The bacterial community in the gut of the cockroach 1

Shelfordella lateralis reflects the close evolutionary 2

relatedness of cockroaches and termites 3

4

Christine Schauer, Claire L. Thompson, Andreas Brune* 5

Department of Biogeochemistry, Max Planck Institute for Terrestrial Microbiology, 6

Karl-von-Frisch-Str. 10, 35043 Marburg, Germany 7

*Author for correspondence 8

Phone: +49-6421-178-701 9

Fax: +49-6421-178-709 10

Email: brune@mpi-marburg.mpg.de 11

Running title: Bacterial diversity in the cockroach gut 12

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.07788-11 AEM Accepts, published online ahead of print on 10 February 2012

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 2

Abstract

Termites and cockroaches are closely related, with molecular phylogenetic analyses 13

even placing termites within the radiation of cockroaches. The intestinal tract of 14

wood-feeding termites harbors a remarkably diverse microbial community that is 15

essential for the digestion of lignocellulose. However, surprisingly little is known 16

about the gut microbiota of their closest relatives, the omnivorous cockroaches. Here, 17

we present a combined characterization of physiological parameters, metabolic 18

activities, and bacterial microbiota in the gut of Shelfordella lateralis, a representative 19

of the cockroach family Blattidae, the sister group of termites. We compared the 20

bacterial communities within each gut compartment using terminal-restriction 21

fragment length polymorphism (T-RFLP) analysis and made a 16S rRNA gene clone 22

library of the microbiota in the colon—the dilated part of the hindgut with the highest 23

density and diversity of bacteria. The colonic community was dominated by members 24

of the Bacteroidetes, Firmicutes (mainly Clostridia), and some Deltaproteobacteria. 25

Spirochaetes and Fibrobacteres, which are abundant members of termite gut 26

communities, were conspicuously absent. Nevertheless, detailed phylogenetic 27

analysis revealed that many of the clones from the cockroach colon clustered with 28

sequences previously obtained from the termite gut, which indicated that the 29

composition of the bacterial community reflects at least in part the phylogeny of the 30

host. 31

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 3

Introduction

Termites and cockroaches share a close common ancestor. Although they are 32

traditionally considered separate insect orders, recent molecular phylogenetic 33

analyses revealed that termites (Isoptera) and cockroach family Blattidae are sister 34

groups (e.g., 38, 27, 71). Since termites fall within the radiation of cockroaches 35

(Blattodea) – a fact that is not yet reflected in the current taxonomy –, the 36

cockroaches are rendered paraphyletic and termites should be considered merely a 37

family of social cockroaches (for a review, see 70). 38

There are, however, fundamental differences in the diet of termites and cockroaches. 39

While termites feed almost exclusively on lignocellulose in various stages of decay, 40

many cockroaches subsist on a highly variable diet. The ability of termites to digest 41

lignocellulose is attributed largely to the metabolic activities of their gut microbiota 42

(14), and both structure and diversity the intestinal microbial communities have been 43

examined for representatives of all major feeding guilds (11, 49). However, an in-44

depth analysis of the gut microbiota of an omnivorous cockroach is so far lacking. 45

The only cockroach whose gut microbiota has been characterized with cultivation-46

independent molecular methods is the wood-feeding Cryptocercus punctulatus (1, 47

17), which – based on its phylogenetic position, xylophagous lifestyle, and the 48

presence of gut flagellates otherwise restricted to termites – can be actually regarded 49

as the most primitive termite (38, 40, 51). 50

The limited current knowledge of the gut microbiota of omnivorous cockroaches 51

comes from older, cultivation-based studies of mostly three species: Periplaneta 52

americana and Blatta orientalis (Blattidae), and Eublaberus posticus (Blaberidae) 53

(e.g. 15, 21, 22), yielded isolates of the genera Enterobacter, Klebsiella, and 54

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 4

Citrobacter (from the midgut), and Clostridium, Fusobacterium, Bacteroides, 55

Serratia, and Streptococcus (from the hindgut). A similar inventory of isolates has 56

been obtained also in earlier cultivation-based studies of termite guts (e.g., 60, 65). 57

However, cultivation-independent studies of the termite gut microbiota have since 58

shown that these genera represent at most only small populations (see 9). Here, we 59

used a cultivation-independent approach to examine the gut microbiota of 60

Shelfordella lateralis, a member of the cockroach family Blattidae, and a close but 61

less invasive relative of the pest species P. americana and B. orientalis. We 62

characterized the gut physiochemical parameters of this cockroach and conducted a 63

detailed analysis of the structure, phylogeny and metabolic activities of the bacterial 64

community in the colon, the gut compartment with the highest bacterial density and 65

diversity. The objectives of the study were to analyze the bacterial gut microbiota of 66

an omnivorous cockroach and to test the hypothesis that host phylogeny, particularly 67

in closely related taxa, is reflected in the structure of the gut microbial community. 68 on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 5

Materials and Methods

Cockroaches and dissection. Shelfordella lateralis was obtained from a commercial 69

breeder (J. Bernhard, Helbigsdorf, Germany) and maintained on a diet of chicken feed 70

(Gold Plus, Versele-Laga, Deinze, Belgium) at 25 °C and 50% humidity with a 12 h 71

light-dark cycle. The gut was dissected, the fat body was removed, and the weight of 72

the whole gut was recorded. For analysis of individual compartments, the gut was 73

divided into several sections: crop (with gizzard), midgut (gastric caeca were 74

analyzed separately for community structure), colon, and rectum. All guts used for the 75

T-RFLP profiles and clone libraries were sampled within a period of 2 days. Adult 76

females were used for all experiments to exclude factors like developmental stage and 77

sex that potentially add to individual variability. 78

Gut metabolites. Metabolites within the different gut compartments were measured 79

by high-pressure liquid chromatography (HPLC). Each gut compartment was 80

homogenized in 200 μl water and centrifuged for 10 min at 20,000 × g. The 81

supernatant was acidified with 1 volume 100 mM H2SO4 and filtered (0.2 µm, ReZist, 82

Whatman). Glucose and microbial fermentation products were quantified by ion-83

exclusion chromatography using an HLPC system equipped with a Grom Resin IEX 84

column (8 µm, 250 × 4.6 mm i.d., Grom, Rottenburg, Germany) and a refractive 85

index detector (RID-10A, Shimadzu) with a mobile phase of 5 mM H2SO4 and a 86

column temperature of 60 °C. Peak identity was verified using external standards. The 87

presence of glucose was confirmed using a glucose oxidase assay kit (Sigma Aldrich). 88

Prior to the assay, samples were deproteinized according to the protocol of Zeidler et 89

al. (74). 90

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 6

Microsensor measurements. Oxygen and hydrogen concentrations, pH, and redox 91

potential within the gut of S. lateralis were measured using microsensors (Unisense, 92

Aarhus, Denmark). Oxygen (Ox-10) microsensors had tip diameters of 10 µm and 93

were calibrated in Ringer’s solution at oxygen partial pressures of 0 and 21 kPa as 94

previously described (12). Hydrogen microsensors (H2-50) had tip diameters of 50 95

µm and were calibrated in Ringer’s solution at hydrogen partial pressures ranging 96

from 0 to 100 kPa (28). The redox microelectrode (RD-10) had a tip diameter of 10 97

µm and was calibrated using saturated quinhydrone solutions in pH standard solutions 98

of pH 4.0 and 7.0 (28); the pH microelectrode (PH-50) had a tip diameter of 50 µm 99

and a sensitive tip length of 200–300 µm and was calibrated using standard solutions 100

of pH 4.0, 7.0, and 9.0 (13). In both cases, the electrode potentials were measured 101

against Ag–AgCl reference electrodes using a high-impedance voltmeter (Ri > 1014 102

Ω). 103

Microsensor measurements were done in glass-faced microchambers using the 104

general setup as previously described (12). The dissected guts were fixed with insect 105

pins onto a bottom layer of 2% agarose and irrigated with air-saturated Ringer’s 106

solution using a peristaltic pump (16). For pH measurements, the guts were dissected 107

and immediately embedded in Ringer’s solution solidified with 0.5% agarose. 108

DNA extraction of different gut compartments. Prior to DNA extraction, each gut 109

compartment was frozen in liquid nitrogen and homogenized in 0.75 ml sodium 110

phosphate buffer (200 mM, pH 8.0). DNA was prepared using a bead-beating 111

protocol combined with phenol–chloroform extraction and ethanol precipitation as 112

previously described (36); extraction and precipitation was repeated twice to remove 113

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 7

substances inhibiting PCR. DNA was dissolved in 10 mM Tris buffer (pH 8) and 114

stored at –20 °C. 115

Microbial cell counts. Microbial cells were counted as previously described (59). 116

Briefly, gut contents were diluted 1:100 in phosphate-buffered saline (pH 7.2), stained 117

with 4',6-diamidino-2-phenylindole (DAPI), and applied to polycarbonate filters (0.2 118

μm, GTTP, Millipore) using a vacuum pump. For quantification, each filter was 119

divided into quarters, and five fields per quarter were counted using an 120

epifluorescence microscope (Axiophot, Zeiss). 121

T-RFLP analysis. T-RFLP profiles of 16S rRNA genes were generated as previously 122

described (30). Briefly, 16S rRNA genes were amplified using universal bacterial 123

primers: a 6-carboxyfluorescein-labeled forward primer (27f: 124

GAGTTTG(AC)TCCTGGCTCAG; 41) and an unlabelled reverse primer (907r; 125

CCGTCAATTCCTTT(AG)AGTTT; 46). Following digestion with MspI, samples 126

were analyzed on an automatic sequence analyzer (ABI 3130, Applied Biosystems, 127

Carlsbad, Calif., USA). All samples were analyzed in triplicate. The percentage peak 128

area was calculated for each terminal-restriction fragment (T-RF). T-RFs with a 129

relative height of < 1% of the total peak height were excluded. Bacterial phylotype 130

richness was expressed as the total number of peaks within each profile. Diversity and 131

community similarity were assessed by calculating the Shannon index (61) and the 132

Morisita-Horn index (34). Evenness was assessed using the Pielou index (54). 133

Nonmetric multidimensional scaling (NMDS) analysis was performed using R 134

(version 2.10; 56) and the VEGAN software package (26). Reproducibility was 135

determined to exclude that measured differences in the T-RFLP profiles were due to 136

technical artifacts. Samples that were analyzed in triplicate had a Morisita-Horn 137

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 8

similarity of 0.92 ± 0.08, which indicated that profiles were reproducible and reflected 138

the bacterial gut composition. 139

Clone library of bacterial 16S rRNA genes. A clone library was constructed from 140

colon DNA pooled from six adult female cockroaches. 16S rRNA genes were 141

amplified using the universal bacterial primers, 27f and 1492r 142

(TACGG(CT)TACCTTGTTACGACTT; 41), and the PCR products were purified 143

and cloned following the protocol described by Strassert et al. (64). Clones were 144

screened for the correct insert size and inserts were sequenced using vector primers 145

(GATC Biotech, Konstanz, Germany). 16S rRNA gene sequences were aligned with 146

the SINA aligner tool and imported into the SILVA database (release 102; 55) using 147

the ARB software package (version 5.1; 45). Phylogenetic trees were calculated from 148

almost-full-length sequences (>1400 bp) using RAxML, a maximum likelihood 149

method implemented in ARB (63). Chimeric sequences were identified using 150

Bellerophon (35) and ‘fractional treeing’ (44). Tree topology was tested using 151

RAxML and maximum-parsimony analysis (DNAPARS) implemented in ARB (100 152

or 1000 bootstraps, respectively). Rarefaction analysis was performed using 153

MOTHUR (version 1.23; 58) 154

Nucleotide sequence accession numbers. 16S rRNA gene sequences of clones were 155

deposited in Genbank under the accession numbers JN680560– JN680689. 156

Results

Gut structure. The intestinal tract of S. lateralis consists of several morphologically 157

distinct gut compartments (Fig. 1) and closely resembles in size and morphology the 158

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 9

gut of its close relative Periplaneta americana (6). The major gut compartments are 159

formed by a large crop, including a chitinized gizzard, a tubular midgut carrying a 160

crown of gastric caeca, and a hindgut composed of an enlarged colon followed by the 161

rectum. On the standard diet used in this study, gut weight contributed about 13% to 162

the total weight of the insect (597 ± 110 mg, n = 30), with the colon weighing slightly 163

less than the crop and the midgut (Table 1). 164

Physicochemical conditions. Axial profiles of redox potential in intact guts irrigated 165

with Ringer’s solution indicated reducing conditions at the center of all compartments 166

(Fig. 1). This was in agreement with the oxygen microsensor measurements, which 167

revealed that these regions were completely anoxic (not shown). Most profiles 168

showed only low concentrations of hydrogen throughout the entire gut, but in three of 169

eight individuals, hydrogen strongly accumulated in the midgut and hindgut with up 170

to 23 kPa in the anterior colon (Fig. 1). Gut pH increased only slightly from proximal 171

(pH 5.8) to distal compartments (pH 6.8), with the highest variability in the colon 172

(Fig. 1). 173

Metabolite pools. The metabolite pools in the different gut compartments strongly 174

differed (Fig. 2). Glucose was the most abundant metabolite in the crop and decreased 175

in the posterior sections, with lowest values found in the colon. Acetate was the 176

prominent fermentation product in all compartments. Other products were distributed 177

unevenly. Lactate was the second most abundant product in the crop and the midgut, 178

but it was only of minor importance in the hindgut compartments. The second most 179

abundant product in both colon and rectum was propionate, which was restricted to 180

these hindgut regions. Small amounts of succinate were present in the crop and the 181

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 10

midgut. Substantial amounts of ethanol were found in the crop, but only in half of the 182

animals tested (Fig. 2). 183

Bacterial diversity in different compartments. Each gut compartment contained 184

high numbers of microorganisms (1.5–5.6 × 107 cells), with the highest density in the 185

colon (Table 1). T-RFLP profiles of gut homogenates of four individuals yielded a 186

total of 184 distinct T-RFs, with phylotype richness varying considerably between 187

different animals and gut compartments (Table 1). The highest number of T-RFs was 188

present in the hindgut profiles (Table 1; Fig. 3). The colon and the rectum also 189

displayed a high evenness of T-RF distribution, but similarity indices among 190

individuals were low. Conversely, the crop, gastric caeca, and midgut showed less 191

diversity and lower evenness but a higher similarity among individuals. Although not 192

all T-RFs were represented in each individual (Table 1), a small number of T-RFs 193

were common in the profiles of the crop, midgut, and colon (Fig. 3). A comparison of 194

the community structure using nonmetric multidimensional scaling (NMDS) showed 195

that profiles of the midgut and gastric caeca clustered and were closer to the profiles 196

of the crop. The profiles of the colon clustered tightly and overlapped with those of 197

the rectum. The hindgut profiles were well separated from the foregut and midgut 198

profiles (Fig. 4). 199

Bacterial community of the colon. We pooled the colon samples of six 200

representative cockroaches (Fig. S1) and constructed a clone library of 16S rRNA 201

genes. A total of 265 randomly selected clones were sequenced; 14 were putative 202

chimerae and were excluded from further analysis. The remaining clones were 203

assigned to 132 different phylotypes (> 97% sequence similarity). Phylogenetic 204

analysis revealed that the clones represented 11 bacterial phyla. Almost half of clones 205

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 11

belonged to Bacteroidetes (49 phylotypes), followed by Firmicutes (58 phylotypes, 206

mostly Clostridia), and diverse Proteobacteria (12 phylotypes). Three phylotypes 207

clustered among the Planctomycetes, two each among Deferribacteres, Elusimicrobia, 208

Actinobacteria, and only a single phylotype each among Fusobacteria, Chloroflexi, 209

Synergistetes, and the candidate division TM7. Members of the Spirochaetes and 210

Fibrobacteres, which are consistently found in the gut of termites, were absent from 211

the clone library. Rarefaction analysis indicated that the number of clones analyzed 212

was not sufficient to describe bacterial diversity within the cockroach gut at either 213

genus (95% sequence similarity) or species level (97% sequences similarity) (Fig. S2): 214

However, reasonable coverage was obtained at a sequence similarity threshold of 215

90%, indicating that the most abundant groups within the six individuals sampled 216

were adequately covered. 217

Clones assigned to Bacteroidetes. The 120 clones assigned to Bacteroidetes fell 218

mostly within the order Bacteroidales (Fig. 5). The most abundant phylotype was 219

SL41 (5.2% of the library), which clustered together with SL35 (3.2%) and other 220

phylotypes among a group consisting exclusively of termite and Cryptocercus clones 221

in the Termite Group IV of Bacteroidales. They represented a total of 28 clones in the 222

library and were distantly related to bacteria in the genus Parabacteroides (92–94% 223

sequence similarity to Parabacteroides distasonis from the mammalian gut). Another 224

abundant group, represented by SL14 (4.0%), was loosely affiliated with clones from 225

other intestinal sources, including Bacteroides cellulosilyticus (90–94% sequence 226

similarity). Several phylotypes, with SL20 being the most abundant, formed a large 227

cluster with clones in Cluster V of Bacteroidales (4.4% of clones), which consists 228

exclusively of clones from termites and Cryptocercus punctulatus, many of which 229

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 12

represent symbionts of gut flagellates from Candidatus Azobacteroides 230

pseudotrichonymphae (33). Phylotypes SL48–SL51 clustered with sequences 231

originating from the guts of higher termites within Bacteroidales Cluster I (50), 232

distantly related (91–93% sequence identity) to bacteria in the genus Alistipes. 233

Numerous phylotypes represented minor clusters affiliated with the genera 234

Dysgonomonas and Tannerella, and with other clones or isolates from human or 235

animal intestines, often with clones from termite guts as closest relatives. The clone 236

library also contained three clones (phylotype SL65) affiliated with Blattabacterium 237

sequences—obligate endosymbionts present in all cockroaches (24)—with the 238

sequence from Blatta orientalis as closest relative. 239

Clones assigned to Firmicutes. The majority of the 95 clones that belonged to 240

Firmicutes fell into the class Clostridia (Fig. 6). The most abundant group was 241

represented by phylotypes SL84–SL90 (5.6% of the library), which clustered with 242

uncultivated members of Clostridiaceae from termite, ruminant, and mammalian guts. 243

Within the family Lachnospiraceae, two phylotypes (SL82 and SL83, 2.8%) were 244

affiliated with Clostridium piliforme and clustered with sequences from termite, 245

millipede, and scarab beetle guts. Five phylotypes (SL76–SL80, 2%) formed a cluster 246

with Clostridium hylemonae as their closest relative and several clones from the gut 247

of scarab beetle larvae (Pachnoda epphipiata). Within the family Ruminococcaeceae, 248

the most abundant cluster consisted of phylotypes SL99–SL104 (4.4%), which were 249

affiliated with Clostridium leptum (88–90% similarity) and sequences from the 250

mammalian gut. Within the family Veillonellaceae, two phylotypes, SL128 and 251

SL129 (2%), were neighbored by sequences from higher termites and a ground beetle, 252

with Succinispira mobilis as their closest relative. Five phylotypes (SL117–SL121, 253

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 13

2.8%) belonged to the Enterococcaceae, with Enterococcus asini as the closest 254

cultivated relative. Phylotypes SL122 and SL124, and phylotype SL125 formed two 255

clusters (2% total) within the Erysipelotrichi, with Erysipelothrix inopinata as their 256

next cultivated relative. 257

Clones assigned to other phyla. The majority of the remaining clones (5.6% of the 258

library) belonged to the phylum Proteobacteria. Most of the clones were 259

Deltaproteobacteria of the family Desulfovibrionaceae and clustered with sequences 260

previously obtained from the termite gut (Fig. 7). One clone affiliated with 261

Gammaproteobacteria had 99% sequence similarity to Escherichia blattae, a species 262

previously isolated from the cockroach gut (15). 263

Other phyla were only scarcely represented. Phylotype SL13 (2%) belonged to the 264

phylum Fusobacteria and showed > 99% sequence similarity with Fusobacterium 265

varium. Phylotypes SL66–SL68 (1.6%) belonged to an uncultured cluster of 266

Planctomycetes from the termite gut. Two clones (phylotypes SL69 and SL70) were 267

assigned to Deferribacteres, with Mucispirillum schaedleri as next cultivated 268

neighbor. Two clones (phylotypes SL135 and SL136) were affiliated with the phylum 269

Elusimicrobia and fell into a lineage of putatively free-living Endomicrobia from 270

termites and other cockroaches (37). Two clones (phylotypes SL130 and SL131) were 271

assigned to Actinobacteria, with Propionibacterium granulosum or sequences from 272

the termite gut as closest relatives. Two others (phylotypes SL133 and SL134) 273

clustered with clones from mammalian guts among the candidate division TM7 or 274

with clones from termite guts among the Synergistetes. 275

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 14

Discussion

The results of the molecular characterization revealed that the hindgut of Shelfordella 276

lateralis harbors a diverse community of mostly obligately anaerobic bacteria. This is 277

in contrast to earlier perceptions of the cockroach gut microbiota, which were based 278

on cultivation-based studies that yielded mostly facultatively anaerobic bacteria (15, 279

21). The only isolates that were represented in the clone library were Escherichia 280

blattae and Fusobacterium varium; the others were apparently too rare to be detected. 281

Moreover, our results agree with the findings of Bracke and colleagues (5), who 282

reported a strong reduction of the hindgut microbiota of Periplaneta americana by 283

metronidazole, which indicates that obligate anaerobes constitute a significant 284

proportion of the gut microbial community. The majority of the microbiota in S. 285

lateralis consists of hitherto uncultivated bacterial lineages found also in other 286

intestinal habitats. Many sequences clustered with those previously obtained from the 287

guts of termites, indicating that the composition of the bacterial community reflects at 288

least in part the phylogeny of the host. 289

The gut environment of S. lateralis is typical of blattid cockroaches. The gut 290

morphology of S. lateralis is typical of other blattid cockroaches, such as P. 291

americana (2, 6). Also the physiochemical characteristics of the gut, such as pH and 292

redox potential, are conserved between different species of the same cockroach family 293

(69, this study). Similar pH conditions have also been observed in lower termites (12). 294

Previous studies of cockroaches have reported a decrease in redox potential along the 295

gut, with oxidizing conditions in crop and midgut (4, 69) and reducing conditions in 296

the hindgut (25, 72). We observed that the center of each gut compartment was anoxic 297

and possessed a low redox potential (Fig. 1). By contrast, in lower termites, oxygen 298

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 15

has been detected in all gut compartments with exception of the paunch (12, 68). The 299

low redox potential in the hindgut lumen is consistent with the accumulation of 300

hydrogen and the presence of a large and diverse community of Clostridiales. 301

The fermentation products in the gut of S. lateralis are similar to those previously 302

detected in other cockroaches, with acetate prevailing in all compartments (39). Also 303

the high lactate concentration in the crop (Fig. 2) has been previously reported for P. 304

americana, where the foregut was determined to be a site of considerable lactate 305

production owing to the abundance of lactic acid bacteria (39). The presence of lactic 306

acid bacteria in the crop and midgut of S. lateralis is documented by the relative 307

abundance of the T-RFs corresponding to Lactobacillales (555 and 579 bp; Fig. 3). 308

Lactate concentration was lowest in the colon, the main site of absorption of 309

fermentation products in P. americana, including lactate (3, 7), but it is possible that 310

it is also subject to rapid turnover, as in the hindgut of lower termites (53, 66). 311

312

The hindgut harbors a diverse and individualistic community. Phylogenetic 313

analysis of 16S rRNA genes revealed that the cockroach gut contains a highly diverse 314

microbial community consisting mainly of uncultivated species. Each gut region 315

contained its own characteristic assemblage, with highest similarities in structure 316

between adjacent compartments (Fig. 4). The colon of S. lateralis was found to 317

contain the highest abundance of microorganisms; this in agreement with previous 318

studies of P. americana that used approaches based on cultivation (2) and electron 319

microscopy (6). 320

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 16

When the bacterial community in the colon of S. lateralis is compared to that of 321

termites, it most closely resembles the hindgut microbiota of a fungus-growing 322

species (62). A predominance of Bacteroidetes and Firmicutes is not typical for 323

wood-feeding termites, but is a common feature of omnivorous mammals (29, 42). 324

Moreover, our results underline that the potential pathogens isolated from other 325

blattid cockroaches in past cultivation attempts (e.g., Escherichia coli, Klebsiella 326

pneumoniae, Staphylococcus aureus, and various Salmonella species; see 15, 22, 57), 327

which gave rise to the concept of the cockroach gut as a reservoir for human disease, 328

in fact do not represent large populations. 329

The large differences in bacterial community structure among individual cockroaches 330

corresponds to the large variations of those gut parameters that are influenced by 331

microbial activities, such as the accumulation of hydrogen (Fig. 1) and the other 332

microbial products (Fig. 2). Individual variations among bacterial communities 333

colonizing identical habitats have been reported also for the gut communities of many 334

omnivorous mammals, including humans (75) and pigs (67), and are believed to arise 335

from the random acquisition of microorganisms from the environment (23). 336

The hindgut microbiota reflects the phylogeny of the host. Besides gut 337

morphology and diet, host phylogeny is considered an important factor that shapes 338

structure and diversity of the gut communities of mammals (43). Termites and 339

cockroaches are known to share a common evolutionary origin, with termites having 340

descended from an omnivorous cockroach ancestor (38). Evidence for a shared 341

evolutionary history has been previously observed already in the bacterial lineages 342

colonizing the gut of the wood-feeding cockroach Cryptocercus punctulatus (1), 343

although this is hardly surprising since this species has – besides the close 344

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 17

phylogenetic relatedness – also many other features of a primitive termite (e.g., diet, 345

proctodeal trophallaxis, and presence of cellulolytic gut flagellates; see 47). The 346

impact of host phylogeny on the composition of the gut microbiota is more evident in 347

S. lateralis. It is a representative of the Blattidae, the sister group of termites, but has 348

an omnivorous diet and lacks both sociality and gut flagellates. Despite these 349

differences, the gut microbiota of S. lateralis shares many phylogenetic lineages with 350

that of termites. Approximately 30% of the clones in the bacterial clone library of the 351

colon clustered with sequences previously obtained from termite guts. These 352

cockroach–termite clusters were unevenly distributed among the bacterial phyla. 353

While the fraction of clones affiliated with termite clusters were rather low among the 354

dominant phyla (Firmicutes, 27%; Bacteroidetes, 24%), they made up the majority of 355

clones among Proteobacteria (67%), consisting mostly of clones affiliated with 356

Desulfovibrio spp. (Fig. 7). Also the phyla Elusimicrobia, Synergistetes, and 357

Actinobacteria were represented by clones that fell into termite clusters, whereas 358

clones affiliated with Fusobacteria, Deferribacteres, Planctomycetes, and the 359

candidate division TM7 had no closest relatives among the termite gut microbiota. 360

Some of the cockroach–termite clusters represent bacterial lineages previously 361

identified as termite specific, suggesting that the respective lineages were present 362

already in the common ancestor of blattid cockroaches and termites. Of particular 363

interest are several clones from the phyla Bacteroidetes and Elusimicrobia, which are 364

closely related to bacterial symbionts that specifically colonize the surface or interior 365

of termite gut flagellates (36, 48). The sequences detected in S. lateralis (this study) 366

and other cockroaches (37) are likely to represent free-living relatives because 367

cockroaches, with the exception of Cryptocercus punctulatus, lack gut flagellates. 368

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 18

Habitat-specific elements of the hindgut microbiota. In addition to being reflective 369

of host phylogeny, the gut microbiota of S. lateralis also has many elements in 370

common with the gut communities of more distantly related animals. Noticeably, the 371

majority of clones (63%) in the library clustered with sequences previously obtained 372

from other gut environments, such as the cow rumen and the mouse and human 373

intestinal tracts. Only a few clones (7%) clustered with sequences from non-gut 374

environments. This suggests the existence of common environmental factors that 375

determine the composition of the resident microbial communities in the intestinal 376

habitats. 377

Members of the phylum Spirochaetes form an abundant group among the gut 378

microbiota of most termites (8) and may constitute up to half of all bacteria in wood-379

feeding higher termites (32, 52). They are present among the gut microbiota of C. 380

punctulatus (1) and have been observed in the hindgut of the blaberid cockroach 381

Eublaberus posticus (20), but seem to be absent from the hindgut of S. lateralis. Also 382

members of the phylum Fibrobacteres, which are abundant in the hindgut of wood-383

feeding higher termites (31, 73) and – like spirochetes – have been implicated in 384

cellulose digestion (see also 10), were not encountered in the clone library. A 385

previous study of P. americana and E. posticus reported high viable counts of 386

carboxymethyl cellulose-degrading obligate anaerobes in the hindgut of both 387

cockroaches (19). Since the predominant groups were tentatively identified as 388

members of the genera Clostridium and Eubacterium, it is possible that members of 389

the diverse clostridial populations also contribute to cellulose degradation in the 390

hindgut of S. lateralis. 391

392

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 19

In conclusion, our results show that S. lateralis harbors a diverse gut microbiota 393

whose community contains many habitat-specific elements, including the high level 394

of individual variation typical of the gut communities of other omnivorous animals. 395

On the other hand, there is also a clear phylogenetic element. The presence of 396

numerous bacterial lineages that were likely present already in the common ancestor 397

of blattid cockroaches and termites underlines that relatedness is an important factor 398

shaping the structure of the gut microbial community. 399

Acknowledgements 400

This work was supported within the LOEWE program of the 401

State of Hesse (Center for Synthetic Microbiology) and by the Max Planck Society. 402

CLT received a postdoctoral fellowship of the Alexander von Humboldt Foundation. 403

We thank Katja Meuser for the excellent technical assistance and Karen A. Brune for 404

linguistic comments on the manuscript. 405

References

1. Berlanga, M., B. J. Paster, and R. Guerrero. 2009. The taxophysiological 406

paradox: changes in the intestinal microbiota of the xylophagous cockroach 407

Cryptocercus punctulatus depending on the physiological state of the host. 408

Int. Microbiol. 12:227–236. 409

2. Bignell, D. E. 1977. Some observations on the distribution of gut flora in the 410

American cockroach, Periplaneta americana. J. Invertebr. Pathol. 29:338–411

343. 412

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 20

3. Bignell, D.E. 1980. An ultrastructural study and stereological analysis of the 413

colon wall in the cockroach, Periplaneta americana. Tissue Cell. 12:153–164. 414

4. Bignell, D. E. 1984. Direct potentiometric determination of redox potentials 415

of the gut contents in the termites Zootermopsis nevadensis and Cubitermes 416

severus and in three other arthropods. J. Insect Physiol. 30:169–174. 417

5. Bracke, J. W., D. L. Cruden, and A. J. Markovetz. 1978. Effect of 418

metronidazole on the intestinal microflora of the American cockroach, 419

Periplaneta americana L Antimicrob. Agents Chemother. 13:115–120. 420

6. Bracke, J. W., D. L. Cruden, and A. J. Markovetz. 1979. Intestinal 421

microbial flora of the American cockroach. Appl. Environ. Microbiol. 422

38:945–955. 423

7. Bracke, J. W., and A. J. Markovetz. 1980. Transport of bacterial end 424

products from the colon of Periplaneta americana. J. Insect Physiol. 26:85–425

89. 426

8. Breznak, J. A. 1984. Hindgut spirochetes of termites and Cryptocercus 427

puntulatus, p. 67–70. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of 428

systematic bacteriology, vol. 1. Williams & Wilkins, Baltimore, MD. 429

9. Brune, A. 2006. Symbiotic associations between termites and prokaryotes, 430

pp. 439–474. In M. Dworkin, S. Falkow, E. Rosenberg, K-H. Schleifer and E. 431

Stackebrandt (eds.), The Prokaryotes, 3rd ed., Volume 1: Symbiotic 432

associations, Biotechnology, Applied Microbiology, vol. 1. Springer, New 433

York. 434

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 21

10. Brune, A. 2007. Woodworker’s digest. Nature. 450:487–488. 435

11. Brune, A. 2010. Methanogens in the digestive tract of termites, pp. 81–100. 436

In J.H.P. Hackstein (ed.), (Endo)symbiotic Methanogenic Archaea. Springer, 437

Heidelberg. 438

12. Brune, A., D. Emerson, and J. A. Breznak. 1995. The termite gut 439

microflora as an oxygen sink: microelectrode determination of oxygen and 440

pH gradients in guts of lower and higher termites. Appl. Environ. Microbiol. 441

61:2681–2687. 442

13. Brune, A., and M. Kühl. 1996. pH profiles of the extremely alkaline 443

hindguts of soil-feeding termites (Isoptera: Termitidae) determined with 444

microelectrodes. J. Insect Physiol. 42:1121–1127. 445

14. Brune, A., and M. Ohkuma. 2011. Role of the termite gut microbiota in 446

symbiotic digestion, pp 439-475. In D.E. Bignell, Y. Roisin, N. Lo (eds.), 447

Biology of termites: a modern synthesis. Springer, Dordrecht. 448

15. Burgess, N. R. H., S. N. McDermott, and J. Whiting. 1973. Aerobic 449

bacteria occurring in the hind-gut of the cockroach, Blatta orientalis. J. Hyg. 450

(Lond). 71:1–7. 451

16. Charrier, M., and A. Brune. 2003. The gut microenvironment of helicid 452

snails (Gastropoda: Pulmonata): in-situ profiles of pH, oxygen and hydrogen 453

determined by microsensors. Can. J. Zool. 81:928–935. 454

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 22

17. Cleveland, L. R., Hall S. R., Sanders E. P., and Collier J. 1934. The wood-455

feeding roach Cryptocercus, its protozoa and the symbiosis between protozoa 456

and the roach. Mem. Am. Acad. Arts Sci. 17:185–342. 457

18. Collins, M. D., P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez-458

Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow. 1994. The 459

phylogeny of the genus Clostridium: proposal of five new genera and eleven 460

new species combinations. Int. J. Syst. Bacteriol. 44:812–826. 461

19. Cruden, D. L., and A. J. Markovetz. 1979. Carboxymethyl cellulose 462

decomposition by intestinal bacteria of cockroaches. Appl. Environ. 463

Microbiol. 38:369–372. 464

20. Cruden, D. L., and A. J. Markovetz. 1981. Relative numbers of selected 465

bacterial forms in different regions of the cockroach hindgut. Arch. 466

Microbiol. 129:129–134. 467

21. Cruden, D. L., and A. J. Markovetz. 1984. Microbial aspects of the 468

cockroach hindgut. Arch. Microbiol. 138:131–139 469

22. Cruden, D. L., and A. J. Markovetz. 1987. Microbial ecology of the 470

cockroach gut. Annu. Rev. Microbiol. 41:617–643. 471

23. Curtis, T. P., and W. T. Sloan. 2004. Prokaryotic diversity and its limits: 472

microbial community structure in nature and implications for microbial 473

ecology. Curr. Opin. Microbiol. 7:221–226. 474

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 23

24. Dasch, G. A., E. Weiss, and K. P. Chang. 1984. Endosymbionts of insects, 475

p. 811–833. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of 476

systematic bacteriology, vol. 1, Williams and Wilkins, Baltimore, MD. 477

25. Day, M. F. and D. F. Waterhouse. 1953. The mechanism of digestion, p. 478

311–330. In K. D. Roeder (ed.), Insect Physiology. John Wiley & Sons, New 479

York. 480

26. Dixon, P. 2003. VEGAN, a package of R functions for community ecology. 481

J. Veg. Sci. 14:927–930. 482

27. Djernæs, M., K.-D. Klass, M.D. Picker, and J. Damgaard. 2012. 483

Phylogeny of cockroaches (Insecta, Dictyoptera, Blattodea), with placement 484

of aberrant taxa and exploration of out-group sampling. System. Entomol. 485

37:65–83. 486

28. Ebert, A., and A. Brune. 1997. Hydrogen concentration profiles at the oxic-487

anoxic interface: a microsensor study of the hindgut of the wood-feeding 488

lower termite Reticulitermes flavipes (Kollar). Appl. Environ. Microbiol. 489

63:4039–4046. 490

29. Eckburg, P. B., E. M. Bik, C. N. Bernstein, E. Purdom, L. Dethlefsen, M. 491

Sargent, S. R. Gill, K. E. Nelson, and D. A. Relman. 2005. Diversity of the 492

human intestinal microbial flora. Science. 308:1635–1638. 493

30. Egert, M., B. Wagner, T. Lemke, A. Brune, and M. W. Friedrich. 2003. 494

Microbial community structure in midgut and hindgut of the humus-feeding 495

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 24

larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae). Appl. Environ. 496

Microbiol. 69:6659–6668. 497

31. Hongoh, Y., P. Deevong, S. Hattori, T. Inoue, S. Noda, N. 498

Noparatnaraporn, T. Kudo, and M. Ohkuma. 2006. Phylogenetic 499

diversity, localization and cell morphologies of the candidate phylum TG3 500

and a subphylum in the phylum Fibrobacteres, recently found bacterial 501

groups dominant in termite guts. Appl. Environ. Microbiol. 72:6780–6788. 502

32. Hongoh, Y., P. Deevong, T. Inoue, S. Moriya, S. Trakulnaleamsai, M. 503

Ohkuma, C. Vongkaluang, N. Noparatnaraporn, and T. Kudo. 2005. 504

Intra- and interspecific comparisons of bacterial diversity and community 505

structure support coevolution of gut microbiota and termite host. Appl. 506

Environ. Microbiol. 71:6590–6599. 507

33. Hongoh, Y., V. K. Sharma, T. Prakash, S. Noda, H. Toh, T. D. Taylor, T. 508

Kudo, Y. Sakaki, A. Toyoda, M. Hattori, and M. Ohkuma. 2008. Genome 509

of an endosymbiont coupling N2 fixation to cellulolysis within protist cells in 510

termite gut. Science. 322:1108–1109. 511

34. Horn, H. S. 1966. Measurement of "Overlap" in comparative ecological 512

studies. Am. Nat. 100:419–424. 513

35. Huber, T., G. Faulkner, and P. Hugenholtz. 2004. Bellerophon: a program 514

to detect chimeric sequences in multiple sequence alignments. 515

Bioinformatics. 20:2317–2319. 516

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 25

36. Ikeda-Ohtsubo, W., M. Desai, U. Stingl, and A. Brune. 2007. Phylogenetic 517

diversity of "Endomicrobia" and their specific affiliation with termite gut 518

flagellates. Microbiology. 153:3458–3465. 519

37. Ikeda-Ohtsubo, W., N. Faivre, and A. Brune. 2010. Putatively free-living 520

‘Endomicrobia’– ancestors of the intracellular symbionts of termite gut 521

flagellates? Environ. Microbiol. Rep. 2:554–559. 522

38. Inward, D., G. Beccaloni, and P. Eggleton. 2007. Death of an order: a 523

comprehensive molecular phylogenetic study confirms that termites are 524

eusocial cockroaches. Biol. Lett. 3:331–335. 525

39. Kane, M. D., and J. A. Breznak. 1991. Effect of host diet on production of 526

organic acids and methane by cockroach gut bacteria. Appl. Environ. 527

Microbiol. 57:2628–2634. 528

40. Klass, K-D., C. Nalepa, and N. Lo. 2008. Wood-feeding cockroaches as 529

models for termite evolution (Insecta: Dictyoptera): Cryptocercus vs. 530

Parasphaeria boleiriana. Mol. Phylogenet. Evol. 46:809–817. 531

41. Lane, D. J. 1991. 16S/23S rRNA sequencing, p. 115–175. In E. Stackebrandt 532

and M. Goodfellow (ed.), Nucleic acid techniques in bacterial systematics. 533

John Wiley and Sons Ltd., New York, N.Y. 534

42. Leser, T. D., J. Z. Amenuvor, T. K. Jensen, R. H. Lindecrona, M. Boye, 535

and K. Møller. 2002. Culture-independent analysis of gut bacteria: the pig 536

gastrointestinal tract microbiota revisited. Appl. Environ. Microbiol. 68:673–537

690. 538

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 26

43. Ley, R. E., M. Hamady, C. Lozupone, P. J. Turnbaugh, R. R. Ramey, J. 539

S. Bircher, M. L. Schlegel, T. A. Tucker, M. D. Schrenzel, R. Knight, and 540

J. I. Gordon. 2008. Evolution of mammals and their gut microbes. Science. 541

320:1647–1651. 542

44. Ludwig, W., O. Strunk, S. Klugbauer, N. Klugbauer, M. Weizenegger, J. 543

Neu-maier, M. Bachleitner, and K.H. Schleifer.1998. Bacterial phylogeny 544

based on comparative sequence analysis. Electrophoresis 19:554–568. 545

45. Ludwig, W., O. Strunk, R. Westram, L. Richter, H. Meier, Yadhukumar, 546

A. Buchner, T. Lai, S. Steppi, G. Jobb, W. Förster, I. Brettske, S. Gerber, 547

A. W. Ginhart, O. Gross, S. Grumann, S. Hermann, R. Jost, A. König, T. 548

Liss, R. Lüssmann, M. May, B. Nonhoff, B. Reichel, R. Strehlow, A. 549

Stamatakis, N. Stuckmann, A. Vilbig, M. Lenke, T. Ludwig, A. Bode, and 550

K-H. Schleifer. 2004. ARB: a software environment for sequence data. 551

Nucleic Acids Res.32:1363–1371. 552

46. Muyzer, G. A. Teske, C. O. Wirsen, and H. W Jannasch. 1995. 553

Phylogenetic relationships of Thiomicrospira species and their identification 554

in deep-sea hydrothermal vent samples by denaturing gradient gel 555

electrophoresis of 16S rDNA fragments. Arch. Microbiol. 164:165–172. 556

47. Nalepa, C. A., D. E. Bignell, and C. Bandi. 2001. Detritivory, coprophagy, 557

and the evolution of digestive mutualisms in Dictyoptera. Insectes Soc 558

48:194–201. 559

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 27

48. Noda, S., T. Inoue, Y. Hongoh, M. Kawai, C. A. Nalepa, C. Vongkaluang, 560

T. Kudo, and M. Ohkuma. 2006. Identification and characterization of 561

ectosymbionts of distinct lineages in Bacteroidales attached to flagellated 562

protists in the gut of termites and a wood-feeding cockroach. Environ. 563

Microbiol. 8:11–20. 564

49. Ohkuma, M. and A. Brune, 2011. Diversity, structure, and evolution of the 565

termite gut microbial community, pp 413-438. In D.E. Bignell, Y. Roisin, N. 566

Lo (eds.), Biology of termites: a modern synthesis. Springer, Dordrecht. 567

50. Ohkuma, M., S. Noda, Y. Hongoh, and T. Kudo. 2002. Diverse bacteria 568

related to the Bacteroides subgroup of the CFB phylum within the gut 569

symbiotic communities of various termites. Biosci. Biotechnol. Biochem. 570

66:78–84. 571

51. Ohkuma, M., S. Noda, Y. Hongoh, C. A. Nalepa, and T. Inoue. 2008. 572

Inheritance and diversification of symbiotic trichonymphid flagellates from a 573

common ancestor of termites and the cockroach Cryptocercus. Proc. R. Soc. 574

B 276:239–245. 575

52. Paster, B. J, F. E. Dewhirst, W. G. Weisburg, L. A. Tordoff, G. J. Fraser, 576

R. B. Hespell, T. B. Stanton, L. Zablen, L. Mandelco, and C. R. Woese. 577

1991. Phylogenetic analysis of the spirochetes. J. Bacteriol. 173:6101-6109. 578

53. Pester, M., and A. Brune. 2007. Hydrogen is the central free intermediate 579

during lignocellulose degradation by termite gut symbionts. ISME J. 1:551–580

565. 581

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 28

54. Pielou, E. C. 1966. The measurement of diversity in different types of 582

biological collections. J. Theor. Biol. 13:131–144. 583

55. Pruesse, E., C. Quast, K. Knittel, B. Fuchs, W. Ludwig, J. Peplies, and F. 584

O. Glöckner. 2007. SILVA: a comprehensive online resource for quality 585

checked and aligned ribosomal RNA sequence data compatible with ARB. 586

Nucleic Acids Res. 35:7188–7196. 587

56. R Development Core Team. 2009. R: a language and environment for 588

statistical computing. R Foundation for Statistical Computing, Vienna, 589

Austria. http://www.R-project.org. 590

57. Roth, L. M., and E. R. Willis. 1957. The medical and veterinary importance 591

of cockroaches. Smithsonian Misc. Coll. 134:1–147. 592

58. Schloss, P.D., S. L. Westcott, T. Ryabin, J. R. Hall, M. Hartmann, E. B. 593

Hollister, R. A. Lesniewski, B. B. Oakley, D. H. Parks, C. J. Robinson, J. 594

W. Sahl, B. Stres, G. G. Thallinger, D. J. Van Horn, and C. F. Weber. 595

2009. Introducing mothur: Open-source, platform-independent, community-596

supported software for describing and comparing microbial communities. 597

Appl. Environ. Microbiol. 75:7537–7541. 598

59. Schmitt-Wagner, D., M. W. Friedrich, B. Wagner, and A. Brune. 2003. 599

Axial dynamics, stability, and interspecies similarity of bacterial community 600

structure in the highly compartmentalized gut of soil-feeding termites 601

(Cubitermes spp.). Appl. Environ. Microbiol. 69:6018–6024. 602

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 29

60. Schultz, J. E., and J. A. Breznak. 1979. Cross-feeding of lactate between 603

Streptococcus lactis and Bacteroides sp. isolated from termite hindguts. Appl. 604

Environ. Microbiol. 37:1206-1210. 605

61. Shannon, C. E., and W. Weaver. 1963. The mathematical theory of 606

communication. University of Illinois Press, Urbana, Ill. 607

62. Shinzato, N., M. Muramatsu, T. Matsui, and Y. Watanabe. 2007. 608

Phylogenetic analysis of the gut bacterial microflora of the fungus-growing 609

termite Odontotermes formosanus. Biosci. Biotechnol. Biochem. 71:906–915. 610

63. Stamatakis, A. 2006. RAxML-VI-HPC: maximum likelihood-based 611

phylogenetic analyses with thousands of taxa and mixed models. 612

Bioinformatics 22:2688–2690. 613

64. Strassert, J. F., M. S. Desai, R. Radek, and A. Brune. 2010. Identification 614

and localization of the multiple bacterial symbionts of the termite gut 615

flagellate Joenia annectens. Microbiology. 156:2068–2079. 616

65. Thayer, D.W. 1976. Facultative wood-digesting bacteria from the hind-gut of 617

the termite Reticulitermes hesperus. J. Gen. Microbiol. 95:287–296. 618

66. Tholen, A., and A. Brune. 2000. Impact of oxygen on metabolic fluxes and 619

in situ rates of reductive acetogenesis in the hindgut of the wood-feeding 620

termite Reticulitermes flavipes. Environ. Microbiol. 2:436–449. 621

67. Thompson, C. L., B. Wang, and A. J. Holmes. 2008. The immediate 622

environment during postnatal development has long-term impact on gut 623

community structure in pigs. ISME J. 2:739–748. 624

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 30

68. Veivers, P. C., R. W. O’Brien, and M. Slaytor. 1980. The redox state of the 625

gut of termites. J. Insect Physiol. 26:75–77 626

69. Vinokurov, K., Y. Taranushenko, N. Krishnan, and F. Sehnal. 2007. 627

Proteinase, amylase, and proteinase-inhibitor activities in the gut of six 628

cockroach species. J. Insect Physiol. 53:794–802. 629

70. Trautwein, M.D., B.M. Wiegmann, R. Beutel, K.M. Kjer, and D.K. 630

Yeates. 2012. Advances in insect phylogeny at the dawn of the postgenomic 631

era. Annu. Rev. Entomol. 57:449–468. 632

71. Ware, J., J. Litman, K.-D. Klass, and L.A. Spearman. 2008. Relationships 633

among the major lineages of Dictyoptera: the effect of outgroup selection on 634

dictyopteran tree topology. Syst. Entom. 33:429–450. 635

72. Warhurst, D.C. 1964. Growth and survival, in vitro and in vivo of Endolimax 636

blattae, an entozoic amoeba of cockroaches. Ph. D. Thesis. University of 637

Leicester, U.K. 638

73. Warnecke, F., P. Luginbühl, N. Ivanova, M. Ghassemian, T. H. 639

Richardson, J. T. Stege, M. Cayouette, A. C. McHardy, G. Djordjevic, N. 640

Aboushadi, R. Sorek, S. G. Tringe, M. Podar, H. G. Martin, V. Kunin, D. 641

Dalevi, J. Madejska, E. Kirton, D. Platt, E. Szeto, A. Salamov, K. Barry, 642

N. Mikhailova, N. C. Kyrpides, E. G. Matson, E. A. Ottesen, X. Zhang, 643

M. Hernández, C. Murillo, L. G. Acosta, I. Rigoutsos, G. Tamayo, B. D. 644

Green, C. Chang, E. M. Rubin, E. J. Mathur, D. E. Robertson, P. 645

Hugenholtz, and J. R. Leadbetter 2007. Metagenomic and functional 646

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 31

analysis of hindgut microbiota of a wood-feeding higher termite. Nature. 647

450:560–565. 648

74. Zeidler, R. B., P. Lee, and H .D. Kim. 1976. Kinetics of 3-O-methyl glucose 649

transport in red blood cells of newborn pigs. J. Gen. Physiol. 67:67–80. 650

75. Zoetendal, E. G., A. D. L. Akkermans, and W. M. De Vos. 1998. 651

Temperature gradient gel electrophoresis analysis of 16S rRNA from human 652

fecal samples reveals stable and host-specific communities of active bacteria. 653

Appl. Environ. Microbiol. 64:3854–3859. 654

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Table 1. Fresh weight, microbial cell density and bacterial diversity in the gut compartments of S. lateralis (mean ± standard deviation). 655

Crop Gastric caeca Midgut Colon Rectum

Fresh weight (mg)a 22.8 ± 17.3 9.8 ± 5.2 20.5 ± 9.5 16.0 ± 6.6 7.5 ± 3.6

Microbial cell density (106 mg–1) 2.2 ± 1.7 2.4 ± 1.2 2.0 ± 1.3 22.3 ± 15.9 15.3 ± 6.3

Average T-RFsb 8 ± 6 8 ± 5 8 ± 4 45 ± 20 24 ± 19

Shared T-RFsc 1 3 2 15 0

Morisita-Horn similarity index 0.51 ± 0.42 0.96 ± 0.02 0.54 ± 0.25 0.35 ± 0.19 0.19 ± 0.27

Shannon index 0.51 ± 0.17 0.43 ± 0.22 0.48 ± 0.15 1.55 ± 0.11 1.14 ± 0.35

Pielou index 0.65 ± 0.21 0.39 ± 0.10 0.42 ± 0.10 0.86 ± 0.02 0.69 ± 0.14

a Fresh weight per compartment (n = 30) 656 b Total number of distinct T-RFs in profiles (n = 4) 657 c Number of T-RFs occurring in all profiles from the same gut compartment658

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Figure legends

Fig. 1. Microsensor profiles of physiochemical conditions in the gut of S. lateralis. 659

The gut comprises the foregut (mainly crop and gizzard, g), the midgut with gastric 660

caeca (gc), and the hindgut (colon and rectum). Axial profiles of pH (n = 3), redox 661

potential (n = 3), and hydrogen concentration (n = 5; filled squares) were measured 662

within these compartments as indicated using microsensors. In the larger 663

compartments, measurements were taken at the anterior (a), middle (m), and posterior 664

(p) regions. The open squares indicate the typical profile of an individual 665

accumulating large amounts of hydrogen. All deviations are given as standard error of 666

the mean. 667

Fig. 2. Microbial fermentation products within the different gut compartments of S. 668

lateralis. Deviations are given as standard error of the mean (n = 8). 669

Fig. 3. T-RFLP analysis of bacterial diversity within the gut compartments of S. 670

lateralis. Each column represents an average of T-RFLP profiles from four 671

individuals, and shading indicates the proportion of individuals containing a particular 672

T-RF. T-RFs present in all compartments are labeled with their size in base pairs. 673

Fig. 4. Nonmetric multidimensional scaling plot of Bray–Curtis similarities showing 674

clustering of gut microbiota by compartments and the degree of individual variation. 675

Symbols represent different gut compartments; numbers represent gut compartments 676

from the same individual. The stress value of the plot was 0.15, which indicated that 677

the plot provides a good representation of the samples. 678

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 34

Fig. 5. Phylogenetic position of the phylotypes obtained in this study belonging to the 679

phylum Bacteroidetes. Clusters identified by Ohkuma et al. (50) are shaded. The tree 680

was constructed using maximum likelihood and is based on the analysis of 1,815 valid 681

alignment positions. The tree was rooted using sequences selected from other phyla. 682

Phylotypes from this study are shown in bold. Numbers in parentheses indicate the 683

number of clones assigned to each phylotype or cluster. Circles indicate bootstraps 684

values above 90% () and 70% ().The scale bar represents a 10% estimated 685

sequence divergence. 686

Fig. 6. Phylogenetic position of the phylotypes obtained in this study belonging to the 687

phylum Firmicutes, inferred by maximum-likelihood analysis of 941 valid alignment 688

positions. Major clostridial clusters defined by Collins et al. (18) are shaded. For 689

details on tree calculation and notation, refer to Fig. 5. 690

Fig. 7. Phylogenetic position of the phylotypes obtained in this study belonging to the 691

phylum Proteobacteria, inferred by maximum likelihood analysis of 1,420 valid 692

alignment positions. Clusters affiliated with Beta-, Delta-, and Gammaproteobacteria 693

are indicated. For details on tree calculation and notation, refer to Fig. 5. 694

Supplementary Figures 695

Fig. S1. T-RFLP profile of 16S rRNA genes amplified from pooled colon DNA of S. 696

lateralis (n = 6). The horizontal axis indicates the size (nucleotide base pairs) of the 697

T-RFs. This sample was used to construct the 16S rRNA gene clone library of the 698

colon. The peaks were identified by in silico digestion of the clone sequences. 699

Fig. S2. Rarefaction analysis of all bacterial 16S rRNA gene clones recovered from 700

the colon of Shelfordella lateralis. The expected number of clones was calculated 701

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Schauer et al. 35

from the number of clones analyzed at the species level with 97% sequence similarity 702

() and at a sequence similarity level of 95% () and 90% (). 703

704

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

0

5

10

15

20

25

a m p a m p a m p

Midgut Colon Rectum

Hyd

roge

n (k

Pa)

5.0

6.0

7.0

pH

-300

-200

-100

0

Red

ox p

oten

tial (

mV)

Crop

gc

g

Hindgut

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

0

200

400

600

800

1000

1200

1400

Crop Midgut Colon Rectum

GlucoseLactateAcetatePropionateEthanolSuccinate

Am

ount

(nm

ol)

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

95

95

555

555

555

9595

555 555

0

10

20

30

40

50

60

70

80

90

100

Crop Gastriccaeca

Midgut Colon Rectum

R

elat

ive

abun

danc

e (%

)

95

1/42/43/44/4

579579

579 579

Frequency

579

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

2

4

1

2

3

1

1

11

2

2

2

33

3

3

4

4

4

−1.5 −1.0 −0.5 0 0.5 1.0 1.5

−1.5

−1.0

−0.5

0

0.5

1.0

NMDS1

NM

DS2

4

CropGastric caecaMidgut Colon

Rectum

5

6

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

I

Rikenellaceae

V

III

Porphyromonadaceae

Clones SL20-23, SL25 (11) JN680565-69, 20 clones from lower termites and Cryptocercus punctulatus

16 Clones from Candidatus Azobacteroides pseudotrichonympha

Clone SL24 (2) JN680570 and 2 clones from lower termites 2 Clones from Cryptocercus punctulatus

Clone SL27 (1) JN680571 and clone from lower termiteClone SL26 (1) JN680572

5 Clones from human gutBacteroides cellulosilyticus, AJ583243

Clone SL19 (1) JN6805603 Clones from scarab beetle larvaClone SL16 (7) JN680561

Clones SL14−15 (11) JN680562-63Clone SL18 (1) JN680564

Dysgonomonas mossii, AJ319867Clone from lower termite, AB055736

Clone SL28 (3) JN680573Clone SL29 (2) JN680574Clone from scarab beetle larva, AJ576373

Clone SL30 (2) JN680575Clone from scarab beetle larva, AJ576361

Clone SL32 (1) JN680576Clone from crab gut, DQ856511

Clone SL31 (2) JN680577Dysgonomonas capnocytophagoides, U41355

Clone SL33 (3) JN680578

21 Clones from mammalian gut

8 Clones from human gutClone SL34 (1) JN680579, Candidatus Vestibaculum illigatum, 3 clones from Cryptocercus punctulatus

Parabacteroides distasonis, AB238922

Clones SL35−36, SL40−41 (28) JN680580-83, 2 clones from higher termite and Cryptocercus punctulatus

Clones SL38 and SL42 (8) JN680584-85, 3 clones from lower termites

Clone SL44 (2) JN680586Clone from scarab beetle larva, AJ576330 Tannerella forsythensis, DQ344916Clone SL45 (1) JN680587Clone from scarab beetle larva, AJ576344

Clones SL46−47 (4) JN680588-89, 2 clones from higher termitesClone from subsurface groundwater, AB237700

Paludibacter propionicigenes, AB078842

Clones SL62−64 (3) JN680590-92, 9 clones from mammalian gut,Butyricimonas synergistica, AB443948

5 Clones from mammalian gutClone SL61 (1) JN680593Clone from lower termite, AY571432

Clone SL59 (4) JN680594Clone from fish intestine, EU884946

Cytophaga fermentans, D12661

5 Clones from higher termitesClone SL60 (1) JN680595

Clone SL43 (1) JN680596

Clones SL48−51 (15) JN680597-600, 3 clones from higher termites Clone from ground beetle gut, EF608547

Alistipes putredinis, NR_025909Clone from higher termite, AB055725

5 Clones from human gutClone SL53 (2) JN680601

Clone from higher termite, AB055728Clone SL52 (3) JN680602Clone from higher termite, AB234415

Clones SL54−58 (7) JN680603-07, 4 clones from lower and higher termites

0.10

IV

Bacteroidaceae

II

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Clone SL127 (1) JN680639, 3 clones from ruminants

Clone SL121 (1) JN680640Clone from lion feces, EU776671

Enterococcus cecorum, AF061009 Clone SL119 (1) JN680641

Enterococcus canintestini, AJ888906 Clone SL118 (3) JN680642, Enterococcus asini Y11621, clone from anaerobic sludge Clone SL117 (1) JN680643

Clone SL120 (1) JN680644, Enterococcus avium AF061008, clone from ant lion larva

Clones SL115−116 (4) JN680645-46 , Lactobacillus parabuchneri, AJ970317 Peptococcus niger, X55797

Clones SL128−129 (5) JN680647-48, clone from higher termite Clone from ground beetle gut, EF608543

Succinispira mobilis, AJ006980

Clones SL75 and SL81 (2) JN680649-50, 2 clones from mammalian gut Catonella morbi, X87151

Clones SL73−74 (2) JN680651-52, 3 clones from scarab beetle larva gut Clones SL76−80 (5) JN680653-57, Clostridium hylemonae AB023972, 7 clones Pei041 from scarab beetle larva gut

Robinsoniella peoriensis, AF445285 Clones SL71−72 (7) JN680658-59, 5 clones from rumens and mammalian guts

Clone SL110 (1) JN680660, 3 clones from bird and mammalian guts Clones SL82−83 (7) JN680661-62, 3 clones from lower termites

Anaerofustis stercorihominis, AJ518871 Clone SL114 (1) JN680663

Clone SL112 (2) JN680664, clone from lower termite Eubacterium brachy, U13038

Clone SL113 (2) JN680665 Clostridium aminobutyricum, X76161

0.10

Veillonellaceae

Lactobacillaceae

Enterococcaceae

Eubacteriaceae

Lachnospiraceae

Clones SL100−103 (8) JN680608-11, clone from anaerobic digestorClone SL99 (2) JN680612, 3 clones from cow rumen and sheep feces

Clostridium leptum Clone SL104 (1) JN680613

Clone SL105 (1) JN680614Clostridium viride, X81125 Clone SL106 (1) JN680615, Bacteroides capillosus AY136666, 3 clones from human guts Clone SL109 (1) JN680616, 3 clones from herbivore guts Clone SL107 (2) JN680617, 3 clones from various environments Clone SL108 (1) JN680618, 3 clones from various environments

Eubacterium desmolans, L34618 Clostridium islandicum, EF088328 Clone SL98 (2) JN680619

Clones SL96-97 (2) JN680620-21, 3 clones from higher termites Anaerotruncus colihominis, AJ315980

Clones SL91−95 (5) JN680622-26, 5 clones from higher termites Clone from digestive tract of ground beetle, EF608549Anaerofilum pentosovorans, X97852

Clones SL84−90 (14) JN680627-33, 9 clones from termites and various guts Clostridium botulinum, X68317

Clone SL111 (1) JN680634 Clostridium butyricum, AB075768

Clone SL125 (1) JN680635, Erysipelothrix inopinata AJ550617, 4 clones from various environments

Clones SL122 (1), SL124 (4) JN680636-37, 3 clones from termite and scarab beetle larvae gutsSpiroplasma chrysopicola, AY189127

Clone SL126 (1) JN680638, clones from rumen and mammalian guts

Erysipelotrichi

Clostridiaceae

Ruminococcaceae

Mollicutes

I

IV

XIV

IX

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from

Desulfovibrio sp. strain ZIRB−2 from termite gut, AY532164 Clone SL11 (1) JN680666

Clone from higher termite gut, AB234536 Clone from higher termite gut, AB234528

Clone SL7 (1) JN680667Clone from higher termite gut, AB234538

Clone SL8 (2) JN680668Clone SL6 (5) JN680669

Desulfovibrio cuneatus, X99501

3 Clones from termite gutClone SL10 (1) JN680670Clone SL9 (1) JN680671

Clone from higher termite gut, DQ307713 Clone from higher termite gut, DQ307712

Desulfovibrio litoralis, X99504 Clone SL12 (1) JN680672Clone from higher termite gut, EF454852 Clone from squirrel feces, EU459956

Clone SL4 (1) JN680673Clone from fish gut, EU885093

Clone from higher termite gut, EF454453 Clone from higher termite gut, EF454754

Clone SL5 (1) JN680674Clone from fish gut, EU885097

Desulfatiferula olefinivorans, DQ826724

Clone from mangrove soil, DQ811840 Clone from hypersaline microbial mat, DQ330949 Clone SL3 (1) JN680676

Undibacterium pigrum, AM397630 Clone from higher termite gut, AB234524

Clone SL2 (1) JN680675

Escherichia blattae from cockroach hindgut, EU868610 Clone SL1 (1) JN680677

Escherichia coli, GQ273515 0.10

δ

γ Enterobacteriaceae

Oxalobacteriaceae

Coxiellaceae

Desulfobacteraceae

Desulfovibrionaceae

β

on March 17, 2020 by guest

http://aem.asm

.org/D

ownloaded from