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Compartmentalized microbial composition, oxygen gradients and nitrogen fixation in the gut of Odontotaenius disjunctus

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Coarse woody debris is an important biomass pool in forest ecosystems that numerous groups of insects have evolved to take advantage of. These insects are ecologically important and represent useful natural analogs for biomass to biofuel conversion. Using a range of molecular approaches combined with microelectrode measurements of oxygen, we have characterized the gut microbiome and physiology of Odontotaenius disjunctus, a wood-feeding beetle native to the eastern United States. We hypothesized that morphological and physiological differences among gut regions would correspond to distinct microbial populations and activities. In fact, significantly different communities were found in the foregut (FG), midgut (MG)/posterior hindgut (PHG) and anterior hindgut (AHG), with Actinobacteria and Rhizobiales being more abundant toward the FG and PHG. Conversely, fermentative bacteria such as Bacteroidetes and Clostridia were more abundant in the AHG, and also the sole region where methanogenic Archaea were detected. Although each gut region possessed an anaerobic core, micron-scale profiling identified radial gradients in oxygen concentration in all regions. Nitrogen fixation was confirmed by (15)N2 incorporation, and nitrogenase gene (nifH) expression was greatest in the AHG. Phylogenetic analysis of nifH identified the most abundant transcript as related to Ni-Fe nitrogenase of a Bacteroidetes species, Paludibacter propionicigenes. Overall, we demonstrate not only a compartmentalized microbiome in this beetle digestive tract but also sharp oxygen gradients that may permit aerobic and anaerobic metabolism to occur within the same regions in close proximity. We provide evidence for the microbial fixation of N2 that is important for this beetle to subsist on woody biomass.
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ORIGINAL ARTICLE
Compartmentalized microbial composition, oxygen
gradients and nitrogen fixation in the gut of
Odontotaenius disjunctus
Javier A Ceja-Navarro
1,6
, Nhu H Nguyen
2,6
, Ulas Karaoz
1
, Stephanie R Gross
3
,
Donald J Herman
4
, Gary L Andersen
1
, Thomas D Bruns
1
, Jennifer Pett-Ridge
5
,
Meredith Blackwell
3
and Eoin L Brodie
1,4
1
Ecology Department, Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA;
2
Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA;
3
Department of
Biological Sciences, Louisiana State University, Baton Rouge, LA, USA;
4
Department of Environmental
Science Policy and Management, University of California, Berkeley, CA, USA and
5
Chemical Sciences
Division, Lawrence Livermore National Laboratory, Livermore, CA, USA
Coarse woody debris is an important biomass pool in forest ecosystems that numerous groups of
insects have evolved to take advantage of. These insects are ecologically important and represent
useful natural analogs for biomass to biofuel conversion. Using a range of molecular approaches
combined with microelectrode measurements of oxygen, we have characterized the gut microbiome
and physiology of Odontotaenius disjunctus, a wood-feeding beetle native to the eastern United
States. We hypothesized that morphological and physiological differences among gut regions
would correspond to distinct microbial populations and activities. In fact, significantly different
communities were found in the foregut (FG), midgut (MG)/posterior hindgut (PHG) and anterior
hindgut (AHG), with Actinobacteria and Rhizobiales being more abundant toward the FG and PHG.
Conversely, fermentative bacteria such as Bacteroidetes and Clostridia were more abundant in the
AHG, and also the sole region where methanogenic Archaea were detected. Although each gut
region possessed an anaerobic core, micron-scale profiling identified radial gradients in oxygen
concentration in all regions. Nitrogen fixation was confirmed by
15
N
2
incorporation, and nitrogenase
gene (nifH) expression was greatest in the AHG. Phylogenetic analysis of nifH identified the most
abundant transcript as related to Ni–Fe nitrogenase of a Bacteroidetes species, Paludibacter
propionicigenes. Overall, we demonstrate not only a compartmentalized microbiome in this beetle
digestive tract but also sharp oxygen gradients that may permit aerobic and anaerobic metabolism
to occur within the same regions in close proximity. We provide evidence for the microbial fixation
of N
2
that is important for this beetle to subsist on woody biomass.
The ISME Journal advance online publication, 29 August 2013; doi:10.1038/ismej.2013.134
Subject Category:
Microbial population and community ecology
Keywords: symbiosis; microbial diversity; gut microbiome; insect; Passalidae; cellulose
Introduction
Coarse woody debris represents a significant frac-
tion of the total forest carbon pool, for example, 14%
in the United States alone (USDA, 2010). As a result,
a number of insect groups have evolved and adapted
to take advantage of this abundant resource, forming
specialist wood-feeding (xylophagous) guilds.
Among the most prominent groups of xylophagous
insects that have specialized in this manner are the
termites (Isoptera) and specific groups of beetles
(Coleoptera). These groups are considered econom-
ically important insects because of the destructive-
ness of some of their members, including
subterranean termites (Rhinotermitidae), powder-
post beetles (Bostrichidae), ambrosia and
bark beetles such as the mountain pine beetle
(Scolytinae) and longhorn beetles (Cerambycidae).
The ability of these insects to perform mechanical
and enzymatic breakdown of coarse woody biomass
is of critical importance to the ecology of many
ecosystems (Kaufman et al., 2000; Moran, 2007)
and may also hold the potential for bioenergy
applications because of the efficiency and diversity
of novel gut biota with unexplored capabilities
Correspondence: JA Ceja-Navarro or EL Brodie, Ecology Depart-
ment, Earth Sciences Division, Lawrence Berkeley National
Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA.
E-mail: JCNavarro@lbl.gov or ELBrodie@lbl.gov
6
These authors contributed equally to this work.
Received 10 September 2012; revised 3 July 2013; accepted 13
July 2013
The ISME Journal (2013), 1–13
&
2013 International Society for Microbial Ecology All rights reserved 1751-7362/13
www.nature.com/ismej
(Hongoh et al., 2003; Stingl et al., 2004; Ohkuma
et al., 2007; Ohkuma and Brune, 2011).
The ability to subsist on woody biomass is due in
large part to the symbiotic associations between
wood-eating insects and their gut microorganisms
(Ohkuma, 2003; Tokuda and Watanabe, 2007). The
diversity and functions of these gut communities
have been primarily described in termites, which
form two primary groups, the lower and higher
termites (Wood, 1986). Termites rely on their gut
microbiota to aid in digestion. Despite their ability
to break down woody biomass into simple carbon
compounds, fermentable sugars and hydrogen gas
for use in both catabolic and anabolic processes, the
high carbon(C):nitrogen(N) ratio of the substrate
results in N limitation (Machida et al., 2001).
To alleviate N limitation, wood-feeding termites
harbor N-fixing microorganisms in anaerobic gut
regions (e.g. reviewed in Brune and Ohkuma,
(2011)). The gut microbiome of wood-feeding beetles
likely overcomes similar N constraints, and certain
beetles have received recent attention regarding
their digestive tract morphology and culturable
fungal communities (Slaytor, 1992; Suh et al.,
2003; Nardi et al., 2006; Klass et al., 2008).
One of the most ubiquitous members of the beetle
wood-feeding guild in the eastern United States is
Odontotaenius disjunctus (Passalidae), a large beetle
that can reach B30 mm in length and is common
from Florida northward to southern Canada and
westward to the Rocky Mountains. These insects
spend their entire lives (14–16 months) within
decayed wood of class III or above (visual and
physical signs of decay present) where they feed and
raise their larvae in subsocial communities. Adult
beetles feed larvae a mixture of macerated wood and
frass to replenish the microbes lost while shedding
of the sclerotized hindgut during larval develop-
ment (Pearse et al., 1936; Gray, 1946). Observations
of this behavior, and the high degree of genetic
similarity of fungal associates found in widely
dispersed beetle specimens (Suh et al., 2003),
provides an initial indication of the importance of
microbial symbionts to these beetles.
Despite over a century of studies, the ecological
roles of many of the hindgut microbes of
O. disjunctus remain unknown. The first reported
gut microorganism of this beetle was the protozoan
Gregarina passali-cornuti (Leidy, 1852). Later, the
communities of phoretic arthropods and eukaryotic
gut organisms were more extensively reported by
Pearse et al. (1936), followed a decade later by life
history studies by Gray (1946). More recently,
a number of yeasts and other eukaryotic organisms,
including parabasalids, have been cultured or
cloned and identified from the O. disjunctus gut
(Suh et al., 2003; Zhang et al., 2003; Nguyen et al.,
2006). One of these yeasts is the potentially
commercially important species, Scheffersomyces
stipitis (syn. Pichia stipitis), that has the ability
to ferment xylose, a significant component of
hemicellulose (Shi et al., 2010). In a detailed study
of O. disjunctus gut morphology and gut inhabi-
tants, Nardi et al. (2006) divided each gut into four
morphologically distinct regions where the
gut biota appears to be differentiated as noted
previously for fungus-like organisms known as
trichomycetes (Lichtwardt et al., 1999).
Here we describe the bacterial, archaeal, includ-
ing N-fixing populations across the four gut regions
of O. disjunctus, and characterize the physicochem-
ical properties (pH, O
2
) and nitrogenase gene
expression activity of each. We show that microbial
diversity and phylogenetic clustering are differen-
tiated by gut region, that nitrogen fixation occurs
and that nitrogenase-expressing organisms and over-
all gene expression vary by gut region. Our phylo-
genetic analyses showed that many bacterial taxa are
related to well-characterized cellulolytic organisms,
and we show that both acetoclastic and hydrogeno-
trophic methanogens are confined to a single gut
region. Unlike termite digestive tracts, all gut
regions of the passalid beetle have anaerobic cores,
although steep radial O
2
gradients may permit both
aerobic and anaerobic processes to occur in close
proximity in each region.
Materials and methods
Specimens
Specimens of common size—measuring approxi-
mately 30 mm in length—were collected from a
single oak log in Baton Rouge, LA, USA (30124.98
0
N
9117.18
0
W) and kept in individual containers
together with the wood pieces on which they were
collected. Dissection and bacterial/archaeal commu-
nity identification were made approximately 2
weeks after the collection date.
Beetle dissection, DNA extraction and pH
measurements
Beetles were surface sterilized by immersion in 95%
ethanol for 2 min, followed by a wash in sterile
phosphate-buffered saline. Individual beetles were
dissected in sterile phosphate-buffered saline by
first removing the elytra to expose the membranous
dorsal side. Subsequently, the wings were removed
and the cuticular membrane was dissected to expose
the natural gut alignment within the abdominal
cavity (Figure 1). The whole gut was then removed,
stretched out and cut into four regions (foregut—FG,
midgut—MG, anterior hindgut—AHG, posterior
hindgut—PHG; Figure 1b). Each region was placed
in 1 ml RNALater (Qiagen, Valencia, CA, USA) and
stored overnight at 4 1C before extraction. Crude
extracts were prepared by bead beating each gut
region in 750 ml RLT (guanidine thiocyanate) buffer
(Qiagen) in Lysing Matrix E tube (Qbiogene Inc.,
Carlsbad, CA, USA) for 30 s at 5.5 m s
1
, cooling for
1 min and repeating the process for another 30 s.
Nucleic acids were separated from the mixture by
Diversity and functions of gut communities in
O. disjunctus
JA Ceja-Navarro et al
2
The ISME Journal
using standard phenol–chloroform phase-separation
techniques and precipitated with ethanol. Crude
nucleic extracts were further purified using the
AllPrep DNA/RNA Kit (Qiagen), according to the
manufacturer’s instructions, to simultaneously sepa-
rate the DNA and RNA fractions. In another set of
four beetles, the pH of each gut region was measured
by dissecting out each portion and homogenizing
the gut contents in 30% water (w v
1
).
PCR amplification for PhyloChip analyses
Our experiments consisted of four beetles (repli-
cates) and four gut regions per beetle (16 samples).
Polymerase chain reaction (PCR) amplifications
were performed in triplicated reactions per sample
with three different annealing temperatures using
5 ml1 Takara ExTaq PCR buffer with MgCl
2
, 300 pM
of primers 27F (5
0
-GTTTGATCCTGGCTCAG-3
0
) and
1492R (5
0
-GGTTACCTTGTTACGACTT-3
0
) for bacteria,
1492R and 4F (5
0
-TCCGGTTGATCCTGCCRG-3
0
)for
Archaea, 50 mg bovine serum albumin, 200 m
M
dNTPs, 2.5 U ExTaq DNA polymerase (Takara Mirus
Bio Inc., Madison, WI, USA), 5 ng template and
milliQ H
2
O to complete 50 ml volume. PCR cycling
was performed with an initial denaturation at 95 1C
for 3 min, followed by 25 cycles at 95 1C for 30 s,
annealing at 48 1C, 53 1C and 56 1C for 25 s, extension
at 72 1C for 2 min and a final extension of 72 1C for
10 min. Products from the three different annealing
temperatures (150 ml) were combined and concen-
trated using ethanol precipitation (15 ml3
M sodium
acetate and 300 ml 100% ethanol). Pellets were
resuspended in TE (Tris-EDTA) buffer and purified
using a PCR Purification Kit (Qiagen) before
quantification by gel electrophoresis.
PhyloChip hybridization and sample detection
Microarray construction, methods for labeling,
hybridization, detection and quantification were
described in detail by Brodie et al. (2006). The G2
PhyloChip contains more than 300 000 probes that
target 8741 bacterial and archaeal taxa. From the
combined PCR reactions, 200 ng of bacterial and
50 ng of archaeal PCR products were fragmented
with DNAse I, biotin labeled, hybridized, washed
and stained according to the manufacturer’s recom-
mended protocol. In total, 16 microarrays were
analyzed. Each PhyloChip was scanned and
recorded as a pixel image, and initial data acquisi-
tion and intensity determination were performed
using standard Affymetrix software (GeneChip
microarray analysis suite, version 5.1 Santa Clara,
CA, USA). A taxon/OTU (Operational Taxonomic
Unit) was considered present in the sample when
over 90% of its assigned probe pairs were positive in
at least three of the four replicates per gut region.
Statistical analysis of PhyloChip assays
All statistical analyses were carried out in the R
programming environment (The R Development
Core Team, 2010). Corrections for variation asso-
ciated with quantification of amplicon target and
downstream variation associated with target frag-
mentation, labeling, hybridization, washing, stain-
ing and scanning were performed as detailed in
Ivanov et al. (2009). Intensities of each taxon/OTU
across the four gut regions were tested for signifi-
cance using analysis of variance. The resulting
P-values were corrected for multiple testing using
the Benjamini–Hochberg false discovery rate proce-
dure (Benjamini and Hochberg, 1995). A phyloge-
netic tree for the bacterial taxa detected was
generated as detailed in Goldfarb et al. (2011) and
visualized and annotated using the interactive tree
of life web server (Letunic and Bork, 2007).
Phylogenetic community analysis
Phylogenetic community analyses were performed
using the Picante R package (Kembel et al., 2010).
A maximum-likelihood RA ML (Stamatakis et al.,
2005) tree of sequences targeted by the G2 Phylo-
Chip was used as the initial tree. Taxa not detected
in the gut regions or with ambiguous designations
were trimmed from the tree, resulting in a tree with
1382 taxa. We used the tip shuffling null model
(model 1) running 10 000 replicates (Webb et al.,
2008). In this model, the tips (individual taxa) are
shuffled throughout the phylogeny and the resulting
metrics were compared with the observed value
with a ¼ 0.05. The tip shuffling model was deter-
mined to be robust for Phylocom analysis with a low
level of Type I error (false positives; Hardy, 2008).
We used the net relatedness index (NRI) metric
to compare phylogenetic clustering of microbial
communities across the distinct gut regions.
Figure 1 (a) Dorsal view of a dissected passalid beetle showing
the gut arrangement within the abdominal cavity. (b) Dissected
beetle gut showing entire gut removed and extended.
Diversity and functions of gut communities in
O. disjunctus
JA Ceja-Navarro et al
3
The ISME Journal
We also determined the phylogenetic diversity (PD)
(Faith, 1992; Kembel et al., 2010) and the Shannon
diversity index (H
0
; Table 1).
Confirmation of nitrogen fixation by
15
N
2
incorporation
Four beetles were placed in an anesthetizing box
connected to a balloon filled with 66 ml of O
2
,and
flushed with 99 atom %
15
N
2
to achieve an N
2
concentration of approximately 78%. After 12 days
of incubation, the box was purged and beetles were
removed for dissection and extraction of nucleic
acids as described above. RNA extracts were pre-
pared for isotopic analysis by pipetting the extract
onto Chromosorb W (Advanced Minerals, Santa
Clara, CA, USA) in tin capsules. Samples were
analyzed for N content and d
15
N with isotope ratio
mass spectrometry using a RoboPrep-CN analyzer
coupled to a model 20–20 isotope ratio mass spectro-
meter (Sercon Ltd., Cheshire, UK).
NifH expression
We used 100 ng of total RNA from each gut region of
three beetles for the synthesis of cDNA. Total RNA,
300 ng of random hexamer primers and 20 nmol of
each dNTP were combined to a total volume of 11 ml
and the mixture incubated at 65 1C for 5 min. Then,
5 mlof5 first-strand buffer was added to
the mixture, together with 20 U of SUPERase-ln
(Invitrogen, Grand Island, NY, USA) and incubated
at 25 1C for 2 min. After incubating, 200 U of Super-
Script III reverse transcriptase (Invitrogen) was
added to the mixture and incubated at 50 1C for
50 min. The reaction was inactivated at 70 1C
for 10 min. cDNA was then used as template for
quantitiative PCR (qPCR) of the nifH (nitrogenase
gene) and rpoB (RNA polymerase b-subunit) genes
using the primers PolF and PolR, and 1689F and
2041R (Dahllo
¨
f et al., 2000; Poly et al., 2001). qPCR
conditions were optimized using a temperature
gradient. A single band of the expected size
(360 bp for nifH and 390 bp for rpoB) was considered
indicative of accurate amplification, and cloned and
sequenced for validation and for further use as a
qPCR standard. Amplification reactions were car-
ried out in a total volume of 25 ml, which consisted
of 1 ml of a diluted (1:10) cDNA template, 12.5 mlof
2 iQ SYBR green super mix (Bio-Rad, Hercules,
CA, USA), 0.4 m
M of each primer and 9.5 ml of water.
The PCR conditions were 95 1C for 3 min, then 45
cycles of 95 1C for 10 s, followed by 59 and 56 1C for
30 s with the iQ system (Bio-Rad) for nifH and rpoB,
respectively. DNA standards (cloned rpoB and nifH
fragments) were quantified by fluorescence using
the Qubit system (Invitrogen). Threshold cycle (C
T
)
values were determined in triplicate for each
sample. For all qPCR assays, there was a linear
relationship between the log of the standard DNA
copy number and the calculated threshold cycle
across the specified concentration (r
2
¼ 0.98). PCR
amplification efficiencies of standards and samples
were determined to be within the range of 98–105%
across all assays with a lower detection limit of 20
molecules per ml. Runs that fell outside of these
ranges (r
2
o0.98 and efficiency o0.98 or 41.1) were
repeated. qPCR efficiency and sample threshold
values were used to calculate the normalized
expression ratio of nifH in reference to rpoB using
the 2
DDCT
method (Livak and Schmittgen, 2001).
NifH cloning, sequencing and phylogenetic analysis
qPCR products from each gut region were pooled
and purified using the QIAquick PCR Purification
Kit (Qiagen). Purified products were used as
template for a second PCR using the nifH primers
described above. PCR was conducted in a final
reaction volume of 25 ml, containing 5 mlof10
ExTaq buffer, 200 m
M of dNTPs, 2.5 U ExTaq DNA
polymerase (Takara Mirus Bio Inc.), 800 ng ml
1
of
bovine serum albumin, 10 ng of template and
diethylpyrocarbonate-treated water. PCR products
were cloned into competent Escherichia coli DH10B
cells using the pGEM-T Easy Vector Kit (Promega,
Madison, WI, USA) according to the manufacturers
instructions. Transformants were incubated in mod-
ified lysogeny broth media (0.4% glycerol and
1.7 m
M KH
2
PO
4
and 7.2 mM K
2
HPO
4
)at371C and
300 r.p.m. for 19 h. Plasmids were extracted using
the SeqPrep 96 HP Plasmid Prep Kit (EdgeBio,
Gaithersburg, MD, USA) and sequenced with an
ABI377 sequencer (Applied Biosystems, Grand
Island, NY, USA) using M13 universal forward and
reverse sequencing primers. Sequences were visua-
lized using 4Peaks and edited with the Seaview
software (Gouy et al., 2010). DNA sequences were
translated in all frames using the CLC sequence Viewer
6 (CLC Bio, Cambridge, MA, USA). Translated nifH
sequences were aligned using Clustal X (Thompson,
1997) with corresponding nifH reference sequences
from National Center for Biotechnology Information
and phylogenetic analysis was carried out with
maximum parsimony criteria in PAUP (Phylogenetic
Analysis Using Parsimony) (Swofford, 1998). Heuristic
tree searches were performed using a tree bisection
Table 1 Phylogenetic community metrics for each region of the
passalid beetle gut
Gut region OTU
a
H
0
PD NRI P
NRI
FG 1179 7.08 208.9 7.34 0.0001
MG 825 6.72 172.5 6.50 0.0001
AHG 730 6.56 169.8 3.99 0.0002
PHG 937 6.85 183.9 5.52 0.0001
Abbreviations: AHG, anterior hindgut foregut; FG, foregut;
H
0
, Shannon diversity index; MG, midgut; NRI, net relatedness index;
OTU, Operational Taxonomic Unit; PD, phylogenetic diversity;
PHG, posterior hindgut; P
NRI
, P-value of NRI metric.
Positive NRI values indicate phylogenetic clustering.
a
Relative richness across gut regions, these patterns were insensitive
to pf threshold (see Supplementary Table 2).
Diversity and functions of gut communities in
O. disjunctus
JA Ceja-Navarro et al
4
The ISME Journal
reconnection model and a branch-swapping algorithm
with 100 random stepwise swaps. One hundred trees
were calculated for each pseudoreplicate. A rescaled
consistency index, derived from trees and obtained by
unweighted analysis, was used to generate an a
posteriori weighted data set. The same heuristic search
conditions as used for the unweighted data were used
to analyze the weighted data set. Branch support was
obtained with 100 bootstrap replicates. Nitrosomonas
marina was used as outgroup in each phylogenetic
reconstruction.
O
2
measurements using microelectrodes
A set of three beetles was used to measure the profiles
of O
2
at each gut region. Clark-type oxygen microelec-
trodes (OX-25; Unisense, Aarhus N, Denmark) were
used for the measurement of O
2
concentration. Before
use, the electrodes were polarized overnight and
calibrated in water saturated with air in the CAL 300
calibration chamber (Unisense), as well as in an anoxic
solution consisting of 0.1
M sodium hydroxide and
0.1
M sodium ascorbate. Calibration was carried out
before each experiment. The current was measured
with a Unisense microsensor multimeter and recorded
using SensorTracePRO software (Unisense). Before
microelectrode measurements, 30 ml of low melting
point agarose consisting of 1% agarose in insect
Ringer’s solution (111 m
M NaCl, 3.3 mM KCl, 4.5 mM
CaCl
2
,2.8mM Na
2
CO
3
) was cast into a microchamber. A
freshly dissected gut was placed on this layer of
agarose, fully extended and immediately covered with
a second layer of molten agarose at 30 1C. Microelec-
trodes were positioned using a motorized micromani-
pulator (MXU2; PyroScience, Aachen, Germany).
Measurements were performed radially starting at the
surface of the gut wall (0 mm) through the beetle gut
until the tip completely penetrated the whole tissue.
The progress of the tip was followed with a digital
microscope (44032; Celestron, Torrance, CA, USA)
connected to a computer. All measurements were
carried out at room temperature.
Microarray and nucleotide accession numbers
The microarray data and nifH gene sequences
obtained in this study have been deposited in the
GEO repository and GenBank under accession num-
bers GSE40067, and JX523423–JX523605 respectively.
Results
Beetle internal anatomy
Figure 1a shows the dorsal gut alignment within the
abdominal cavity of O. disjunctus. The FG resides
within the head and part of the thorax (not
dissected). The convoluted MG, the AHG and the
PHG are located within the abdominal cavity.
The coiled MG (Figure 1b) extends from the gastric
ceca to the Malpighian tubules. To the posterior, the
gut abruptly widens into an area of pouches,
the AHG, which lies above a layer of tracheal tubes
(not visible); the slender PHG lies immediately
posterior to the AHG, which also is surrounded by
tracheal tubes. The entire dorsal abdominal cavity is
covered by a thick cuticle layer protected by a pair of
wings (removed) that lock together and only open
during flight, which rarely occurs in this species.
The typical length of the whole gut measured ca.
11 cm, with the FG being the shortest region and the
MG being the longest region.
The gut segments from the four dissected beetles for
our array analysis were uniform in weight: FG ¼ 13.3
±
2.1 mg, MG ¼ 263.5
±
37.9 mg, AHG ¼ 125.5
±
21.4
mg and PHG ¼ 47
±
6.5 mg. All individual gut seg-
ments yielded at least 200 ng of total (prokaryotic and
eukaryotic) DNA/RNA for PCR amplification and
cDNA synthesis. Bacterial 16S rRNA gene amplifica-
tion was successful for all gut regions. However,
archaeal 16S rRNA genes were only successfully
amplified in the AHG. pH was measured in a different
set of beetles and the average pH of each gut region
was: FG ¼ 7.44
±
0.21, MG ¼ 8.38
±
0.12, AHG ¼ 7.21
±
0.05 and PHG ¼ 6.89
±
0.18.
Oxygen profiles
The passalid beetle gut presents a set of complex
microenvironments given the existence of at
least four morphologically differentiated regions
(Figure 1b). To better understand any compartmen-
talization of microbial composition or activity, O
2
profiles were measured at different locations within
each gut region with the use of microelectrodes
(Figure 2). During each measurement, the tip was
driven to the surface of the gut wall with a
micromanipulator and monitored using a digital
microscope, and it was maintained in that position
until steady-state readings of O
2
were measured
(B1 min). In each case, once the tip of the electrode
penetrated the gut wall, oxygen concentrations
declined rapidly, with steep gradients apparent
until complete oxygen depletion was observed.
The anaerobic regions dominated in volume not
only in the AHG but also in all four gut regions
(Figure 2). In many termites, similar anaerobic zones
are confined to the hindgut paunch (Brune et al.,
1995; Ebert and Brune, 1997; Schmitt-Wagner and
Brune, 1999). The continuous movement of the
electrode tip through the beetle gut until it reached
the opposite side showed profiles of O
2
that
mirrored those obtained when entering the gut. By
measuring the gut wall thickness of the AHG and
PHG from the images reported by Nardi et al. (2006)
and according to the O
2
profiles determined in this
paper, all gut regions are characterized by a rapid
transition from microaerophilic to anaerobic condi-
tions in the lumen. The extent of oxygen availability
varied by gut region, being 4300 mm into the lumen
in the FG and MG when considering gut wall
thickness o100 mm compared with approximately
50 and 280 mm for the AHG and PHG, respectively,
where gut wall thicknesses were 200 and 110 mm.
Diversity and functions of gut communities in
O. disjunctus
JA Ceja-Navarro et al
5
The ISME Journal
Gut region community assemblage
For community composition analyses, a taxon was
considered present when above the detection
threshold (pf4 ¼ 0.90) in at least three of the four
replicates per gut region. On the basis of these
criteria, the FG contained the highest richness,
followed by the PHG, and then MG and AHG. This
pattern was observed across a range of pf thresholds
(0.90–1.0; Supplementary Table 2), where richness
patterns correlated between R of 0.98 and 0.99. PD
and Shannon diversity indices support the patterns
suggested by the richness measurements, that is,
that the FG was the most diverse gut region,
followed by the PHG, MG and AHG.
Hierarchical clustering (Figure 3) of all 16 gut
regions demonstrated that the FG and AHG formed
two distinct clusters, whereas the MG and PHG
communities were not resolved, and clustered
together by individual beetle. The relative abun-
dance (probe-set intensity) of specific taxa was also
statistically different across gut regions. The phylo-
genetic tree of the taxa detected shows the average
relative abundance of bacterial taxa across the four
gut regions (white-to-red gradient rings indicating
increasing relative abundance; Figure 4). The outer-
most ring (black) shows taxa that had statistically
significant differences in relative abundance
between at least two gut regions (Po0.05, analysis
of variance following BH correction for multiple
comparisons). The majority of taxa detected (75%)
showed significant differences in distribution across
gut regions.
To test the hypothesis that each gut region serves
as a distinct environmental filter, we assessed
phylogenetic clustering in each gut region. Phylo-
genetic clustering is indicated by the NRI where
increasing positive values represent communities
that are more clustered by phylogenetic relatedness,
and increasing negative values represent more
dispersed phylogenetic signals. Our results show
that the community within each gut region is
significantly clustered with the FG and PHG regions
showing the greatest clustering (Po0.001; Table 1).
The adjacent regions of the O. disjunctus digestive
tract showed changes in their bacterial community
composition (Figure 5). Aerobic groups such as the
Actinomycetales, Burkholderiales, Rhizobiales,
Chromatiales, Azospirillales, Bradyrhizobiales and
Figure 3 Hierarchical cluster analysis of the microbial commu-
nity from each of the four gut regions from four individual beetles
(1–4). (A)–(D) indicate the clades of the tree. Each terminal leaf
represents an array from a single gut region
Figure 2 Profiles of O
2
concentration for (a) FG, (b) MG, (c) AHG and (d) PHG. Note the anaerobic core of the lumen in each region. The
figure shows the oxygen profiles of three different beetle guts, indicated with a circle, triangle or a square.
Diversity and functions of gut communities in
O. disjunctus
JA Ceja-Navarro et al
6
The ISME Journal
Nitrosomonadales were enriched at both ends of the
gut (FG and PHG) and less abundant in the central
regions (MG and AHG). Anaerobic bacterial groups
including the Bacteroidales, Clostridiales, Spiro-
chaetales, Desulfobacterales, Desulfomonadales,
Syntrophobacterales and Thermotogales were
enriched in the AHG relative to the MG and PHG.
Methanogens, including the Methanomicrobiaceae,
Methanospirillaceae and Methanosarcinaceae, were
only detected in the AHG (Figures 6a and b).
The microbial communities associated with the
different regions of the passalid beetle were also
assessed by 454 pyrosequencing of the 16S rRNA
gene V9 region (see Supplementary Methods). This
analysis confirmed the results obtained with the
PhyloChip, in terms of the distribution of the
microbial groups, that is, aerobic bacteria more
abundant at FG and PHG, anaerobic bacteria dom-
inating the AHG and MG (Supplementary Figure S1)
and the AHG harboring methanogenic archaea.
UPGMA (Unweighted Pair Group Method with
Arithmetic Mean) clustering of the communities’
b-diversity displayed similar patterns of clustering
as the PhyloChip, with the FG and AHG clustering
in well-differentiated clades, and MG and PHG
clustering together but primarily according to beetle
rather than gut region as observed with PhyloChip
analysis (Supplementary Figure S2).
NifH expression and phylogeny of abundant nifH
transcripts
Nitrogen fixation was initially analyzed by incubat-
ing beetles in a
15
N
2
headspace for 12 days, followed
by dissection and bulk isotope ratio mass spectro-
metry of the extracted gut RNA. All four regions
showed
15
N
2
enrichment (Supplementary Figure
S4), although the location of N
2
-fixation activity
Figure 4 Phylogenetic tree of all taxa detected in the passalid beetle gut. Branches are colored based on the phyla. The four rings colored
in various shades of red are dependent on intensity measures for which the brightest red color represents the greatest taxon intensities.
Starting from the inside, each red ring represents different gut regions in order: FG, MG, AHG and PHG. The outer most ring in black
shows taxa that have statistically different intensities in any one of the gut regions. Each phylogenetic group is labeled based on either
Phylum or Class level. g, g-Proteobacteria; Clos, Clostridia; Spiro, Spirochetes; Cyan, Cyanobacteria; D ¼ Deinococcus-Thermus;
e, e-Proteobacteria; Acido ¼ , Acidobacteria; N, Nitrospira; Chloro, Chloroflexi; others(Aquificae, Caldithrix, Chlamydiae, Chlorobi,
Coprothermobacteria, Deferribacteres, Gemmatinomonadetes, Lentisphaerae, Natronoanaerobium, Synergistes, Thermotogae, Thermo-
desulfobacteria, Verrucomicrobia), make up 13.2% of the taxa depicted on the tree.
Diversity and functions of gut communities in
O. disjunctus
JA Ceja-Navarro et al
7
The ISME Journal
was difficult to confirm from these analyses because
of the lengthy incubation times (B12 days) required
to achieve
15
N detection. To estimate the N
2
-fixation
potential in each gut region, nifH gene expression
was assessed by qPCR and normalized to expression
of the rpoB gene. Across the gut, the lowest
expression levels were found in the FG; and its C
T
values were used as calibrator for relative quantifi-
cation (Table 2). Relative to the FG, nifH expression
was found to be 26.7 times higher in the AHG, 4.7
times higher in the MG and 2.4 times higher in the
PHG. N
2
-fixing groups were identified by cloning and
phylogenetic analysis (Supplementary Figure S3).
The dominant group in all gut regions was a group
of sequences related (93–96% similarity) to the
Ni–Fe nitrogenase of Paludibacter propionicigenes.
In addition, nifH sequences related to Clostridium
cellobioparum, Azoarcus sp. and Bradyrhizobium
japonicum were identified in the FG, whereas the
MG contained sequences related to Azorhizobium
doebereineae, Bradyrhizobium denitrificans and
Beijerinckia sp. As in all other gut regions, most of
the sequences identified in the AHG were also
related to P. propionicigenes, with some sequences
related to C. lentocellum. The PHG showed three
main groups of nifH sequences, (1) the one related to
Figure 5 Comparative cluster analysis of the number of bacterial taxa within families that had significantly greater relative abundance
between any two adjacent gut regions.
Diversity and functions of gut communities in
O. disjunctus
JA Ceja-Navarro et al
8
The ISME Journal
P. propionicigenes, (2) another related to the aerobic
N
2
-fixing organisms Bradyrhizobium sp. and (3) the
one related to Methylocapsa sp.
Discussion
O. disjunctus, a passalid beetle, is a large and well-
known arthropod detritivore with a broad distribu-
tion throughout eastern North America and southern
Canada (Pearse et al., 1936). These beetles show
a subsocial behavior inhabiting decaying hardwoods
and surviving on a low-nutrient diet, a feature that
has been shown in termites to require the presence
of symbiotic microorganisms such as bacteria and
flagellates that contribute to nitrogen fixation and
lignocellulose digestion.
The passalid beetle provides a useful natural
model to study the role of microbial communities
in plant cell wall degradation because of its ability
to subsist on a low-nutrient diet of decaying wood,
for which many of the involved degradation pro-
cesses require the activity of microbial enzymes.
In this study, we provide the first insight into microbial
community assembly, one aspect of its function
(N-fixation) and physiological gradients (O
2
, pH) that
may regulate microbial composition and activity in
this beetle’s digestive tract. We hypothesized that
the morphological and physiological differences
observed among gut regions (Nardi et al., 2006)
Figure 6 (a) Average probe-set positive fraction for methanogenic groups at each gut region (n ¼ 4). (b) Average of the normalized probe-
set intensity for methanogenic groups at the AHG (n ¼ 4). A taxon/OTU was considered present when over 90% of assigned probe pairs
were positive in at least three of the four replicates per gut region.
Table 2 C
T
values and normalized nifH expression ratios
Gut
region
Threshold nifH
cycle values
a
C
T(target, nifH)
Threshold rpoB
cycle values
C
T(ref, rpoB)
Normalized nifH
expression ratio in
reference to
rpoB
b
2
DDCT
FG 33.4 (0.46) 29.7 (0.18) 1.00
MG 30.37 (3.40) 28.92 (1.94) 4.76
AHG 27.27 (0.81) 28.31 (0.88) 26.72
PHG 32.13 (1.41) 29.71 (0.59) 2.43
Abbreviations: AHG, anterior hindgut foregut; C
T
, threshold cycle;
FG, foregut; MG, midgut; niFH, nitrogenase gene; pf, detection
threshold; PHG, posterior hindgut; rpOB, RNA polymerase b-subunit.
a
C
T
values for nifH and rpoB representing 100 ng of total RNA isolated
from the four gut regions of the gut of Odontotaenius disjunctus;
within parenthesis is the standard deviation values for the assay in
three beetles.
b
2
DDCT
is the expression ratio of the nitrogenase gene nifH,
normalized in reference to the single copy bacterial gene of the rpoB.
Expression ratios for all regions are expressed relative to the lowest
values detected C
T
values (from the FG).
Diversity and functions of gut communities in
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JA Ceja-Navarro et al
9
The ISME Journal
allow for the existence of distinct microbial
populations and activities within each gut region.
Some factors that can affect hindgut microbial
community assemblages include availability of
oxygen, pH, redox potential and biotic interactions
among the organisms (Dillon and Dillon, 2004). The
analysis of O
2
profiles showed the formation of O
2
gradients in all gut regions that were characterized
by a rapid transition from microaerophilic to
anaerobic conditions, with a variation in the depth
of the zones with oxygen availability that were
larger in the FG and MG than in the AHG and PHG.
Oxygen gradients have been extensively character-
ized in termites; however, unlike the passalid beetle,
termites such as Microcerotermes parvus and
Reticulitermes flavipes are known to act as ‘sinks
of oxygen’ only at the paunch, a digestive structure
analogous to the AHG in O. disjunctus, or also at the
posterior portion of the posterior half of the hindgut
in the case of Nasutitermes lujae (Brune et al., 1995;
Ebert and Brune, 1997). The presence of a gradient
of O
2
that reaches anaerobic conditions in all gut
regions makes the passalid beetle a unique system
for the study of the arrangement of microbial
populations and metabolic processes such as the
degradation of wood macromolecules and potential
production of biofuels such as CH
4
and H
2
along and
across gut regions. To characterize pH variations in
the O. disjunctus gut, we made bulk pH measure-
ments of the gut contents comparable to the methods
used to determine termite gut pH by Bignell and
Eggleton (1995). Unlike the higher termites in which
at least one gut region (hindgut or paunch) is
extremely alkaline (as high as 10.5), we found the
beetle gut environment is less extreme, although the
MG is alkaline (pH 8.38; Table 1). This is in contrast
to pH conditions found in many other insect and
mammalian guts where highly alkaline or acidic
environments are required for proper food digestion
(Brune and Ku
¨
hl, 1996; Fallingborg, 1999; Valencia
et al., 2000).
Overall, the phylogenetic microarray and
454-pyrosequencing data showed that in most
gut regions, richness within phyla was in the
order of Proteobacteria4Firmicutes4Actinobacteria4
Bacteriodetes, with the exception of the AHG region.
Here richness was in the order Firmicutes4Proteo-
bacteria4Bacteroidetes4Actinobacteria, suggesting
selection for a distinct population. The observed
similarity of both diversity and community structure
between the MG and PHG may be related to the
morphology of these regions, both MG and PHG lack
the elaborate gut structures that are seen in the AHG
that may support the establishment of microbial
reservoirs (Nardi et al., 2006). The highly clustered
microbial assemblages and the diversity patterns of
the FG and AHG may also be related to O
2
availability. A greater distance of O
2
penetration in
the FG together with a reduced anaerobic zone
compared with the AHG (Figure 2) may explain the
different distributions of specific microbial groups.
For example, aerobic N
2
-fixing bacteria and Actino-
bacteria were relatively more abundant in the FG
and either not detected or of low relative abundance
in the AHG. Although the FG is smaller by weight
than other regions, its bacterial diversity was the
highest. By comparison, the AHG had the lowest
diversity as well as the lowest O
2
availability
(Figure 2). All gut regions were phylogenetically
clustered, indicating that habitat filtering has
occurred (Webb et al., 2002; Horner-Devine and
Bohannan, 2006). Therefore, although the FG region
was most diverse in terms of numbers of taxa
detected, Shannon diversity and total phylogenetic
distance (Faith’s PD), the organisms in the FG were
more closely related to each other than those in the
AHG (Table 1). This finding was not expected but
may be related to the relative abundance of O
2
as a
terminal electron acceptor.
Unlike the other gut regions, the AHG is highly
morphologically differentiated, showing internal
pouches and projections (Nardi et al., 2006) that
would allow spatial separation, promoting a higher
diversity of niches, and thus, a higher taxonomic
diversity as observed in the guts of other insects
(Breznak, 2000). However, the lower O
2
penetration
and more extensive anaerobic zones may lead to
lower electron acceptor diversity that could counter-
act the impact of spatial separation on diversity in
this region (Figure 5). When comparing the micro-
bial composition of all gut regions, the AHG
was characterized by the enrichment of anaerobic
groups that may contribute to important metabolic
processes such as transformation of lignocellulosic
materials, N
2
fixation, H
2
and CH
4
production. It was
only within this region that methanogens such
as Methanosarcina were detected. Interestingly,
methane oxidizers such as the Methylococcales
were detected in relatively greater abundance in
regions adjacent to the AHG—the likely source of
methane (Figure 5). In the AHG, we detected some
representatives of anaerobic groups that have been
extensively studied because of their importance in
biotechnology, for example H
2
-producing C. tyrobu-
tyricum, or aromatic o-demethylating homoacetogen
C. methoxybenzovorans (Mechichi et al., 1999; Jo
et al., 2008). Spirochetes, also enriched in the AHG,
have been reported as an important component of
the termite gut biota because of their N
2
-fixation and
cellulose degradation abilities (Lilburn et al., 2001),
although we saw no evidence in this case of nif H
gene expression in this group. In the AHG, the
Bacteroidetes including the family Porphyromona-
daceae were enriched relative to adjacent regions
(Figure 4 and Supplementary Table 1). The FG and
PHG regions were characterized by a higher relative
abundance of aerobic bacteria and notably cellulo-
lytic taxa such as Cellulophaga , Cellulosimicrobium
and aerobic N
2
-fixing organisms such as Brad-
yrhizobium, and Beijerinckia (Kaneko, 2002;
Nedashkovskaya, 2004; Lo et al., 2009). Clearly, the
beetle gut can be seen as a complex system,
Diversity and functions of gut communities in
O. disjunctus
JA Ceja-Navarro et al
10
The ISME Journal
where metabolic processes appear to be spatially
dispersed.
The presence of radial O
2
gradients to full O
2
depletion in all gut regions and the high C:N ratio of
the woody biomass on which these beetle subsist
would be expected to select for N
2
fixation. We first
assessed this possibility by means of
15
N
2
-isotope
ratio mass spectrometry and demonstrated that
15
N
2
enrichment occurred in all gut regions. To identify
the likely location of nitrogen fixation, nitrogenase
gene expression was quantified by using nifH-qPCR.
This demonstrated that nifH expression was highest
in the AHG (27-fold higher than the FG; Table 2),
with the predominant nifH transcript sequences
related to the Ni–Fe nitrogenase of the member of
the Porphyromonadales, P. propionicigenes (Gronow
et al., 2011) as well as the Ni-Fe nitrogenase of
Clostridiales member, C. lentocellum, a cellulolytic
N
2
-fixing bacterium (Leschine and Canale-Parola,
1989; Leschine, 1995). NifH expression results
confirmed our hypothesis of microbial composi-
tional and functional segregation because of differ-
ential physiological conditions, that is, variation in
O
2
concentrations at each gut region. The sequence
analysis of nifH transcripts agreed well with
phylogenetic microarray analyses, confirming the
presence of aerobic N
2
-fixing bacteria at the FG, MG
and PHG, as well as anaerobic organisms such as the
Clostridiales and Porphyromonadales. Importantly,
nifH expression was detected in all gut regions and
the FG, MG and PHG not only contained transcripts
of N
2
-fixing bacteria related to P. propionicigenes
but also groups of aerobic N
2
-fixing bacteria.
This co-occurrence of aerobic and anaerobic N-fixers
is likely related to presence of microaerophilic
regions throughout the beetle gut and indicates
some degree of functional redundancy in N
2
fixation
(Bowen et al., 2011). As discussed earlier, no
spirochete-related nif H transcripts were detected;
this observation is likely not related to primer bias,
as the same nifH primers were used to identify N
2
-fixing
spirochetes in termites (Reid and Lloyd-Jones,
2009). This suggests that unlike termite hindguts
where spirochetes are the primary N-fixing group,
this role is filled mainly by a Bacteroidetes species
(Paludibacter) and other organisms in the passalid
beetle gut.
Our multiscale approach has shown that the gut of
the passalid beetle is a highly compartmentalized
environment with well-stratified microbial commu-
nities, ranging from areas with a high diversity of
aerobic groups (FG, PHG) to the AHG mainly
dominated by anaerobic bacteria. The passalid
beetle gut possesses a highly diverse community of
N
2
-fixing bacteria, dominated by organisms related
to P. propionicigenes rather than the spirochetes that
perform this role in termites. The beetle gut is
characterized by a radial gradient of O
2
reaching
anaerobic conditions that dominate in area within
the lumen of all gut regions. On the basis of this, it is
likely that aerobic and anaerobic processes occur
within close proximity and contribute to the
efficiency of lignocellulose degradation in this
insect. Understanding how these populations inter-
act between regions and across gradients to con-
tribute to this insects nutrition is a topic of further
research and will improve our ability to optimize
lignocellulosic biofuel production processes.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements
This work was supported by the Department of Energy,
Office of Biological and Environmental Research, Geno-
mic Sciences program through the Lawrence Livermore
National Laboratory Biofuels Scientific Focus Area (SFA)
award SCW1039. Part of this work (ELB, JAC-N, UK, GLA)
was performed at Lawrence Berkeley National Laboratory
under the Department of Energy contract number
DE-AC02-05CH11231. Contributions of JPR are under the
auspices of the US DOE at LLNL (DE-AC52-07NA27344).
Additional support was provided from the NSF-GRFP
program to NHN, the LSU Boyd Professor Research fund
to MB, and JAC-N was also supported in part by a grant
from ‘Consejo Nacional de Ciencia y Tecnologı
´
a’ (CON-
ACyT, Mexico). We thank Hector Urbina for collection of
beetles; George Stanley for help in conducting the nitrogen
uptake experiments; Doug Wendell and Gail Ackermann
form the QIIME-DB for their support with the submission
of the sequencing data to the EBI.
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Diversity and functions of gut communities in
O. disjunctus
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The ISME Journal
... Bess beetles (Passalidae) are key players in forest decomposition and ecosystem turnover in North America, consuming more than four times their body weight in woody debris each day (25). Passalids deconstruct the complex plant polymers within their diet through contributions from their spatially structured gut microbiome, whose members perform complementary metabolic Specialized symbioses with bacterial symbionts promote beetle fitness and adaptation by contributing to plant biomass digestion, facilitating resistance against plant secondary compounds, conferring defense against predators and pathogens, ensuring niche preservation, and/or supplementing nutrition. ...
... Illustration by Julie Johnson. functions (24,25). This is facilitated by morphologically differentiated gut compartments varying in cuticle thickness and physiochemical conditions (25). ...
... functions (24,25). This is facilitated by morphologically differentiated gut compartments varying in cuticle thickness and physiochemical conditions (25). Symbiont depolymerization of lignocellulose is achieved in aerobic gut regions (midgut, posterior hindgut), while fermentation, acetogenesis, and methanogenesis are carried out by bacteria in the anaerobic hindgut (24). ...
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... Actinomycetes with antimicrobial properties are widespread in O. disjunctus galleries Passalid beetles of the species O. disjunctus (formerly known as Passalus cornutus, and commonly referred to as 'bessbugs', Figure 1A) are widely distributed across eastern North America, where they are important decomposers of rotting timber (Ceja-Navarro et al., 2014;Ceja-Navarro et al., 2019;Gray, 1946;Pearse et al., 1936). This role has prompted interest in the O. disjunctus gut microbiota as a potential source of lignocellulose-processing microbes for biofuel efforts (Ceja-Navarro et al., 2014;Ceja-Navarro et al., 2019;Nguyen et al., 2006;Suh et al., 2003;Suh et al., 2005;Urbina et al., 2013). ...
... Actinomycetes with antimicrobial properties are widespread in O. disjunctus galleries Passalid beetles of the species O. disjunctus (formerly known as Passalus cornutus, and commonly referred to as 'bessbugs', Figure 1A) are widely distributed across eastern North America, where they are important decomposers of rotting timber (Ceja-Navarro et al., 2014;Ceja-Navarro et al., 2019;Gray, 1946;Pearse et al., 1936). This role has prompted interest in the O. disjunctus gut microbiota as a potential source of lignocellulose-processing microbes for biofuel efforts (Ceja-Navarro et al., 2014;Ceja-Navarro et al., 2019;Nguyen et al., 2006;Suh et al., 2003;Suh et al., 2005;Urbina et al., 2013). O. disjunctus is subsocial, with mating pairs establishing galleries within decaying logs where they rear their larvae (Schuster and Schuster, 1985;Wicknick and Miskelly, 2009). ...
... The frass found in O. disjunctus galleries constitutes an important commodity in the lifestyle of this beetle. The frass itself is composed of partially digested wood with high organic carbon content, and nitrogen fixation by microbes in the gut of O. disjunctus enhances its bioavailable nitrogen content as well (Ceja-Navarro et al., 2014;Ceja-Navarro et al., 2019). Thus, frass represents a valuable nutrient source for these beetles, any associated microbes, and potential pathogenic invaders. ...
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... Nevertheless, the culture-dependent method may detect only a limited number of diazotrophic species because of the inability to culture most of environmental isolates. As the gene nifH encoding dinitrogenase reductase is genetically conserved [59], the culture-independent molecular techniques based on the amplification of nifH gene has been used to detect an unexpected diversity of nitrogen-fixing bacteria within guts of these nitrogenfixing insects [3,12,14,16,33,34,52]. However, the successful amplifications of nifH gene do not always indicate that the encoding dinitrogenase reductase is actually expressed by the corresponding bacteria, because the expressions of nifH genes are regulated at the transcriptional and post-transcriptional level [59]. ...
... However, the successful amplifications of nifH gene do not always indicate that the encoding dinitrogenase reductase is actually expressed by the corresponding bacteria, because the expressions of nifH genes are regulated at the transcriptional and post-transcriptional level [59]. Thus, detection of mRNA by a reverse transcription polymerase chain reaction (RT-PCR) is usually used to further support the expression of nifH genes and determine the actual activity of specific gut microbes in nitrogen fixation [3,12,14,31,32]. ...
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... The microbiota components that mediate these interactions consist primarily of fungi and bacteria. However, the gut microbial assemblages and functions of borers may vary considerably depending on gut morphology, insect species, and host plant (Warnecke et al., 2007;Schauer et al., 2012;Ceja-Navarro et al., 2013;Mikaelyan et al., 2017). ...
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... Usually, samples are placed in an airtight container, in which the N 15 -labeled atmospheric nitrogen is injected moderately. After that, using isotope mass spectrometer to detect the content of N 14 and N 15 in samples within a certain time and calculate the values of δ 15 N, which reflects the efficiency of nitrogen fixation [47]. Similarly, N 15 -labeled nitrogenous waste compounds, such as uric acid [48] and urea [30,49], are usually added to the diets when evaluating the efficiency of NWR, the values of δ 15 N in samples are calculated after feeding a period of time. ...
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Nitrogen is usually a restrictive nutrient that affects the growth and development of insects, especially of those living in low nitrogen nutrient niches. In response to the low nitrogen stress, insects have gradually developed symbiont-based stress response strategies—biological nitrogen fixation and nitrogenous waste recycling—to optimize dietary nitrogen intake. Based on the above two patterns, atmospheric nitrogen or nitrogenous waste (e.g., uric acid, urea) is converted into ammonia, which in turn is incorporated into the organism via the glutamine synthetase and glutamate synthase pathways. This review summarized the reaction mechanisms, conventional research methods and the various applications of biological nitrogen fixation and nitrogenous waste recycling strategies. Further, we compared the bio-reaction characteristics and conditions of two strategies, then proposed a model for nitrogen provisioning based on different strategies.
... Based on the data in the phenotype prediction analysis of the intestinal microbiome of the crabs, we also detected apparent nitrogenase activity in the intestinal bacteria of E. versicolor and N. smithi (Fig 6C). In animals, nitrogenase activity has been reported in the symbiotic microbes in termites, wood-boring beetles, shipworms, sponges, corals, and fiddler crabs, which allow them to survive in nitrogen-poor environments [26,[80][81][82][83]. Generally, nitrogen-containing compounds are partly released as feces, while other compounds are incorporated into the body of the host animal [65]. ...
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Mangrove ecosystems, where litter and organic components are degraded and converted into detrital materials, support rich coastal fisheries resources. Sesarmid (Grapsidae) crabs, which feed on mangrove litter, play a crucial role in material flow in carbon-rich and nitrogen-limited mangrove ecosystems; however, the process of assimilation and conversion into detritus has not been well studied. In this study, we performed microbiome analyses of intestinal bacteria from three species of mangrove crab and five sediment positions in the mud lobster mounds, including the crab burrow wall, to study the interactive roles of crabs and sediment in metabolism. Metagenome analysis revealed species-dependent intestinal profiles, especially in Neosarmatium smithi , while the sediment microbiome was similar in all positions, albeit with some regional dependency. The microbiome profiles of crab intestines and sediments were significantly different in the MDS analysis based on OTU similarity; however, 579 OTUs (about 70% of reads in the crab intestinal microbiome) were identical between the intestinal and sediment bacteria. In the phenotype prediction, cellulose degradation was observed in the crab intestine. Cellulase activity was detected in both crab intestine and sediment. This could be mainly ascribed to Demequinaceae , which was predominantly found in the crab intestines and burrow walls. Nitrogen fixation was also enriched in both the crab intestines and sediments, and was supported by the nitrogenase assay. Similar to earlier reports, sulfur-related families were highly enriched in the sediment, presumably degrading organic compounds as terminal electron acceptors under anaerobic conditions. These results suggest that mangrove crabs and habitat sediment both contribute to carbon and nitrogen cycling in the mangrove ecosystem via these two key reactions.
... In contrast, the coleopteran gut is highly segmented, with certain modifications and enlargements based on their diet. They show marked variations in gut microbial communities [55,56]. In comparison to holometabolous insects, hemipterans gut tissues and microbiomes show significant modifications. ...
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Insects nurture a panoply of microbial populations that are often obligatory and exist mutually with their hosts. Symbionts not only impact their host fitness but also shape the trajectory of their phenotype. This co-constructed niche successfully evolved long in the past to mark advanced ecological specialization. The resident microbes regulate insect nutrition by controlling their host plant specialization and immunity. It enhances the host fitness and performance by detoxifying toxins secreted by the predators and abstains them. The profound effect of a microbial population on insect physiology and behaviour is exploited to understand the host-microbial system in diverse taxa. Emergent research of insect-associated microbes has revealed their potential to modulate insect brain functions and, ultimately, control their behaviours, including social interactions. The revelation of the gut microbiota-brain axis has now unravelled insects as a cost-effective potential model to study neurodegenerative disorders and behavioural dysfunctions in humans. This article reviewed our knowledge about the insect-microbial system, an exquisite network of interactions operating between insects and microbes, its mechanistic insight that holds intricate multi-organismal systems in harmony, and its future perspectives. The demystification of molecular networks governing insect-microbial symbiosis will reveal the perplexing behaviours of insects that could be utilized in managing insect pests.
... Insights into the drivers of nitrogen fixation in the environment are of interest in microbial physiology, ecology, and agriculture, and they are useful in modeling and predicting the dynamics of nitrogen cycling (4,5). Importantly, nitrogen fixation in gut symbionts has been linked to nitrogen acquisition by the host, which has led to much attention in animal biology studies (6). ...
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Nitrogen fixation, a distinct process incorporating the inactive atmospheric nitrogen into the active biological processes, has been a major topic in biological and geochemical studies. Currently, insights into diversity and distribution of nitrogen-fixing microbes are dependent upon homology-based analyses of nitro-genase genes, especially the nifH gene, which are broadly conserved in nitrogen-fixing microbes. Here, we report the pitfall of using nifH as a marker of microbial nitrogen fixation. We exhaustively analyzed genomes in RefSeq (231,908 genomes) and KEGG (6,509 genomes) and cooccurrence and gene order patterns of nitroge-nase genes (including nifH) therein. Up to 20% of nifH-harboring genomes lacked nifD and nifK, which encode essential subunits of nitrogenase, within 10 coding sequences upstream or downstream of nifH or on the same genome. According to a phenotypic database of prokaryotes, no species and strains harboring only nifH possess nitrogen-fixing activities, which shows that these nifH genes are "pseudo"-nifH genes. Pseudo-nifH sequences mainly belong to anaerobic microbes, including members of the class Clostridia and methanogens. We also detected many pseudo-nifH reads from metagenomic sequences of anaerobic environments such as animal guts, wastewater, paddy soils, and sediments. In some samples, pseudo-nifH overwhelmed the number of "true" nifH reads by 50% or 10 times. Because of the high sequence similarity between pseudo-and true-nifH, pronounced amounts of nifH-like reads were not confidently classified. Overall, our results encourage reconsideration of the conventional use of nifH for detecting nitrogen-fixing microbes, while suggesting that nifD or nifK would be a more reliable marker. IMPORTANCE Nitrogen-fixing microbes affect biogeochemical cycling, agricultural productivity , and microbial ecosystems, and their distributions have been investigated intensively using genomic and metagenomic sequencing. Currently, insights into nitrogen fixers in the environment have been acquired by homology searches against nitrogenase genes, particularly the nifH gene, in public databases. Here, we report that public databases include a significant amount of incorrectly annotated nifH sequences (pseudo-nifH). We exhaustively investigated the genomic structures of nifH-harboring genomes and found hundreds of pseudo-nifH sequences in RefSeq and KEGG. Over half of these pseudo-nifH sequences belonged to members of the class Clostridia, which is supposed to be a prominent nitrogen-fixing clade. We also found that the abundance of nitrogen fixers in metagenomes could be overestimated by 1.5 to .10 times due to pseudo-nifH recorded in public databases. Our results encourage reconsideration of the prevalent use of nifH as a marker of nitrogen-fixing microbes.
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Mealworms, the larvae of a coleopteran insect Tenebrio molitor L., are capable of eating, living on and degrading the non-hydrolyzable vinyl plastics as sole diet. However, vinyl plastics are carbon-rich but nitrogen-deficient. It remains puzzling how plastic-eating mealworms overcome the nutritional obstacle of nitrogen limitation. Here, we provide the evidence for nitrogen-fixation activity within plastic-eating mealworms. Acetylene reduction assays illustrate that the nitrogen-fixing activity ranges from 12.3 ± 0.7 to 32.9 ± 9.3 nmol ethylene·h − 1 ·gut − 1 and the corresponding fixed nitrogen equivalents of protein are estimated as 8.6 to 23.0 µg per day per mealworm. Nature nitrogen isotopic analyses of plastic-eating mealworms provide further evidence for the importance of nitrogen fixation as a new nitrogen source. Eliminating the gut microbial microbiota with antibiotics impairs the mealworm’s ability to fix nitrogen from atmosphere, indicating the contribution of gut microbiota to nitrogen fixation. By using the traditional culture-dependent technique, PCR and RT-PCR of nifH gene, nitrogen-fixing bacteria diversity within the gut was detected and the genus Klebsiella was demonstrated to be an important nitrogen-fixing symbiont. These findings first build the relationship between the plastic degradation (carbon metabolism) and nitrogen fixation (nitrogen metabolism) within mealworms. Combined with previously reported plastic-degrading capability and nitrogen-fixing activity, mealworms may be potential candidates for up-recycling of plastic waste to produce protein sources.
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Among the many parasitic or commensalistic symbionts of Passalidae (Coleoptera) are fungi that live within their hindgut and on the exoskeleton of the beetles and their parasitic mites. Three Eccrinales (Trichomycetes) include Leidyomyces attenuatus (= Enterobryus attenuatus), Passalomyces compressus (= Enterobryus compressus), and an unnamed species originally described by Heymons and Heymons in 1934. Leidyomyces attenuatus has been found in populations throughout the range of Passalidae in the Americas, whereas P. compressus and the Heymons' eccrinid are reported only from Neotropical passalid beetles. A new genus and species of branched fungus, Enteroramus dimorphus, lives in the hindgut of the common eastern North American passalid, Odontotaenius disjunctus. In axenic culture the fungus converts to a yeastlike growth form. Externally, both the beetles and their mites carry parasitic thalli of many species of Rickia (Ascomycota: Laboulbeniales). The probability that many fungi from Passalidae remain unreported worldwide is discussed.
Chapter
The gut of wood- and litter-feeding termites harbors a dense and diverse community of prokaryotes that contribute to the carbon, nitrogen and energy requirements of the insects. Acetogenesis from H2 plus CO2 by hindgut prokaryotes supports up to 1/3 of the respiratory requirement of some termite species; and N2-fixing and uric acid-degrading microbes can have a significant impact on termite N economy. Microelectrode studies reveal that hindguts consist of an anoxic lumen surrounded by a microoxic periphery — a finding consistent with the occurrence of both anaerobic and O2-dependent microbial metabolism in hindguts. They also suggest that the enigmatic dominance of acetogens over methanogens as an H2 “sink” reflects a spatial separation of these H2-consuming populations, with the former being closer to sources of H2 production. Isolation of a number of the prokaryotes (including spirochetes, which have proven to be H2/CO2-acetogens) reveals that termite guts are a source of novel microbial diversity. However, molecular biological analyses indicate that much of that diversity is still poorly represented in culture.
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We have regularly cultured yeasts from the gut of certain beetles in our ongoing research. In this study cloned PCR products amplified from the gut contents of certain mushroom-feeding and wood-ingesting beetles in four families (Erotylidae, Tenebrionidae, Ciidae, and Passalidae) were sequenced and compared with culture results. Cultural techniques detected some yeasts present in the gut of the beetles, including a Pichia stipitis-like yeast associated with wood-ingesting passalid beetles. Clone sequences similar to several ascomycete yeasts and Malassezia restricta, a fastidious basidiomycetous yeast requiring special growth media, however, were not detected by culturing. Unexpectedly, phylogenetic analysis of additional clone sequences discovered from passalid beetles showed similarity to members of the Parabasalia, protists known from other wood-ingesting insects, termites, and wood roaches. Examination of all gut regions of living passalids, however, failed to reveal parabasalids, and it is possible that they were parasites in the gut tissue present in low numbers.
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The complete nucleotide sequence of the genome of a symbiotic bacterium Bradyrhizobium japonicum USDA110 was determined. The genome of B. japonicum was a single circular chromosome 9,105,828 bp in length with an average GC content of 64.1%. No plasmid was detected. The chromosome comprises 8317 potential protein-coding genes, one set of rRNA genes and 50 tRNA genes. Fifty-two percent of the potential protein genes showed sequence similarity to genes of known function and 30% to hypothetical genes. The remaining 18% had no apparent similarity to reported genes. Thirty-four percent of the B. japonicum genes showed significant sequence similarity to those of both Mesorhizobium loti and Sinorhizobium meliloti , while 23% were unique to this species. A presumptive symbiosis island 681 kb in length, which includes a 410-kb symbiotic region previously reported by Göttfert et al., was identified. Six hundred fifty-five putative protein-coding genes were assigned in this region, and the functions of 301 genes, including those related to symbiotic nitrogen fixation and DNA transmission, were deduced. A total of 167 genes for transposases/104 copies of insertion sequences were identified in the genome. It was remarkable that 100 out of 167 transposase genes are located in the presumptive symbiotic island. DNA segments of 4 to 97 kb inserted into tRNA genes were found at 14 locations in the genome, which generates partial duplication of the target tRNA genes. These observations suggest plasticity of the B. japonicum genome, which is probably due to complex genome rearrangements such as horizontal transfer and insertion of various DNA elements, and to homologous recombination.
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— We studied sequence variation in 16S rDNA in 204 individuals from 37 populations of the land snail Candidula unifasciata (Poiret 1801) across the core species range in France, Switzerland, and Germany. Phylogeographic, nested clade, and coalescence analyses were used to elucidate the species evolutionary history. The study revealed the presence of two major evolutionary lineages that evolved in separate refuges in southeast France as result of previous fragmentation during the Pleistocene. Applying a recent extension of the nested clade analysis (Templeton 2001), we inferred that range expansions along river valleys in independent corridors to the north led eventually to a secondary contact zone of the major clades around the Geneva Basin. There is evidence supporting the idea that the formation of the secondary contact zone and the colonization of Germany might be postglacial events. The phylogeographic history inferred for C. unifasciata differs from general biogeographic patterns of postglacial colonization previously identified for other taxa, and it might represent a common model for species with restricted dispersal.