1
Do Insect Host Diet and Taxonomy Influence Their Gut Bacterial Communities?
2
D.R. Colman, E.C. Toolson, C.D. Takacs-Vesbach
3
Department of Biology, University of New Mexico, 167 Castetter Hall MSC02 2020, 1
4
University of New Mexico, Albuquerque, NM 87131-0001
5
Keywords: Insect Microbial Community, Intestinal Microbiota, Gut Ecology,
6
Corresponding Author: Cristina D. Takacs-Vesbach
7
Cristina D. Takacs-Vesbach
8
UNM Biology Department
9
MSC03 2020
10
1 University of New Mexico
11
Albuquerque, NM 87131-0001
12
Fax: (505) 277 - 3418
13
cvesbach@unm.edu
14
15
exhibit, show Fig. or Figure, figure numbers, should order be capilatilized?
16
17
Abstract
Many insects contain diverse and rich gut microbial communities. While a majority of
18
studies have focused on a single or small group of species, comparative studies of
19
phylogenetically diverse hosts are necessary to understand the association of intestinal
20
communities and their hosts. In this study, we tested the hypotheses that 1) host diet and 2) host
21
phylogeny influences intestinal community composition. We used published 16S rRNA gene
22
sequence data for 58 insect species in addition to four beetle species sampled from the Sevilleta
23
National Wildlife Refuge to test these hypotheses. Phylogenetic and statistical analysis suggested
24
a prominent role of both host diet and host taxonomic affiliation in explaining gut community
25
variation. Host orders and diet guilds were variable in community membership conformity.
26
Hymenopterans and termites were among the orders with the most similar communities within
27
their groups. Diet guilds that were similar included xylophagous insects (both live tree and
28
decayed wood groups), detritivores, herbivores and pollenivores. The analysis suggests groups
29
with a diet reliant on cellulolysis maintain lower gut community variation than other guilds.
30
Overall, gut bacterial species richness in these insects was much lower than has been reported for
31
other animals such as XXXX. Xylophagous termites were the guild with the most diverse
32
bacterial gut flora. Our analysis provides a baseline comparison of insect gut bacterial
33
communities from which to test further hypotheses concerning ultimate and proximate causes of
34
these associations.
35
2
36
37
Introduction
Intestinal tracts harbor rich, dense communities of microorganisms (Dillon & Dillon
38
2004; Hongoh 2010; Ley et al. 2006). A single insect gut can harbor up to 107-109 prokaryotic
39
cells mL-1 of gut fluid (Broderick et al. 2004; Egert et al. 2005; Hongoh et al. 2006a). Intestinal
40
microbes can participate in many relationships with their hosts, though most are nutritional
41
commensals or symbionts. Nutritional symbionts have been shown to aid nitrogen cycling in
42
Tetraponera ants among others, lignocellulose metabolism in termites, and may facilitate
43
granivory in Carabid beetles (Breznak & Brune 1994; Lundgren & Lehman 2010; van Borm et
44
al. 2002). While many studies have described insect gut microbial communities, a synthesis
45
explaining the dynamics and causes of variation between and among different insect groups is
46
still lacking. A further understanding of variation between insect hosts could potentially reveal
47
insights into the proximate and ultimate causes of these associations. In addition, understanding
48
relationship dynamics broadly across insects may aid efforts in biocontrol of insect pests, which
49
is a focus of much insect gut microbiology (Broderick et al. 2004; Geib et al. 2009; Lundgren et
50
al. 2007; Vasanthakumar et al. 2006).
51
There are likely many factors influencing insect gut communities. Diet, pH, host-
52
specificity (e.g. coevolutionary effects), life stage, and host environment have all been reported
53
to influence community structure (Behar et al. 2008; Hongoh et al. 2005; Mohr & Tebbe 2006;
54
Santo Domingo et al. 1998; Schmitt-Wagner et al. 2003). These different factors are not
55
necessarily exclusive of one another, but there is strong evidence that both diet and taxonomic
56
affiliation of the host can strongly affect an organism’s gut microbial community.
57
Experimental evidence suggests that altering a host’s diet can change not only the
58
metabolic functioning of gut communities, but could also the community structure (Broderick et
3
59
al. 2004; Kane & Breznak 1991; Santo Domingo et al. 1998). One study of the fungus-growing
60
termite Macrotermes gilvus indicated a strong effect of host diet on intraspecies gut community
61
variation (Hongoh et al. 2006b). Termites, as a whole, have different domain level gut
62
microbiota compositions which is coincident with dietary differences (Brauman et al. 2001). In
63
addition, mammals of similar diet share more similar gut communities, though the similarity of
64
communities could also be explained by host taxonomic affiliation (Ley et al. 2008). Though diet
65
does indeed appear to be a strong influence on gut communities, there also appears to be a strong
66
relationship between communities and host taxonomy.
67
Of the few studies that have compared disparate hosts by diet, there has been little
68
evidence to exclude effects resulting from coevolutionary processes. While domain level
69
microbial compositions are coincident with dietary differences in termites, they also reflect an
70
influence of their host’s taxonomy (Brauman et al. 2001). Additionally, termite gut communities
71
exhibit stronger host-specificity than would be expected if diet was solely influencing
72
community structure (Hongoh et al. 2005). A similar result was reported for mammalian gut
73
microbiota comparisons, where similarities were significantly associated with host taxonomy,
74
and the clustering of gut communities reflected the hosts’ phylogeny (Ley et al. 2008). Diet may
75
be inexorably linked to host taxonomic affiliation due to diet-driven evolution of hosts. Thus, the
76
relative influence of diet and taxonomy on host gut communities remains unclear.
77
Our study compared the bacterial community composition (based on the 16S rRNA gene)
78
associated with the intestinal tracts of a wide diversity of insects to test two hypotheses: 1) host
79
diet influences community composition and 2) host phylogeny is related to community
80
composition. We found a wide variation in the bacterial species level richness associated with
81
the different hosts and dietsanfdkjdfajdklthatSome finalizing sentences.
4
82
83
Materials & Methods
84
Characterization of insect gut microbial communities
85
Our study is largely a meta-anlysis of published reports comprising 86 bacterial
86
communities from 62 insect species representing 7 taxonomic orders and 9 diet types. We also
87
included new data from four Coleoptera species that represented three diet types to specifically
88
test the effect of diet type within taxonomic order. Published bacterial community 16S rRNA
89
datasets were downloaded from Genbank. Datasets were selected to meet three criteria: 1)
90
comparable methods were used to obtain 16S rRNA gene sequence data (clone library based
91
studies), 2) the entire community dataset, as opposed to only the unique sequences reported in
92
the publishing paper, was publicly available and 3) sufficient host dietary information was given
93
in the publishing paper. If astudy dissected intestines by hindgut, midgut or lumen, the data were
94
combined to represent the entire intestinal tract of the organism so that datasets were comparable
95
across all published samples. Community hosts were classified as belonging to one of nine
96
general feeding strategies: detritivorous, filter-feeding, hematophagous/nectarivorous,
97
herbivorous (foliage and roots), omnivorous, pollenivorous, predacious, live tree xylophagy
98
(phloem, sapwood, bark), and dead or decaying wood xylophagy.
99
The four Coleopteran species we sampled to augment the study were collected from the
100
Nunn Flats area of the Sevilleta National Wildlife Refuge, New Mexico, USA (34° 24´ 24.8' N,
101
106° 36´ 20.5' W) in 2008. The Nunn Flats area is classified as a Chihuahan desert grass
102
dominated ecosystem, and thus all insects collected were from the same ecosystem type. Three
103
adult individuals were collected for each of the four species (n=12), which were chosen based on
104
known or presumptive feeding strategies and presence at the time of sampling. Individual
5
105
samples were immediately stored on ice in the field and frozen at -80oC upon return to the
106
laboratory.
107
Samples were thawed and dissected using sterile instruments and techniques. Cuts were
108
made along the elytra of the individual and the entire intestinal tract (fore, mid and hindgut) was
109
removed and stored in a sucrose lysis buffer (Giovannoni et al. 1990). Within 48 h, total
110
community DNA was extracted from the intestinal sample by using a variation of the CTAB
111
(Cetyl trimethylammonium bromide) extraction method with phenol/chloroform purification
112
(Mitchell & Takacs-Vesbach 2008), modified to include a 30 second bead-beating step with 3
113
mm glass beads to homogenize the sample tissue. The remaining beetle carcasses (???) were
114
preserved in 95% ethanol for taxonomic identification.
115
Beetles were identified by comparison with voucher specimens deposited in the
116
entomology collection at the Museum of Southwestern Biology. Two of the four species,
117
Epicauta longicollis & Megetra cancellata, are foliovores of the Meloidae family (Cartron et al.
118
2008; Toolson). The third beetle, Calosoma peregrinator, is a predacious member of the family
119
Carabidae (Burgess & Collins 1917). The fourth species, Gonasida inferna, is a beetle of the
120
tribe Pimeliinae which exhibit generalist feeding strategies like other members of the
121
Tenebrionidae family (Sanchez-Pinero & Gomez 1995; Thomas 1984).
122
Community Composition Analysis
123
Bacterial 16S rRNA gene sequences were amplified from the DNA extracted from the
124
Nunn Flats beetles in triplicate PCRs using the universal bacterial-specific primers, 8F forward
125
primer: 5’GTTTACCTTGTTACGACTT 3’ (Liu et al. 1997) and 1391R reverse primer: 5’
126
GACGGGCGGTGTGTRCA 3’ (Lane et al. 1985). PCR was performed in 50 µl reactions
127
containing 5 µl 10X buffer (Promega Buffer B w/ 1.5 mM MgCl2), 12.5 mM of each dNTP
6
128
(BioLine USA Inc.), 20 pmol of both the forward and reverse primers, 2.5 units of Taq
129
polymerase (Promega), 3 µl of 2% (w/v) bovine serum albumin and approximately 150 ng of
130
DNA. The PCR thermal cycling program consisted of 30 s at 94oC, 30 s at 50oC, and 90 s at
131
72oC for a total of 30 cycles and was performed on an ABI GeneAmp 2700 (Applied Biosystems
132
Inc.). Replicate PCR products were combined and the 16S rRNA gene amplicons were spin
133
purified using a DNA purification kit (Mo Bio Laboratories). Amplified 16S rRNA genes were
134
ligated and cloned using a TOPO TA cloning kit (Invitrogen corp.). Ninety-six cloned inserts
135
from each individual beetle were randomly chosen for sequencing using the BigDye terminator
136
cycle sequencing kit (PE Applied Biosystems) with the 8F forward primer on an ABI 3130x
137
genetic analyzer (PE Applied Biosystems). Community 16S rRNA gene libraries for each insect
138
species were named as follows: MEG (M. cancellata), EPI (E. longicollis), GON (G. inferna)
139
and CAL (C. peregrinator).
140
Genetic Analysis
141
DNA sequences were edited using CodonCode Aligner (CodonCode Corporation). DNA
142
sequences with less than 400 Phred20 bases were not used for further analysis. The remaining
143
DNA sequences were aligned using the NAST alignment Tool (DeSantis et al. 2006) of
144
Greengenes (http://greengenes.lbl.gov). Sequences were checked for evidence of chimeric
145
properties using the Bellerophon V 3.0 tool (Huber et al. 2004), and suspected chimeras were not
146
included in further analysis. Taxonomic classification of sequences was conducted with the
147
Bayesian classifier function in mothur (Schloss et al. 2009). Sequences with closest identity
148
matches to plant chloroplasts were excluded, as they were likely remnants of undigested plant
149
tissue, and were not useful in describing the intestinal communities.
150
7
151
The beetle and published 16S rRNA gene sequence datasets (aligned with the NAST
152
alignment tool) were combined and imported into ARB (Ludwig et al. 2004) and then parsimony
153
added to a phylogenetic tree consisting of the entire aligned Greengenes 16S rRNA gene
154
database
155
(http://greengenes.lbl.gov/Download/Sequence_Data/Arb_databases/greengenes236469.arb.gz)
156
downloaded on XXXXX, 2011. The tree was exported for clustering and principal coordinate
157
analysis (PCoA) in Unifrac using the Unifrac metric of phylogenetic similarity between samples
158
(Lozupone & Knight 2005). The Unifrac dissimilarity matrix was used to assess hypotheses
159
concerning the source of variation in the distance matrix (e.g. host diet and host taxonomic
160
affiliation) using permutational MANOVA (Anderson 2001) as implemented in the R software
161
package vegan (Oksanen et al. 2011). In addition, perMANOVA was used to test the
162
significance of similarity among gut communities from hosts of the same order or diet category.
163
Taxonomic diversity was assessed via(?) operational taxonomic unit (OTU) analysis
164
using mothur (Schloss et al. 2009). A report of the fully aligned 16S rRNA gene dataset was
165
used to find a region of the alignment covering 400 base pairs that was common to the greatest
166
percentage of sequences. Sequences with less than 300 base pairs in the region encompassed by
167
E. coli base positions 221-421 were screened from the analysis to ensure every sequence
168
comparison utilized at least 200 base pairs. Less than 10% of sequences were filtered from the
169
dataset, with most belonging to single studies. Samples with > 50% of 16S rRNA gene
170
sequences filtered out were not used further in OTU analysis. A distance matrix was created in
171
mothur with the remaining 16S rRNA gene sequences, which were then clustered into OTUs.
172
Bacterial taxonomic diversity is reported as the number of observed OTUs at 3%, 10%, and 20%
173
16S rRNA gene dissimilarity cutoffs, which correspond roughly to commonly used species,
8
174
family/class and phylum level cutoff limits, respectively (Schloss & Handelsman 2004).
175
MATLAB (Mathworks) was used to graphically display the relative abundance of OTUs at the
176
90% (family/class) cutoff level in each insect sample.
177
Congruence of Host Phylogeny and Community Clustering
178
A cladogram was constructed to represent the phylogeny of the gut community hosts
179
used in this analysis. Because genetic data was not available for every host, a cladogram was
180
created to represent simple bifurcations of hosts, as has already been established by published
181
phylogenies (Fig. S1). Recent phylogenies were used to reconstruct an overall topology based on
182
the following studies of taxa: all of Insecta (Kjer 2004), Isoptera (Legendre et al. 2008),
183
Coleoptera (Hunt et al. 2007), Carabidae (Maddison et al. 1999), Harpalinae (Ober & Maddison
184
2008), Cerambycidae (Marvaldi et al. 2009), Lepidoptera (Kristensen et al. 2007), Diptera
185
(Yeates & Wiegmann 1999), Culicidae (Harbach 2007), Apoidea (Danforth et al. 2006), Apidae
186
(Cardinal et al. 2010) and the Apis genus (Arias & Sheppard 2005). Hosts were excluded from
187
this cladogram when taxonomic information was not included in the publishing paper (e.g.
188
species name) that could discern clade relationships to other taxa.
189
TreeMap v 1.0 was used to test the significance of topological congruence between the
190
Unifrac clustering of gut community samples and the phylogeny of their respective hosts (Fig.
191
S1). TreeMap tests the presence of congruence by the use of component analysis to map possible
192
evolutionary histories of a host tree and a hypothesized host-dependent tree (Page 1996).
193
Cospeciation, duplication, and host-switching are used to explain patterns of congruity between
194
host-parasite pairs. The host dependent tree was randomized over 1000 iterations to give a
195
distribution of cospeciation events that were assessed at each iteration. If the intestinal
196
community clustergram is congruent with the host phylogeny, then the majority of the
9
197
randomizations will have had less cospeciation events than what can be deduced from the actual
198
host-host dependent tree reconstruction (Page 1996).
199
Nucleotide Sequence Accession Numbers
200
Partial 16S rRNA gene sequences for the beetle species determined in this study were
201
submitted to Genbank under the accession numbers: HM920248-HM921042.
202
Results
203
Comparison of Community Membership in Relation to Host Diet & Host Phylogeny
204
We curated a dataset that was comprised of 13,295 16S rRNA gene sequences, including
205
the 795 DNA sequences we recovered from the four beetle species reported here, to compare gut
206
communities among 62 insect species. The dataset represents the combination of 84 gut
207
community samples from 36 published reports. Insect species belonged to 27 different families
208
in seven different orders and included diverse dietary strategies that were consistent with nine
209
general categories of diet (Table 1). In addition, a total of 795 DNA sequences were obtained
210
from 12 Nunn Flats beetle gut 16S rRNA gene libraries: three libraries each from M. cancellata,
211
E. longicollis, G. inferna and C. peregrinator. Eight bacterial phyla were detected among the
212
four species. The majority of the bacteria detected were of the phylum Firmicutes (56.98%) or
213
the γ subdivision of Proteobacteria (29.94%). Jk;dsajk;ds included 16S rRNA gene sequences
214
from the broadest taxonomic range (x of the 8 phyla detected), whereas Y only contained X
215
different phyla.
216
The taxonomic composition of OTU richness showed distinctions between the host diet
217
and order groups. The richness of termite and detritivore gut communities is evident in the
218
widespread, deep phylogenetic diversity they contain (Fig. 1). Several bacterial taxa were found
219
almost exclusively in termites, including Spirochaetes, members of the Clostridiales,
10
220
Synergistetes, Deltaproteobacteria and the Bacteroidales. While the Alphaproteobacteria and
221
Betaproteobacteria are present in many of the samples studied here, they appear most prevalent
222
in the Hymenopteran samples that clustered together. The Bacillales/Lactobacillales and
223
Gammaproteobacteria appeared most prevalent in the other insect samples outside the
224
Hymenopterans or termite/detritivore clusters.
225
Based on the Unifrac distance matrix, gut communities clustered by host diet and host
226
evolutionary history (Fig. 1) and in general, both were significantly associated with gut
227
community clustering variation (p < 0.001). However, there was a disparity in which diets and
228
host orders were significantly similar within their groups (Table 2). All Hymenopteran gut
229
samples clustered exclusive of other insects, regardless of their diet. The similarity of
230
Hymenopteran samples to one another was strongly supported statistically (p < 0.001). Within
231
the Hymenoptera cluster, seven of eight samples from the genera Apis and Bombus clustered
232
together, which is consistent with their hosts’ monophyly. Termite communities also clustered
233
together,15 of 17 termite samples formed a single group (p < 0.001). Lower wood-feeding
234
termites (Rhinotermitidae) clustered separately from higher wood-feeding termites (Termitidae).
235
Bacterial communities of higher termites that are principally detritivorous formed amorphous
236
groups with other communities from non-termite detritivores and one root-feeding herbivore,
237
Melolontha melolontha. Higher wood-feeding termites, Microcerotermes sp. and Nasutitermes
238
sp., also clustered together exclusive of other members of the Termitidae family that are
239
detritivorous. There was no evidence that the topology of the gut community clustering was
240
congruous with the overall phylogeny of their insect hosts (p >> 0.05).
241
242
Gut community samples from hosts of the orders Coleoptera and Diptera did not cluster
into discrete groups nor were their libraries more similar within their respective groups (p > 0.05
11
243
for both orders). Diet appeared to explain the community composition for some of the samples
244
within these two orders. Live tree xylophagous Coleopterans were highly similar (p < 0.01). In
245
addition, statistical analysis supported the clustering of detritivores (p < 0.001), which included
246
the detritivorous termites, two detritus feeding Dipterans, and the beetle Pachnoda ephippiata.
247
Communities from herbivorous hosts, which included members of Lepidoptera, Coleoptera and
248
Heteroptera were also significantly similar to each other (p < 0.05). Significant similarities in
249
communities from predatory, omnivorous or hemataphagous/nectarivorous hosts were not
250
supported (p > 0.05). This is also evident in the lack of any distinct clustering for communities
251
from hosts of these three diets (Fig. 1).
252
PCoA plots using Unifrac distances recapitulated the uniqueness of Hymenopterans and
253
termites among the insects analyzed here (Fig. 2b). The clustering of detritivores was also
254
supported, with all but two house fly samples forming a loose cluster. Of the four Carabid beetle
255
gut communities used in this study, PCoA indicated close clustering of the two primarily
256
carnivorous species, Calosoma peregrinator and Poecilus chalcites, which were separated from
257
the two primarily granivorous Carabids, Anisodactylus sanctaecrucis and Harpalus
258
pensylvanicus. Across all predators, clustering was again not evident.
259
Community Richness & Composition
260
Twenty-five phyla of bacteria were represented in the curated dataset and there were
261
2,202 species level OTUs. The average abundance of OTUs differed widely when the DNA
262
sequences were grouped by host diet and taxa (report range) and within group variation was also
263
high in many of the categories (Fig. 3). The lowest level of species level OTU abundance was
264
consistently found among members of the order Hymenoptera (the little black bugs, 12.68),
265
whereas the highest average species level richness was found among the give order name
12
266
(termites, 98.07 OTUs/sample). Dead/decaying wood xylophagous termites had average levels of
267
species richness (116.8 OTUs/sample) over two times as high as detritivores (52.67), which were
268
the next most rich. Live wood xylophagous beetles contained the second least amount of species
269
level OTUs / sample (15.55), which was second to exclusively Hymenopteran pollenivores.
270
Discussion
271
The results reported here demonstrate a clear influence of both host diet and host
272
taxonomic affiliation on insect gut bacterial communities. Of the different host orders analyzed,
273
Hymenopterans, termites, and to a lesser extent, Lepidopterans showed highly significant
274
clustering (Table 2; Fig. 1). Our analyses confirm that bees and wasps harbor gut bacterial
275
communities that may be entirely unique among insects in both levels of richness and
276
community membership. The original analysis of nearly all of the Hymenopterans used here
277
suggested a high degree of bacterial similarity between the genera Apis and Bombus, which our
278
cluster analysis supports (Martinson et al. 2011). It was also suggested that A. mellifera gut
279
communities may be unique among bees, which is supported here by the clustering of three A.
280
mellifera samples from two independent studies (Babendreier et al. 2007; Martinson et al. 2011).
281
The Apis and Bombus clustering, together with the clustering of all Hymenopterans regardless of
282
diet, provide further evidence of strong host specificity of bacterial communities throughout the
283
evolution of wasps and bees. The behaviors, increased antimicrobial defenses, and other immune
284
functions that evolved for sociality may help explain the uniqueness of Apinae bee gut
285
communities among other wasps and bees (Martinson et al. 2011; Mohr & Tebbe 2006; Stow et
286
al. 2007). While Martinson et al. (2011) found no evidence to suggest the presence of ancestrally
287
derived lineages in A. mellifera, our analysis demonstrates that there is some characteristic, aside
13
288
from diet, of bees and wasps that has maintained their highly unique and relatively simple
289
microbiota throughout their evolution.
290
Termite gut communities were also highly similar to one another, although the effect of
291
diet was more prominent in comparison to bees and wasps. Congenerics generally clustered
292
exclusively together, supporting the hypothesis of coevolution between closely related termite
293
taxa and their microbial communities (Hongoh et al. 2005). In addition, the lower wood-feeding
294
termites (Rhinotermitidae) clustered separately from the higher termites (Termitidae). Within the
295
Termitidae clusters, wood-feeding higher termites (Microcerotermes sp. and Nasutitermes sp.)
296
clustered exclusive from the rest of the detritivorous higher termites suggesting a prominent
297
influence of diet nested within host evolution derived effects. These results are in strong
298
agreement with a distinction between Rhinotermitidae and Termitidae domain level gut
299
community compositions, as well as a further distinction between the wood-feeding and
300
detritivorous higher termites (Brauman et al. 2001). We note that the xylophagous termite gut
301
communities are highly similar to each other, while the detritivorous termites share significant
302
similarities with other detritivorous insects from Coleoptera and Diptera. This likely reflects the
303
maintenance of a highly specialized microbiota that is necessary for efficient lignocellulose
304
metabolism, and thus survival in xylophagous termites (Breznak & Brune 1994). The clustering
305
of the Scarabaeidae beetle Melolontha melolontha, a root-feeding herbivore, with detritivores
306
may reflect host-specificity of gut communities in Scarabaeidae family beetles, which are
307
primarily detritivorous (Egert et al. 2005).
308
Bacterial communities from both Coleopterans and Dipterans were not consistent among
309
their respective orders. The variation in community membership of these two groups can be
310
partially explained by a diet-dependent effect. For example, the xylophagous Coleopterans were
14
311
significantly similar to each other within that dietary guild. This may suggest that this specialized
312
dietary niche either requires a distinct microbiota for nutritional supplementing of the host, or
313
that the metabolites present as a result of this diet selects for a similar assemblage of microbial
314
community. It’s unlikely that live tree xylophagous feeding requires a distinct microbiota as may
315
be the case with decayed wood feeding termites because of the large variation in gut community
316
composition of these hosts (Fig. 2a). Previous evidence also indicates loose affiliations between
317
live tree xylophagous beetles and their microbiota both at the intra and interspecific levels of
318
host comparison (Geib et al. 2009; Grunwald et al. 2010; Schloss et al. 2006). The average level
319
of diversity in live tree xylophagous beetles was starkly lower than that of decayed wood feeding
320
termites (Fig. 3), which indicates widely disparate host-microbiota dynamics between the two
321
groups. This variation in community dynamics may be partially explained by the mechanisms of
322
cellulose digestion, which differs between beetles & termites. Cerambycid beetles depend on
323
ingested fungal enzymes to degrade cellulose (Kukor et al. 1988), whereas lower wood-feeding
324
termites employ cellulolytic protists (Cleveland 1924) and higher wood-feeding termites may
325
rely on bacterial cellulolysis (Warnecke et al. 2007). The three approaches to cellulolysis likely
326
have dramatic effects on metabolites produced during this process and thus on gut microbial
327
community dynamics. Alternatively, feeding on live trees exposes both the host and its internal
328
microbiota to tree physiological responses (particularly chemical) (Hanover 1975; Morewood et
329
al. 2004) which may further shape gut community dynamics in live tree xylophagous beetles and
330
not dead wood xylophagous termites.
331
In many cases it is difficult to separate the effects related to host diet from the effects
332
derived from the host’s taxonomic affiliation. Animal speciation and divergence is often related
333
to the filling of a new dietary niche, as is evident in phytophagous insects (Farrell 1998). In
15
334
addition to the already discussed contrast between diet-variable Hymenopterans and higher
335
termites, several closely related insects with varying diet were analyzed and provide insight into
336
dietary influences on gut communities. Two carnivorous Carabid beetles clustered together (Fig.
337
2a), both being more similar to one another than either to the two omnivorous Carabid beetles
338
analyzed. This is particularly striking because one of the predatory species (P. chalcites) is more
339
closely related to the omnivorous Carabids than to the other predatory species (Maddison et al.
340
1999). Of the three fungus-growing termite samples from M. gilvus, the gut community from
341
newly moulted workers appears to deviate from other detritivores and termites, and even from
342
samples of more mature M. gilvus workers, which is concordant with a previous report (Hongoh
343
et al. 2006b). This result may be an effect of diet, since the older M. gilvus consume more
344
cellulosic substances compared to the newly moulted workers (Hongoh et al. 2006b). In contrast,
345
the adult and larvae samples from Agrilus planipennis clustered together despite their having
346
different diets (foliage and cambium/phloem feeding respectively) (Vasanthakumar et al. 2008).
347
The variation in gut community similarity within hosts of similar diet guild may be
348
partially explained by the availability of metabolic niches within the gut due to the host’s food
349
source and/or the level of symbiosis between the gut community and the host. The lack of
350
similarity in predatory, omnivorous, and hematophagous/nectarivorous insect microbiota may be
351
due to the lack of complexity in ingested nutrients. In comparison to organisms that ingest
352
materials that are more refractory to metabolizing, such as those that consume cellulose, these
353
three types of nutrition may provide less metabolic niches due to the relative simplicity of
354
metabolism associated with them. Predatory diets consist mostly of protein, and blood/nectar
355
diets have large inputs of simple carbohydrates and/or protein, which are both metabolized by a
356
wide range of microorganisms. Alternatively, there may not be a need for nutritional symbiosis
16
357
with a specific gut microbiota in these diet guilds due to the ease of nutrient assimilation
358
associated with these three diets. Either of these explanations would be in agreement with high
359
community variation among most members of these diet guilds. Lastly, some community
360
variation may be explained by functional redundancy that is influenced by gut colonization
361
history. It has been demonstrated through gut community transplants between highly disparate
362
hosts (zebrafish and mice) that transplant communities will resemble the endemic community in
363
function, while still being most phylogenetically similar to its original host’s natural community
364
(Rawls et al. 2006), which suggests functional redundancy across disparate microbial taxa. This
365
mechanism may be driving variation in some of the diet guilds whose communities were not
366
significantly similar within their respective groups.
367
We have reported evidence here that both diet and host-specificity shape gut
368
communities in insects to varying extents depending on the nature of the diet and association
369
with the host over evolutionary time scales. While our analysis focused on the bacterial portion
370
of gut communities, it is perceivable that analyzing fungal, archaeal, and protozoan components
371
may give additional insights, as they are all known to be variously important contributors to gut
372
dynamics (Breznak & Brune 1994; Egert et al. 2003; Grunwald et al. 2010; Hongoh et al.
373
2006b). Our analysis also largely contrasts gut richness in insects with that of extensively studied
374
mammals, which appear to be much more rich than what is largely reported here for insects (Ley
375
et al. 2006). It should be noted however, that one limit to our metaanalysis is that the data was
376
comprised from 37 separate studies, and thus the coverage reported on the communities, and
377
methods used to identify the microbiota was likely very different across all studies. We do
378
however note that this analysis was robust enough to confirm properties of insect gut
379
communities that have been suggested previously.
17
380
In conclusion, the goal of this study was to investigate the emergent properties of insect
381
gut communities from the wealth of data that has been published in the previous 10 years. 
382
???Additionally, the contrasts apparent here suggest differences in insect gut community
383
dynamics as compared to a recently published analysis across mammals (Ley et al. 2008). The
384
mechanisms for this difference are beyond the scope of this analysis, but further exploration may
385
yield interesting insights into the fundamental nature of metazoan-microbiota dynamics. Lastly,
386
an understanding of these dynamics across all of Insecta will not only aid efforts in pest
387
management, but will also elucidate the underlying mechanisms responsible for these
388
associations, which has been recently suggested as necessary for furthering insect gut microbial
389
studies (Hongoh et al. 2006b; Kaltenpoth 2011).
390
391
18
392
393
Acknowledgements
We would like to thank Dr. Sandra Brantley at the Museum of Southwest Biology for
394
assistance in insect identification and vouchering. We would also like to thank the 2008 Sevilleta
395
LTER REU participants, Jennifer Johnson and the Sevilleta National Wildlife Refuge
396
administrative team, the U.S. Fish and Wildlife staff at Sevilleta for making this study possible.
397
The Takacs-Vesbach lab, in particular, Justine Hall, provided laboratory assistance, and Nathan
398
Lord assisted in the entomological portion of this study. A portion of the DNA sequencing for
399
this project was performed at the Genome Sequencing Center at Washington University School
400
of Medicine in St. Louis. Additional sequencing was performed at the Molecular Biology
401
Facility at the University of New Mexico, which is supported by NIH Grant Number
402
1P20RR18754 from the Institute Development Award (IDeA) Program of the National Center
403
for Research Resources. This research was supported by the Undergraduate Nurturing
404
Opportunities program (NSF-DEB 0731350), the Sevilleta LTER REU program (NSF-DBI
405
0755059), and the Louise Stokes Alliance for Minority Participation Bridge to the Doctorate
406
program (NSF-EHR 0832947).
407
19
408
Citations
409
Anderson MJ (2001) A new method for non-parametric multivariate analysis of variance. Austral
410
411
Ecology 26, 32-46.
Andert J, Marten A, Brandl R, Brune A (2010) Inter- and intraspecific comparison of the
412
bacterial assemblages in the hindgut of humivorous scarab beetle larvae (Pachnoda spp.).
413
FEMS microbiology ecology 74, 439-449.
414
Arias MC, Sheppard WS (2005) Phylogenetic relationships of honey bees (Hymenoptera :
415
Apinae : Apini) inferred from nuclear and mitochondrial DNA sequence data. Molecular
416
Phylogenetics and Evolution 37, 25-35.
417
Babendreier D, Joller D, Romeis J, Bigler F, Widmer F (2007) Bacterial community structures in
418
honeybee intestines and their response to two insecticidal proteins. FEMS microbiology
419
ecology 59, 600-610.
420
Behar A, Yuval B, Jurkevitch E (2008) Community Structure of the Mediterranean Fruit Fly
421
Microbiota: Seasonal and Spatial Sources of Variation. Israel Journal of Ecology &
422
Evolution 54, 181-191.
423
Brauman A, Dore J, Eggleton P, et al. (2001) Molecular phylogenetic profiling of prokaryotic
424
communities in guts of termites with different feeding habits. FEMS Microbiol Ecol 35,
425
27-36.
426
427
428
Breznak JA, Brune A (1994) Role of Microorganisms in the Digestion of Lignocellulose by
Termites. Annual Review of Entomology 39, 453-487.
Broderick NA, Raffa KF, Goodman RM, Handelsman J (2004) Census of the bacterial
429
community of the gypsy moth larval midgut by using culturing and culture-independent
430
methods. Applied and Environmental Microbiology 70, 293-300.
20
431
Burgess AF, Collins CW (1917) The Genus Calosoma : Including studies of seasonal histories,
432
habits, and economic importance of American species north of Mexico and of several
433
introduced species (ed. Agricuture USDo). Washington Government Printing Office,
434
Washington D.C. .
435
Cardinal S, Straka J, Danforth BN (2010) Comprehensive phylogeny of apid bees reveals the
436
evolutionary origins and antiquity of cleptoparasitism. Proceedings of the National
437
Academy of Sciences of the United States of America 107, 16207-16211.
438
Cartron JE, Lightfoot DC, Mygatt JE, Brantley SL, Lowery TK (2008) A Field Gude to the
439
Plants and Animals of the Middle Rio Grande Bosque University of New Mexico Press,
440
Albuquerque.
441
Cleveland LR (1924) The physiological and symbiotic relationships between the intestinal
442
protozoa of termites and their host, with special reference to Reticulitermes flavipes
443
Kollar. Biological Bulletin 46, 178-201.
444
Cook DM, DeCrescenzo Henriksen E, Upchurch R, Peterson JB (2007) Isolation of polymer-
445
degrading bacteria and characterization of the hindgut bacterial community from the
446
detritus-feeding larvae of Tipula abdominalis (Diptera: Tipulidae). Applied and
447
Environmental Microbiology 73, 5683-5686.
448
Danforth BN, Sipes S, Fang J, Brady SG (2006) The history of early bee diversification based on
449
five genes plus morphology. Proceedings of the National Academy of Sciences of the
450
United States of America 103, 15118-15123.
451
Delalibera I, Vasanthakumar A, Burwitz BJ, et al. (2007) Composition of the bacterial
452
community in the gut of the pine engraver, Ips pini (Say) (Coleoptera) colonizing red
453
pine. Symbiosis 43, 97-104.
21
454
455
456
457
458
DeSantis TZ, Hugenholtz P, Keller K, et al. (2006) NAST: a multiple sequence alignment server
for comparative analysis of 16S rRNA genes. Nucleic Acids Research 34, W394-W399.
Dillon RJ, Dillon VM (2004) The gut bacteria of insects: Nonpathogenic interactions. Annual
Review of Entomology 49, 71-92.
Dunn AK, Stabb EV (2005) Culture-independent characterization of the microbiota of the ant
459
lion Myrmeleon mobilis (Neuroptera: Myrmeleontidae). Applied and Environmental
460
Microbiology 71, 8784-8794.
461
Egert M, Stingl U, Bruun LD, et al. (2005) Structure and topology of microbial communities in
462
the major gut compartments of Melolontha melolontha larvae (Coleoptera: Scarabaeidae).
463
Applied and Environmental Microbiology 71, 4556-4566.
464
Egert M, Wagner B, Lemke T, Brune A, Friedrich MW (2003) Microbial community structure in
465
midgut and hindgut of the humus-feeding larva of Pachnoda ephippiata (Coleoptera:
466
Scarabaeidae). Applied and Environmental Microbiology 69, 6659-6668.
467
468
Farrell BD (1998) "Inordinate fondness" explained: Why are there so many beetles? Science 281,
555-559.
469
Geib SM, Jimenez-Gasco Mdel M, Carlson JE, Tien M, Hoover K (2009) Effect of host tree
470
species on cellulase activity and bacterial community composition in the gut of larval
471
Asian longhorned beetle. Environmental entomology 38, 686-699.
472
Giovannoni SJ, Delong EF, Schmidt TM, Pace NR (1990) Tangential Flow Filtration and
473
Preliminary Phylogenetic Analysis of Marine Picoplankton. Applied and Environmental
474
Microbiology 56, 2572-2575.
22
475
Grunwald S, Pilhofer M, Holl W (2010) Microbial associations in gut systems of wood- and
476
bark-inhabiting longhorned beetles [Coleoptera: Cerambycidae]. Systematic and Applied
477
Microbiology 33, 25-34.
478
Gusmao DS, Santos AV, Marini DC, et al. (2010) Culture-dependent and culture-independent
479
characterization of microorganisms associated with Aedes aegypti (Diptera: Culicidae)
480
(L.) and dynamics of bacterial colonization in the midgut. Acta Tropica 115, 275-281.
481
482
483
484
485
Hanover JW (1975) Physiology of Tree Resistance to Insects. Annual Review of Entomology 20,
75-95.
Harbach RE (2007) The Culicidae (Diptera): a review of taxonomy, classification and
phylogeny. Zootaxa, 591-638.
Hirose E, Panizzi AR, De Souza JT, Cattelan AJ, Aldrich JR (2006) Bacteria in the gut of
486
southern green stink bug (Heteroptera : Pentatomidae). Annals of the Entomological
487
Society of America 99, 91-95.
488
489
490
Hongoh Y (2010) Diversity and genomes of uncultured microbial symbionts in the termite gut.
Biosci Biotechnol Biochem 74, 1145-1151.
Hongoh Y, Deevong P, Hattori S, et al. (2006a) Phylogenetic diversity, localization, and cell
491
morphologies of members of the candidate phylum TG3 and a subphylum in the phylum
492
Fibrobacteres, recently discovered bacterial groups dominant in termite guts. Applied and
493
Environmental Microbiology 72, 6780-6788.
494
Hongoh Y, Deevong P, Inoue T, et al. (2005) Intra- and interspecific comparisons of bacterial
495
diversity and community structure support coevolution of gut microbiota and termite
496
host. Applied and Environmental Microbiology 71, 6590-6599.
23
497
Hongoh Y, Ekpornprasit L, Inoue T, et al. (2006b) Intracolony variation of bacterial gut
498
microbiota among castes and ages in the fungus-growing termite Macrotermes gilvus.
499
Molecular Ecology 15, 505-516.
500
Hongoh Y, Ohkuma M, Kudo T (2003) Molecular analysis of bacterial microbiota in the gut of
501
the termite Reticulitermes speratus (Isoptera; Rhinotermitidae). FEMS microbiology
502
ecology 44, 231-242.
503
504
505
506
507
Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: a program to detect chimeric sequences
in multiple sequence alignments. Bioinformatics 20, 2317-2319.
Hunt T, Bergsten J, Levkanicova Z, et al. (2007) A comprehensive phylogeny of beetles reveals
the evolutionary origins of a superradiation. Science 318, 1913-1916.
Husseneder C, Ho HY, Blackwell M (2010) Comparison of the bacterial symbiont composition
508
of the formosan subterranean termite from its native and introduced range. The open
509
microbiology journal 4, 53-66.
510
511
512
513
Kaltenpoth M (2011) Honeybees and bumblebees share similar bacterial symbionts. Molecular
Ecology 20, 439-440.
Kane MD, Breznak JA (1991) Effect of Host Diet on Production of Organic-Acids and Methane
by Cockroach Gut Bacteria. Applied and Environmental Microbiology 57, 2628-2634.
514
Kjer KM (2004) Aligned 18S and insect phylogeny. Systematic Biology 53, 506-514.
515
Kristensen NP, Scoble MJ, Karsholt O (2007) Lepidoptera phylogeny and systematics: the state
516
517
518
of inventorying moth and butterfly diversity. Zootaxa, 699-747.
Kukor JJ, Cowan DP, Martin MM (1988) The Role of Ingested Fungal Enzymes in Cellulose
Digestion in the Larvae of Cerambycid Beetles. Physiological Zoology 61, 364-371.
24
519
Lane DJ, Pace B, Olsen GJ, et al. (1985) Rapid-Determination of 16s Ribosomal-Rna Sequences
520
for Phylogenetic Analyses. Proceedings of the National Academy of Sciences of the
521
United States of America 82, 6955-6959.
522
Legendre F, Whiting MF, Bordereau C, et al. (2008) The phylogeny of termites (Dictyoptera :
523
Isoptera) based on mitochondrial and nuclear markers: Implications for the evolution of
524
the worker and pseudergate castes, and foraging behaviors. Molecular Phylogenetics and
525
Evolution 48, 615-627.
526
Lehman R, Lundgren J, Petzke L (2009) Bacterial Communities Associated with the Digestive
527
Tract of the Predatory Ground Beetle, Poecilus chalcites, and Their Modification by
528
Laboratory Rearing and Antibiotic Treatment. Microbial ecology 57, 349-358.
529
Ley RE, Hamady M, Lozupone C, et al. (2008) Evolution of mammals and their gut microbes.
530
531
532
533
Science 320, 1647-1651.
Ley RE, Peterson DA, Gordon JI (2006) Ecological and evolutionary forces shaping microbial
diversity in the human intestine. Cell 124, 837-848.
Liu WT, Marsh TL, Cheng H, Forney LJ (1997) Characterization of microbial diversity by
534
determining terminal restriction fragment length polymorphisms of genes encoding 16S
535
rRNA. Applied and Environmental Microbiology 63, 4516-4522.
536
537
538
539
540
541
Lozupone C, Knight R (2005) UniFrac: a new phylogenetic method for comparing microbial
communities. Applied and Environmental Microbiology 71, 8228-8235.
Ludwig W, Strunk O, Westram R, et al. (2004) ARB: a software environment for sequence data.
Nucleic Acids Research 32, 1363-1371.
Lundgren JG, Lehman M (2010) Bacterial Gut Symbionts Contribute to Seed Digestion in an
Omnivorous Beetle. Plos One 5, -.
25
542
Lundgren JG, Lehman RM, Chee-Sanford J (2007) Bacterial communities within digestive tracts
543
of ground beetles (Coleoptera : Carabidae). Annals of the Entomological Society of
544
America 100, 275-282.
545
Mackenzie LM, Muigai AT, Osir EO, et al. (2007) Bacterial diversity in the intestinal tract of the
546
fungus-cultivating termite Macrotermes michaelseni (Sjostedt). African Journal of
547
Biotechnology 6, 658-667.
548
Maddison DR, Baker MD, Ober KA (1999) Phylogeny of carabid beetles as inferred from 18S
549
ribosomal DNA (Coleoptera : Carabidae). Systematic Entomology 24, 103-138.
550
Martinson VG, Danforth BN, Minckley RL, et al. (2011) A simple and distinctive microbiota
551
associated with honey bees and bumble bees. Molecular Ecology 20, 619-628.
552
Marvaldi AE, Duckett CN, Kjer KM, Gillespie JJ (2009) Structural alignment of 18S and 28S
553
rDNA sequences provides insights into phylogeny of Phytophaga (Coleoptera:
554
Curculionoidea and Chrysomeloidea). Zoologica Scripta 38, 63-77.
555
Mitchell KR, Takacs-Vesbach CD (2008) A comparison of methods for total community DNA
556
preservation and extraction from various thermal environments. Journal of Industrial
557
Microbiology & Biotechnology 35, 1139-1147.
558
Mohr KI, Tebbe CC (2006) Diversity and phylotype consistency of bacteria in the guts of three
559
bee species (Apoidea) at an oilseed rape field. Environmental Microbiology 8, 258-272.
560
Morales-Jimenez J, Zuniga G, Villa-Tanaca L, Hernandez-Rodriguez C (2009) Bacterial
561
community and nitrogen fixation in the red turpentine beetle, Dendroctonus valens
562
LeConte (Coleoptera: Curculionidae: Scolytinae). Microbial ecology 58, 879-891.
26
563
Morewood WD, Hoover K, Neiner PR, McNeil JR, Sellmer JC (2004) Host tree resistance
564
against the polyphagous wood-boring beetle Anoplophora glabripennis. Entomologia
565
Experimentalis Et Applicata 110, 79-86.
566
Ober KA, Maddison DR (2008) Phylogenetic relationships of tribes within Harpalinae
567
(Coleoptera: Carabidae) as inferred from 28S ribosomal DNA and the wingless gene.
568
Journal of Insect Science 8, -.
569
Oksanen J, Guillaume Blanchet F, Kindt R, et al. (2011) vegan : Community Ecology Package.
570
Page RDM (1996) Temporal congruence revisited: Comparison of mitochondrial DNA sequence
571
divergence in cospeciating pocket gophers and their chewing lice. Systematic Biology 45,
572
151-167.
573
Pidiyar VJ, Jangid K, Patole MS, Shouche YS (2004) Studies on cultured and uncultured
574
microbiota of wild culex quinquefasciatus mosquito midgut based on 16s ribosomal RNA
575
gene analysis. The American journal of tropical medicine and hygiene 70, 597-603.
576
Rani A, Sharma A, Rajagopal R, Adak T, Bhatnagar RK (2009) Bacterial diversity analysis of
577
larvae and adult midgut microflora using culture-dependent and culture-independent
578
methods in lab-reared and field-collected Anopheles stephensi-an Asian malarial vector.
579
BMC microbiology 9, 96.
580
Rawls JF, Mahowald MA, Ley RE, Gordon JI (2006) Reciprocal gut microbiota transplants from
581
zebrafish and mice to germ-free recipients reveal host habitat selection. Cell 127, 423-
582
433.
583
584
Robinson CJ, Schloss P, Ramos Y, Raffa K, Handelsman J (2010) Robustness of the bacterial
community in the cabbage white butterfly larval midgut. Microbial ecology 59, 199-211.
27
585
Santo Domingo JW, Kaufman MG, Klug MJ, et al. (1998) Influence of diet on the structure and
586
function of the bacterial hindgut community of crickets. Molecular Ecology 7, 761-767.
587
Schloss PD, Delalibera I, Handelsman J, Raffa KF (2006) Bacteria associated with the guts of
588
two wood-boring beetles: Anoplophora glabripennis and Saperda vestita (Cerambycidae).
589
Environmental entomology 35, 625-629.
590
591
Schloss PD, Handelsman J (2004) Status of the microbial census. Microbiol Mol Biol Rev 68,
686-691.
592
Schloss PD, Westcott SL, Ryabin T, et al. (2009) Introducing mothur: Open-Source, Platform-
593
Independent, Community-Supported Software for Describing and Comparing Microbial
594
Communities. Applied and Environmental Microbiology 75, 7537-7541.
595
Schmitt-Wagner D, Friedrich MW, Wagner B, Brune A (2003) Axial dynamics, stability, and
596
interspecies similarity of bacterial community structure in the highly compartmentalized
597
gut of soil-feeding termites (Cubitermes spp.). Applied and Environmental Microbiology
598
69, 6018-6024.
599
Shinzato N, Muramatsu M, Matsui T, Watanabe Y (2005) Molecular phylogenetic diversity of
600
the bacterial community in the gut of the termite Coptotermes formosanus. Bioscience
601
Biotechnology and Biochemistry 69, 1145-1155.
602
Shinzato N, Muramatsu M, Matsui T, Watanabe Y (2007) Phylogenetic analysis of the gut
603
bacterial microflora of the fungus-growing termite Odontotermes formosanus. Bioscience
604
Biotechnology and Biochemistry 71, 906-915.
605
606
Stow A, Briscoe D, Gillings M, et al. (2007) Antimicrobial defences increase with sociality in
bees. Biol Lett 3, 422-424.
28
607
Su Z, Zhang M, Liu X, et al. (2010) Comparison of bacterial diversity in wheat bran and in the
608
gut of larvae and newly emerged adult of Musca domestica (Diptera: Muscidae) by use of
609
ethidium monoazide reveals bacterial colonization. Journal of Economic Entomology
610
103, 1832-1841.
611
Thongaram T, Hongoh Y, Kosono S, et al. (2005) Comparison of bacterial communities in the
612
alkaline gut segment among various species of higher termites. Extremophiles 9, 229-
613
238.
614
Toolson EC Feeding habits of Epicauta longicollis, Unpublished data.
615
van Borm S, Buschinger A, Boomsma JJ, Billen J (2002) Tetraponera ants have gut symbionts
616
related to nitrogen-fixing root-nodule bacteria. Proceedings of the Royal Society of
617
London Series B-Biological Sciences 269, 2023-2027.
618
Vasanthakumar A, Delalibera I, Handelsman J, et al. (2006) Characterization of gut-associated
619
bacteria in larvae and adults of the southern pine beetle, Dendroctonus frontalis
620
Zimmermann. Environmental entomology 35, 1710-1717.
621
Vasanthakumar A, Handelsman J, Schloss PD, Bauer LS, Raffa KF (2008) Gut microbiota of an
622
invasive subcortical beetle, Agrilus planipennis Fairmaire, across various life stages.
623
Environmental entomology 37, 1344-1353.
624
Warnecke F, Luginbuhl P, Ivanova N, et al. (2007) Metagenomic and functional analysis of
625
hindgut microbiota of a wood-feeding higher termite. Nature 450, 560-565.
626
Yang H, Schmitt-Wagner D, Stingl U, Brune A (2005) Niche heterogeneity determines bacterial
627
community structure in the termite gut (Reticulitermes santonensis). Environmental
628
Microbiology 7, 916-932.
29
629
630
631
632
Yeates DK, Wiegmann BM (1999) Congruence and controversy: Toward a higher-level
phylogeny of diptera. Annual Review of Entomology 44, 397-428.
Yu HW, Wang ZK, Liu L, et al. (2008) Analysis of the intestinal microflora in Hepialus
gonggaensis larvae using 16S rRNA sequences. Current Microbiology 56, 391-396.
633
634
30
635
Tables
636
Table 1. Study and diet information for insect species used in the metaanalysis.
Species
Family
Order
Reference
Diet
Label
Sequenc
es
Aedes aegypti
Culicidae
Diptera
Hematophagous/Nectarivorous
AA
57
Agapostemon
virescens
Agrilus
planipennis
(adult and
larvae)
Anisodactylus
sanctaecrucis
Anopheles
stephensi
(female, male
and larvae)
Anoplophora
glabripennis
Anoplophora
glabripennis
Halictidae
Hymenoptera
Pollenivorous
AV
273
Buprestidae
Coleoptera
(Gusmao et al.
2010)
(Martinson et al.
2011)
(Vasanthakumar
et al. 2008)
Herbivorous
Xylophagous : Live Trees
APa
APl
163
188
Carabidae
Coleoptera
Omnivorous
AS
3
Culicidae
Diptera
(Lundgren et al.
2007)
(Rani et al.
2009)
Hematophagous/Nectarivorous
Hematophagous/Nectarivorous
Filter Feeding
ASTf
ASTm
ASTl
138
141
95
Cerambycidae
Coleoptera
Xylophagous : Live Trees
AG
100
Cerambycidae
Coleoptera
(Schloss et al.
2006)
(Geib et al.
2009)
Apis
andreniformis
Apis dorsata
Apidae
Hymenoptera
Xylophagous : Live Trees
Artificial diet
Callery Pear
Horsechestunut
Pin Oak
Silver Maple
Sugar Maple
Sycamore Maple
Pollenivorous
AGgart
AGgcp
AGghc
AGgpo
AGgvm
AGggm
AGgym
AAN
99
126
27
122
147
196
75
78
Apidae
Hymenoptera
Pollenivorous
AD
72
Apis mellifera
Apidae
Hymenoptera
Pollenivorous
AM
38
Apis mellifera
Apidae
Hymenoptera
Pollenivorous
AMm
271
Apis mellifera
hive-wide
sampling
Bombus
impatiens
Bombus
sonorus
Bombus sp.
Apidae
Hymenoptera
Pollenivorous
AMmh
267
Apidae
Hymenoptera
Pollenivorous
BI
71
Apidae
Hymenoptera
Pollenivorous
BSO
78
Apidae
Hymenoptera
Pollenivorous
BS
80
Calliopsis
subalpinus
Calosoma
peregrinator
Caupolicana
yarrowi
Chalybion
Andrenidae
Hymenoptera
Pollenivorous
CS
282
Carabidae
Coleoptera
(Martinson et al.
2011)
(Martinson et al.
2011)
(Martinson et al.
2011)
(Martinson et al.
2011)
Present study
Predacious
CAL
248
Colletidae
Hymenoptera
Pollenivorous
CY
256
Sphecidae
Hymenoptera
(Martinson et al.
2011)
(Martinson et al.
Predacious
CC
204
(Martinson et al.
2011)
(Martinson et al.
2011)
(Babendreier et
al. 2007)
(Martinson et al.
2011)
(Martinson et al.
2011)
31
californicum
2011)
Colletes
inaequalis
Coptotermes
formosanus
Coptotermes
formosanus
Cubitermes
orthognathus
Culex
quinquefasciat
us
Dendroctonus
frontalis
(adult and
larvae)
Dendroctonus
valens
(adult and
larvae)
Diadasia
opuntia
Epicauta
longicollis
Gonasida
inferna
Halictus
patellatus
Harpalus
pensylvanicus
Hepialus
gonggaensis
Hesperapis
cockerelli
Hoplitis
biscutellae
Ips pini
Colletidae
Hymenoptera
(Martinson et al.
2011)
(Shinzato et al.
2005)
(Husseneder et
al. 2010)
(Schmitt-Wagner
et al. 2003)
(Pidiyar et al.
2004)
Pollenivorous
CI
301
Rhinotermitid
ae
Rhinotermitid
ae
Termitidae
Isoptera
Xylophagous : Dead Wood
CFs
51
Xylophagous : Dead Wood
CF
206
Detritivorous
CO
102
Culicidae
Diptera
Hematophagous/Nectarivorous
CQ
168
Curculionidae
Coleoptera
(Vasanthakumar
et al. 2006)
Xylophagous : Live Trees
DFa
DFl
99
91
Curculionidae
Coleoptera
(MoralesJimenez et al.
2009)
Xylophagous : Live Trees
DVa
DVl
32
8
Apidae
Hymenoptera
Pollenivorous
DO
347
Meloidae
Coleoptera
(Martinson et al.
2011)
Present study
Herbivorous
EPI
177
Tenebrionidae
Coleoptera
Present study
Omnivorous
GON
230
Halictidae
Hymenoptera
Pollenivorous
HPA
305
Carabidae
Coleoptera
Omnivorous
HP
6
Hepialidae
Lepidoptera
(Martinson et al.
2011)
(Lundgren et al.
2007)
(Yu et al. 2008)
Herbivorous
HG
35
Dasypodaidae
Hymenoptera
Pollenivorous
HC
349
Megachilidae
Hymenoptera
Pollenivorous
HB
182
Curculionidae
Coleoptera
Xylophagous : Live Trees
IP
77
Leptura rubra
Cerambycidae
Coleoptera
Xylophagous : Live Trees
LR
65
Lymantria
dispar
Lymantriidae
Lepidoptera
(Martinson et al.
2011)
(Martinson et al.
2011)
(Delalibera et al.
2007)
(Grunwald et al.
2010)
(Broderick et al.
2004)
Macrotermes
gilvus
(newly
moulted, old
and young
workers)
Macrotermes
michaelseni
Megachile
odontostoma
Termitidae
Isoptera
(Hongoh et al.
2006b)
LDart
LDasp
LDlar
LDwo
LDwil
MGnw
MGow
MGyw
10
11
16
7
10
26
88
82
Termitidae
Isoptera
Detritivorous
MMI
47
Megachilidae
Hymenoptera
(Mackenzie et al.
2007)
(Martinson et al.
2011)
Pollenivorous
MO
338
Isoptera
Isoptera
Herbivorous
Artificial Diet
Aspen Trees
Larch Trees
White Oak Trees
Willow Trees
Detritivorous
32
Megetra
cancellata
Melolontha
melolontha
Microceroter
mes sp. M1
Microceroter
mes sp. M2
Musca
domestica
(adult and
larvae)
Myrmeleon
mobilis
Nasutitermes
sp.
Nasutitermes
takasagoensis
Nezara
viridula
Odontotermes
formosanus
Pachnoda
ephippiata
Pachnoda
ephippiata
Paragia
vespiformis
Philanthus
gibbosus
Pieris rapae
Meloidae
Coleoptera
Present study
Herbivorous
MEG
140
Scarabaeidae
Coleoptera
Herbivorous
MME
164
Termitidae
Isoptera
Xylophagous : Dead Wood
MS1
217
Termitidae
Isoptera
Xylophagous : Dead Wood
MS2
128
Muscidae
Diptera
(Egert et al.
2005)
(Hongoh et al.
2005)
(Hongoh et al.
2005)
(Su et al. 2010)
Detritivorous
MDa
MDl
4
11
Myrmeleontid
ae
Termitidae
Neuroptera
Predacious
MM
34
Xylophagous : Dead Wood
NS
1252
Termitidae
Isoptera
Xylophagous : Dead Wood
NT
130
Pentatomidae
Heteroptera
Herbivorous
NV
21
Termitidae
Isoptera
Detritivorous
OF
56
Scarabaeidae
Coleoptera
Detritivorous
PEe
108
Scarabaeidae
Coleoptera
Detritivorous
PEa
87
Masaridae
Hymenoptera
Pollenivorous
PV
247
Crabronidae
Hymenoptera
Predacious
PG
360
Pieridae
Lepidoptera
Herbivorous
PR
1207
Plagionotus
arcuatus
Poecilus
chalcites
Rediviva
saetigera
Reticulitermes
santonensis
Reticulitermes
sp. R1
Reticulitermes
speratus
Reticulitermes
speratus
Rhagium
inquisitor
Saperda
vestita
Termes comis
Cerambycidae
Coleoptera
Xylophagous : Live Trees
PA
44
Carabidae
Coleoptera
Predacious
PC
45
Melittidae
Hymenoptera
Pollenivorous
RSA
333
Rhinotermitid
ae
Rhinotermitid
ae
Rhinotermitid
ae
Rhinotermitid
ae
Cerambycidae
Isoptera
Xylophagous : Dead Wood
RST
111
Xylophagous : Dead Wood
RSP
50
Xylophagous : Dead Wood
RS
270
Xylophagous : Dead Wood
RSh
108
Xylophagous : Live Trees
RI
85
Cerambycidae
Coleoptera
Xylophagous : Live Trees
SV
80
Termitidae
Isoptera
Detritivorous
TC
57
Tetropium
castaneum
Tipula
abdominalis
Cerambycidae
Coleoptera
Xylophagous : Live Trees
TCA
74
Tipulidae
Diptera
(Dunn & Stabb
2005)
(Warnecke et al.
2007)
(Hongoh et al.
2006a)
(Hirose et al.
2006)
(Shinzato et al.
2007)
(Egert et al.
2003)
(Andert et al.
2010)
(Martinson et al.
2011)
(Martinson et al.
2011)
(Robinson et al.
2010)
(Grunwald et al.
2010)
(Lehman et al.
2009)
(Martinson et al.
2011)
(Yang et al.
2005)
(Hongoh et al.
2005)
(Hongoh et al.
2003)
(Hongoh et al.
2005)
(Grunwald et al.
2010)
(Schloss et al.
2006)
(Thongaram et
al. 2005)
(Grunwald et al.
2010)
(Cook et al.
2007)
Detritivorous
TA
206
Isoptera
Isoptera
Isoptera
Isoptera
Coleoptera
33
Xylocopa
californica
Apidae
Hymenoptera
(Martinson et al.
2011)
Pollenivorous
XC
305
637
638
Table 2. Results of permutational multivariate analysis of variance (perMANOVA) by host diet
639
and orderab
Category
n
r2
Pr (>F)
Diet
86
0.48308
0.000999
Detritivorous
12
0.08562
0.000999
Hematophagous/Nectarivorous
4
0.00548
0.7712
Herbivorous
12
0.03608
0.02897
Omnivorous
3
0.01337
0.3137
Pollenivorous
20
0.1392
0.000999
Predacious
5
0.0142
0.2957
Xylophagous (primarily
10
0.21221
0.000999
19
0.05558
0.002997
86
0.46941
0.000999
Coleoptera
30
0.02416
0.1129
Diptera
8
0.00701
0.6324
Hymenoptera
22
0.16947
0.000999
Isoptera
17
0.3177
0.000999
Lepidoptera
7
0.06357
0.001998
dead/decaying woods)
Xylophagous (primarily live
trees)
Order
640
a
641
b
Groups with only one representative were excluded from this analysis
Pseudo F statistics are based on 1000 permutations
642
34
643
Figure Legends
644
Fig. 1. Clustering of insect gut bacterial communities by Unifrac distance metric with
645
corresponding OTU relative abundance for each host displayed by bacterial taxa. Clustergram
646
leaves are colored according to host diet. Scale indicates the relative abundance of each OTU
647
within each individual host. Hosts without OTU information were not used in OTU analysis due
648
to incompatible 16S rRNA gene sequence data.
649
Fig. 2. PCoA plots of gut community similarities among host diet (2a) and order (2b). Asterisks
650
in 2a denote Carabid beetles.
651
Fig. 3. Average levels of bacterial OTU richness by host diet (3a) and order (3b). Bars indicate
652
standard deviation within groups. Only groups with more than one representative gut community
653
sample are shown.
654
Fig. S1. TreeMap tanglegram showing the host (based on what) cladogram on left and the
655
Unifrac gut community clustering on right. Red lines indicate community-host relationships.
656
Black dots at nodes are hypothesized ‘cospeciation’ events as inferred in the intial Treemap
657
reconstruction.
35
658
Figures
659
Figure 1.
36
660
661
37
662
Figure 2a.
*
*
*
*
663
664
Figure 2b.
665
666
38
667
Figure 3a.
668
39
669
Figure 3b.
670
671
40
672
Figure S1
HG
DFl
AA
LD
HG
PR
TCA
AGgcp
BI
DVa
BSO
AGghc
MDl
AAN
AD
AM
DO
HP
NV
LDart
LDwil
LDlar
XC
LDwo
MM
HB
AS
MO
MDa
CS
DFa
AV
DVl
PA
HPA
SV
AGgar
CI
RI
CY
AG
RSA
IP
AGgpo
HC
LDasp
CC
AGggm
PG
AGgym
ASTl
PV
CQ
AST
ASTf
ASTm
AA
GON
CQ
EPI
CAL
MD
MEG
TA
PC
MGnw
GON
MMI
EPI
PR
MEG
AGgvm
LR
PA
APa
TCA
APl
AD
LR
AAN
RI
BI
AG
BSO
AM
SV
AMm
IP
AMmh
AV
DF
MO
DV
HC
AP
CS
PG
MME
XC
PEa
CC
CI
AS
HPA
HP
PV
PC
RSA
DO
CAL
CY
MM
HB
TA
NV
CF
TC
CFs
RS
MS1
MS2
RSh
RST
NS
CO
NT
TC
MME
CO
OF
MGow
OF
MG
MMI
MGyw
PEa
PEe
NS
CF
NT
RS
RST
MS1
MS2
Mon May 09 17:36:51 2011
673
41