

RESULTS

Data summary of the MiSeq analysis A total of 4,859,442 16S rDNA V4 region gene sequences were obtained from 47 unique samples comprised of whole body individuals and multiple tissue types

15 dissected from individual specimens (Table 1). After quality control filtering 2,058,389 sequences remained with an average read length of 253 bases. Clustering with USEARCH (Edgar 2013) at 97% sequence similarity returned 422 bacterial OTUs with an average of 65 OTUs per sample. The 47 libraries ranged in size from 4,333 to 421,789 sequences. The samples were first rarified to 12604 sequences per tissue type. Tissue types from individual specimens that contained more than one tissue type were then merged to create a representative whole individual. A second rarefication was performed to create equal sequencing depth across the representative individuals. The final rarefication was to 12,604 sequences per individual. After which 234 bacterial OTUs remained across all 13 individuals with an average of 57 OTUs per sample.

Bacterial taxa associated with Brachinus elongatulus The 10 most abundant OTUs are listed in Table 4, each of these OTUs make up 2% or more of the total reads. A total of 13 bacterial phyla were detected. The most abundant bacterial phyla across all B. elongatulus individuals were (35%), (28%), (22%), Bacteriodetes (13%), and (2%) (Table 4, Figure 2). The most common genus within Tenericutes was Spiroplasma (94%). The most common orders within Proteobacteria were Pseudomonadales (8%), Enterobacteriales (7%), and Pasteurellales (6%). The most common orders within Firmicutes were Lactobacillales (17%) and Clostridales (4%). The most common orders within Bacteriodetes were Bacteroidales (10%) and Flavobacteriales (2%). The most common order within Fuscobacteria was Fusobacteriales (2%). The frequency of phylotypes across the 13 individuals, OTUs with a relative abundance of 5% or greater in at least one individual were plotted (Figure 3). Tenericutes were removed prior to the rarefication steps for this analysis to prevent relative abundances of the phylotypes from being significantly lowered due to the overabundance of Tenericutes reads in some individuals.

16

Figure 2. Most abundant bacterial phyla. Top 5 most abundant file from 2 collection sites (Madera Canyon and Happy Valley. Abundance shown as the percent of total reads per individual (1-13).

Core OTUs For a 16S OTU to be a member of the core microbiome, a stringent definition would require the presence of the OTU in 100% (13/13) of the beetles surveyed. Using this definition, six core phylotypes were found (Table 5) representing Firmicutes (4 phylotypes) and Proteobacteria (2 phylotypes). The core Firmicutes included three species of Lactobacillales, including representatives of the genera Enterococcus and Weissella. For the five beetles collected from Happy Valley, an additional OTU representing Firmicutes was present in all five individuals, but not detected in all the beetles collected in Madera Canyon. For the eight beetles collected in Madera Canyon, six additional OTUs, representing Firmicutes, Burkholderiales, Beikerinckiaceae, Dysgonomonas, Enterobacteriaceae, and Enterococcus, were present in all eight individuals, but not detected in all the beetles collected in Happy Valley.

17

Figure 3. Frequencies of bacterial phylotypes. Each row is an individual beetle and each column in a single bacterial phylotype. Phylotypes with a relative abundance of 5% or greater in any one individual beetle are included. Absence of a phylotype within a specimen is indicated by white. Relative abundance of a phylotype within a specimen ranges from light blue (>0 to less than 5%), dark blue (5% to less than 10%), purple (10% to less than 20%) to red (greater than 20%).

18

19

20 Prevalence of Spiroplasma in Brachinus elongatulus The most abundant phylum was Tenericutes with the genus Spiroplasma accounting for the most read counts across all the individuals (33%); Spiroplasma accounted for 20% or more of the total reads per individual in 9 out of 13 individuals. Spiroplasma was found in individuals from both Happy Valley and Madera Canyon, as well as in both male and female B. elongatulus (Figure 4). OTU1 and OTU5 were the most abundant OTUs classified as Spiroplasma.

Of the 31 B. elongatulus surveyed with Spiroplasma specific primers, 29 samples yielded bands and were sent for Sanger sequencing. Twelve samples produced clean sequences. Of those nine matched Spiroplasma (Table 2) and 3 matched to Streptococcus and Pseudomonas in BLAST searches. Sequences from 17 of the samples were too noisy to identify, suggesting that more than one type of bacteria was present (Spiroplasma or otherwise).

Figure 4. Spiroplasma OTU abundance per individual. Abundance of Spiroplasma shown as percent of total reads per individual (1-13). Males are identified with universal male symbol and specimens without the symbol are female.

Molecular phylogenetic inference of Spiroplasma Seventeen transcriptome sequences ranging in length from 230-612 bases, nine sequences of 410 bases from the Spiroplasma PCR survey, three 253 base pair OTU sequences identified as Spiroplasma and additional Tenericutes sequences from NCBI (Supplementary Table 1) were used to infer the evolutionary relationships our

21 Spiroplasma sequences (Figure 5). Spiroplasma sequences from all three surveys consistently fell out into two distinct - Ixodetis and Citri-Chrysipicola-Mirum (CCM). Ten Spiroplasma sequences, extracted from the B. elongatulus transcriptomes, fell out in the Ixodetis , while the seven other transcriptome sequences fell out in the CCM clade. Six Spiroplasma sequences from the PCR survey fell out in the Ixodetis clade, while the three other PCR survey sequences fell out in the CCM clade. From the MiSeq survey, one OTU (OTU1) fell out in the Ixodetis clade and one OTU (OTU5) fell out in the CCM clade. While it is clear that there are at least two species of Spiroplasma in these samples, it is not possible to determine exactly how many unique species are present without full 16S sequences. Spiroplasma sequences from the PCR survey (410bp) aligned to the V1-V2 region of 16S, while sequences from the MiSeq survey (253bp) aligned to the V4 region. The Spiroplasma sequences from the transcriptome survey (230-612bp) aligned to multiple regions across the entirety of the 16S gene. Therefore many of these sequences may represent the same species. Future work involving cloning will help to resolve this question.

Figure 5. Inferred Molecular Phylogeny of Spiroplasma (RAxML likelihood tree).

22 Spiroplasma sequences from MiSeq (250bp), PCR survey (410bp) and transcriptome survey (230-610bp) fall out in two clades (Ixodetis and Citri-Chrysipicola-Mirum). Sequences were aligned to full- length (~1500bp) 16S rDNA sequences downloaded from NCBI.

DISCUSSION

Our research shows that the microbiome of B. elongatulus (1) includes a core set of six bacterial phylotypes (2) includes the facultative endosymbiont Spiroplasma that may help protect them against nematode infections, and (3) several Lactobacillales species that may influence their aggregation behavior.

The top five most abundant bacterial phyla found in B. elongatulus were Proteobacteria, Firmicutes, , Tenericutes, and Fusobacteria (Figure 2). Proteobacteria, Firmicutes and Bacteroidetes are predominant bacterial phyla detected in gut samples and whole body samples across several families of Coleoptera (Jones 2013, Yun 2014). Fusobacteria have been reported in the digestive tract of predaceous carabid, P. chalcites (Lehman 2009). These types of bacteria have also been found to be common gut symbionts of carnivorous birds (Roggenbuck et al. 2014) and they are common hindgut symbionts of cockroaches (Schauer et al. 2014). They have also been found in low abundance in the midgut of several species of mosquitos (Ephantus et al. 2017) and in the guts of the dipteran Bactrocera minax (Wang 2014), although the function of these gut bacteria is not known. Spiroplasma, belonging to the phylum Tenericutes, are estimated to infect at least 7% of all insects (Duron et al. 2008). Spiroplasma have been found to have commensal, mutualistic or pathogenic relationships with their plant and arthropod hosts (e.g. crustaceans, arachnids, insects). Insects most commonly infected with Spiroplasma include species of Odonata, Hemiptera, Hymenoptera, Diptera, Lepidoptera and Coleoptera (Cisak et al. 2015). Different species of Spiroplasma have been shown (1) to be pathogens of honeybees (Schwarz et al. 2014), (2) to assist in reproductive manipulation in planthoppers and (Sanada- Morimura et al. 2013, Xie 2014), (3) to provide defensive protection against the sterilization affects of nematode infection in (Hamilton et al. 2016), and (4) to be commensals in a wide variety of insects, crustaceans, and arachnids (Cisak 2015).

Two previous studies (Lundgren 2007, Lehman 2009) have examined the microbial communities present in digestive tracts of three species of carabid beetles with two different dietary lifestyles: two granivores- Harpalus pensylvanucus and Ansiodactylus sanctaecrucis and one predator, Poecilus chalcites. All three species, as well as B. elongatulus, had an abundance of Proteobacteria. Weissella sp. was detected in all the carabid species except A. sanctaecrucis. Brachinus elongatulus and P. chalcites had the most similar bacterial community compositions with the most abundant OTUs being represented by Firmicutes, Fusobacteria, Proteobacteria and Bacteroidetes. The most abundant clones with finer taxonomic resolution match to Weissella sp. and two Enterococcus species; these genera were also detected in B.

23 elongatulus. The granivores were found to have lower microbial diversity overall with A. sanctaecrucis containing 3 OTUs, which were all represented by Proteobacteria. Harpalus pensylvanicus contained 6 OTUs represented by Proteobacteria, Firmicutes, and Spiroplasma. The B. elongatulus community composition is likely more similar to P. chalcites due to their predaceous diets. There may also be more similarities between the carabid microbial communities that were not detected, as the cloning technique is less comprehensive than the high throughput sequencing approach. Most insects host gut bacteria consisting of a few bacterial species with some insects hosting fewer than 10 species, such as honeybees, reed beetles and fruit flies (Engel 2013). Other insects, such as the cockroach and termite, regularly host over 1000 OTUs in their gut (Tinker 2016). With an average of 57 OTUs per sample, B. elongatulus has a low to moderate bacterial diversity.

The core microbiome of B. elongatulus consists of 6 OTUs that were present in all thirteen individuals examined, across both localities studied. These OTUs were represented by the Proteobacteria Orbales/Enterobacteriales, and the Firmicutes genera, Enterococcus sp. and Weissella sp (Lactobacillales). Bacteria belonging to the order Orbales have also been found in the guts of honeybees and (Kwong & Moran 2012), as well as in (Kim et al. 2013). The Enterobacteriales is sister to the order Orbales and has been has been found in the digestive tract of wood-boring beetles. The Enterobacteriales has metabolic capabilities, such as the fermentation of carbohydrates and nitrogen fixation (Rizzi et al. 2013). Enterococcus is a common inhabitant of the digestive tracts of mammals, birds, reptiles and insects. Enterococcus has been found across a broad range of insect orders with the most prevalent species being E. faecalis, E. faecium, and E. casseliflavus (Lebreton et al. 2014).

Both Enterococcus and Weissella belong to a group of bacteria known as lactic acid bacteria, which are obligatory heterofermenters. These bacteria ferment carbohydrates, which produce multiple end products such as CO2, volatile acids, and lactic acid (Fusco et al. 2015). These bacteria have been implicated in the production of volatile carboxylic acids (VCAs) that act as fecal aggregation pheromone components, which regulate aggregation in the German cockroach (Wada- Katsumata et al. 2015). Cockroach feces containing isolates and mixtures of Enterococcus avium, Weissella cibaria, and bacteria belonging to the genera Pseudomonas and Acinetobacter, were found to have a significant effect on German cockroach aggregation (when compared to axenic cockroach feces). , Enterobacter cloacae, and fungal isolate Mycotypha microspore were found to affect an aggregation response in firebrat, Thermobia domestica (Woodbury & Gries 2013). Core OTUs have been observed in other gregarious insects, such as the American cockroach which houses at least 67 core OTUs (Tinker 2016). The gregarious bed bug, Cimex lectularius, has a smaller core microbiome consisting of 2 OTUs, a highly abundant Wolbachia (Proteobacteria) and a less abundant and unnamed Gammaproteobacterium (Meriweather et al. 2013).

24

Core OTUs were dominant in some samples, but less common in other samples (Table 5, Figure 3). This may be due to the variety of tissue types used in this study, as well as the varying amount of tissue used. Although the samples were rarified to the same sequencing depth, there was a large range in the sequencing depth of the tissues (4,333 to 421,789 sequences). Due to this range it is also possible that the absence of an OTU or low read counts could be an artifact of varying sequencing depth.

It is difficult to resolve how many species of Spiroplamsa were present in the samples due to the short sequence fragments aligning to various regions across the 16S gene. Sequences from all three surveys fell out into two separate clades (Ixodetis and CCM), therefore it appears that at least two species of Spiroplasma are present. Future sequencing of full length Spiroplasma 16S rDNA would help resolve this issue. It was not possible to derive the function of these Spiroplasma based on their positions in the phylogeny. The CCM clade houses plant and crustacean pathogens, male-killing species of dipteran hosts, as well as a recently identified species that encodes a ribosome-inactivating protein (RIP) that has defensive effects against nematode infection in Drosophila neotestacea (Cisak et al. 2015, Hamilton et al. 2016). The Ixodetis clade houses male-killing species of coleopteran, dipteran and lepidopteran hosts. Both clades also house a suite of commensal species and species with unknown function (DiBlasi et al. 2011).

Due to the presence of both predominant Spiroplasma OTUs in male and female B. elongatulus, we hypothesize these Spiroplasma species do not have reproductive manipulation affects at least via male killing. More in-depth studies would need to be performed to see if parthenogenesis, feminization and/or cytoplasmic incompatibility (CI) could potentially be involved. B. elongatulus live in riparian areas and are commonly infected with nematophorans and nematodes. Further investigation is needed to determine if at least one of the species of Spiroplasma has defensive mechanisms to protect against helminth parasite infection. Transcriptomes were examined for the RIP domains found in the D. neotestacea strain, but yielded no results. This could be because the domains are not present or skewed due to the beetle RNA being optimized for eukaryotic RNA prior to sequencing. A recent study of the digestive tract of Tenebrio molitor (Coleoptera: Tenebrionidae) larvae using culture-dependent methods and denaturing gradient gel electrophoresis (DGGE), detected the presence of Weissella, Enterococcus, Clostridia, and a high abundance of Spiroplasma (Wang & Zhang 2015). In B. elongatulus, we hypothesize that at least one species of Spiroplasma is an intracellular symbiont because it was successfully amplified from the muscle tissue an individual leg; perhaps another species of Spiroplasma inhabits the digestive tract. Co-infection by two or more species of Spiroplasma in arthropods hosts has rarely been reported. Two pathogenic species of Spiroplasma belonging to the Apis clade, S. apis and S. melliferum, have been found to co-infect honeybee colonies in North and South America (Schwarz et al. 2014).

25 This study paves the way for further investigations into the roles of microorganisms in the behavior, immune function, and physiology of B. elongatulus. To resolve the number of Spiroplasma infecting B. elongatulus and to derive their function, future experimentation culturing the bacteria and sequencing their genomes may provide important insight. Microbial cultures of Brachinus feces, treatment of beetles with antibiotics and follow-up behavioral studies would also provide insight into the potential roles bacteria may be playing in Brachinus aggregation behavior.

26 Supplementary Data

Supplementary Table 1: Accession numbers used to infer molecular phylogeny of Spiroplasma Accession numbers from NCBI of the 16S rDNA sequences included in tree

Clade Taxon Accession # Outgroup Esherichia coli HQ286917 Bacillus subtilis HQ228563 Erysipelothrix sp EF050047 Mycoides- neurolyticum M23944 Entomoplasmataceae (ME) Mycoplasma sualvi M23936

Mycoplasma pulmonis M23941

Mycoplasma hominis M24473

Mycoplasma synoviae X52083

Entomoplasma freundtii AF036954

Mesoplasma lactucae AF303132

Mycoplasma sp. M24478

Mesoplasma entomophilum M23931

Mycoplasma ellychnium M24292

Mycoplasma mycoides U26039

Mycoplasma mycoides GQ409972

Mycoplasma capricolum U26047

Mycoplasma putrefaciens NR_025971

Mycoplasma putrefaciens M23938

Mycoplasma yeatsii NR_026037

Mycoplasma cottewii NR_026036

Mycoplasma cottewii U67945

27

Mycoplasma yeatsii U67946

Apis S. culicicola (Diptera) NR_025701

S. chinense (plants) NR_025698

S. velocicrescens AY189311 (Hymenoptera)

S. velocicrescens NR_025713 (Hymenoptera)

S. diabroticae (Coleoptera) GU908490

S. monobiae (Hymenoptera) GU585673

S. cantharicola (Coleoptera) DQ861914

Spiroplasma sp. (plants) EF151267

S. tabanidicola (Diptera) GU585670

S. lineolae (Diptera) DQ860100

S. gladiatoris (Diptera) M24475

S. litorale (Diptera) GU908489

S. turonicum (Diptera) NR_025712

S. corruscae (Coleoptera) NR_025700

S. apis (Hymenoptera) GU993267

S. montanense (Diptera) NR_025709

S. taiwanense (Diptera) HM037992

S. leptinotarasae AY189305 (Coleoptera)

S. alleghenen (Mecoptera) AY189125

S. sabaudiense (Diptera) AY189308

28

Citri-Chrysopicola-Mirum Procambarus clarkia DQ917754 (CCM) (Decapoda)*

Eriocheir sinensis DQ917753 (Decapoda)*

Panaeus vannamei DQ917755 (Decapoda)*

S. mirum (Decapoda) DQ917756

Tabanidae sp. (Diptera)* EF491664

Tababus atratus (Diptera)* AY189314

Haematopota sp. (Diptera)* EF491665

S. chrysopicola (Diptera) NR_025699

S. syrphidicola (Diptera) NR_025711

S.citri (plants) M23942

Spinturnix sp. DQ28984 (Mesostigmata)*

S. phoeniceum (plants) AY772395

S. kunkelii (plants) GU562447

Drosophila mojavensis FJ657217 (Diptera)

Drosophila mojavensis FJ657222 (Diptera)

Drosophila hydei (Diptera) FJ657240

Drosophila wheeleri FJ657225 (Diptera)

Drosophila aldrichi FJ657236 (Diptera)

29 Hippoboscoidea, Streblidae JF266584 (Diptera)*

Hippoboscoidea, Streblidae JF266586 (Diptera)*

S. melliferum NR_025756

S. insolitum NR_025705

Drosophila willistoni M24483 (Diptera)*

Drosophila simulans FJ657181 (Diptera)*

Ixodetis Laelapidae mite JF266582 (Mesostigmata)*

Neriene clathrata EU727102 (Araneae)*

Tetragnatha montana EU727105 (Araneae)*

Meta segmentata EU727104 (Araneae)*

Meta mengei (Araneae)* EU727103

Hepialus gonggaensis EU344951 (Lepidoptera)*

Araneus diadematus EU727099 (Araneae)*

Chrysolina varians EU727100 (Coleoptera)*

Adalia bipunctata AJ006775 (Coleoptera)*

S. ixodetis (Ixodida) GU585671

Laodelphax striatellus AB553862

30 (Hemiptera)*

Antonina crawii AB030022 (Hemiptera)*

Agathemera claraziana JF266577 (Phasmatodea)* (A33 gut)

Drosophila ananassae FJ657247 (Diptera)*

Ctenocephalides felis EF121346 (Siphonaptera)*

Anisosticta AM087471 novemdecimpunctata (Coleoptera)*

Fannia manicata (Diptera)* AY569829

Drosophila atripex FJ657246 (Diptera)*

Drosophila ananassae FJ657248 (Diptera)*

Notostira elongata EU727098 (Hemiptera)*

environmental sample* AY837732

Anopheles sp. (Diptera)* AY837733

Anopheles sp. (Diptera)* AY837731

Ostrinia zaguliaevi AB542741 (Lepidoptera)*

unidentified spider JF266583 (Araneae)*

unidentified spider JF266585 (Araneae)*

31 Tipula oleracea (Diptera)* EU727101

Drosophila tenebrosa FJ657245 (Diptera)*

S. platyhelix (Odonata) DQ860101

S. platyhelix (Odonata) GU993266

Trachymyrmex jamaicensis GQ275127 (Hymenoptera)*

Vollenhovia sp. GQ275136 (Hymenoptera)*

Kerria lacca (Hemiptera)* GU129148

Leptus sayi JF266580 (Trombidiformes)* (Host A33)

Leptus sayi JF266581 (Trombidiformes)* (Host A78)

Agathemera claraziana JF266579 (Phasmatodea)* (A78 gut)

Leptus lomani JF266588 (Trombidiformes)* (Host A39)

Leptus lomani JF266587 (Trombidiformes)* (Host A39)

Agathemera millipunctata JF266578 (Phasmatodea)* (A39 muscle)

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