Hindawi Publishing Corporation
International Journal of Microbiology
Volume 2015, Article ID 285018, 11 pages
http://dx.doi.org/10.1155/2015/285018
Research Article
Isolation of Fungi and Bacteria Associated with
the Guts of Tropical Wood-Feeding Coleoptera and
Determination of Their Lignocellulolytic Activities
Keilor Rojas-Jiménez1,2 andMyriamHernández1
1 Instituto Nacional de Biodiversidad, Apartado Postal 22-3100, Santo Domingo, Heredia, Costa Rica
2Universidad Latina de Costa Rica, Campus San Pedro, Apartado Postal 10138-1000, San José, Costa Rica
Correspondence should be addressed to Keilor Rojas-Jiménez; keilor.rojas@gmail.com
Received 23 May 2015; Accepted 12 August 2015
Academic Editor: Karl Drlica
Copyright © 2015 K. Rojas-Jiménez and M. Hernández. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
The guts of beetle larvae constitute a complex system where relationships among fungi, bacteria, and the insect host occur. In this
study, we collected larvae of five families of wood-feeding Coleoptera in tropical forests of Costa Rica, isolated fungi and bacteria
from their intestinal tracts, and determined the presence of five different pathways for lignocellulolytic activity. The fungal isolates
were assigned to three phyla, 16 orders, 24 families, and 40 genera; Trichoderma was the most abundant genus, detected in all
insect families and at all sites. The bacterial isolates were assigned to five phyla, 13 orders, 22 families, and 35 genera; Bacillus,
Serratia, and Pseudomonas were the dominant genera, present in all the Coleopteran families. Positive results for activities related
to degradation of wood components were determined in 65% and 48% of the fungal and bacterial genera, respectively. Our results
showed that both the fungal and bacterial populations were highly diverse in terms of number of species and their phylogenetic
composition, although the structure of themicrobial communities variedwith insect host family and the surrounding environment.
The recurrent identification of some lignocellulolytic-positive inhabitants suggests that particular microbial groups play important
roles in providing nutritional needs for the Coleopteran host.
1. Introduction microbial-host interaction is highly relevant from several
perspectives. For example, in natural ecosystems, beetles
Plant cell walls are predominantly composed of lignin, cellu- and their associated microorganisms perform important
lose, and hemicellulose. Together, these three polymers repre- functions as prime contributors to the degradation of organic
sent one of themost abundant sources of renewable energy on matter [10, 11].Moreover, some species of beetles have become
Earth [1–3].These polymers also constitute the basic nutrition significant forest pests that cause large-scale economic losses.
source for a large number of terrestrial insects, of which Therefore, a better understanding of their feeding capabilities
the order Coleoptera is perhaps the most representative [4, is relevant for establishing novel management strategies [12–
5]. The adaptation of the coleopteran insects to nutrient- 15]. From the biotechnological point of view, the coleopteran
limited diets, such as wood constituents, is attributed to the gut inhabitants represent a novel source for bioprospecting
establishment of relationships with microorganisms. These of enzymes related to the conversion of plant biomass into
microorganisms, highly prominent in the digestive tracts of biofuels, production of industrial value-added products, and
the host, perform essential functions including digestion of bioremediation of pollutants [16–18].
lignocellulosic biomass, nutrient production, and compound Most of the 300,000 beetle species described to date occur
detoxification [6–9]. in tropical rainforests [19, 20]. Costa Rican rainforests, for
Recently there has been an increasing interest in the example, are known to harbor approximately 10% of the
gut microorganisms of wood-feeding Coleoptera, since this species and 60% of the families of Coleoptera, including
2 International Journal of Microbiology
a number of wood-feeding beetles from the Scarabaeidae, fallen tree found was exhaustively examined for the presence
Passalidae, Cerambycidae, Elateridae, and Tenebrionidae of galleries of wood-feeding beetle larvae, particularly from
families [21]. The life cycles of these insects are highly the Scarabaeidae, Passalidae, Elateridae, Cerambycidae, and
seasonal, with most of the developmental stages occurring Tenebrionidae families. The selected national parks covered
during the rainy season. The feeding sources of the adults most of the natural distribution of the five coleopteran
include dung, carrion, and various plant parts, such as roots, families studied: wet tropical forest ranging from zero to
stems, foliage, flowers, pollen, fruits, and seeds. Conversely, 1,300m altitude (Table 1).
the diets of the larvae are more restricted to roots, decom- The observed frequency of fallen trees and the presence
posing organic matter, and decaying wood [11, 20–22]. of insect galleries within them varied according to the
The substrates on which the insects feed are major conditions of each site.Therefore, sample collection and study
determinants for the gut microbial diversity. However, it results were normalized to unit area. Most of the insect
is also possible that certain microbes have adapted to the galleries contained insects of only one coleopteran family,
endointestinal lifestyle and have developed mutualistic rela- but in few cases, two or three different families were present.
tionships necessary for the host survival. Hence, a fraction of The insect larvae, in the late second or third larval instars,
the endosymbiont microbial community could be vertically were collected, placed in polyethylene boxes together with
transmitted [6, 7, 23]. Among the microbial groups, fungal pieces of their feedingwood, and kept at ambient temperature
and bacterial endosymbionts form complex communities until being transported to the laboratory (Table 2).The initial
that, besides the basic digestive functions, also perform identification of the larval families was performed on site by
nonconventional roles that include synthesis of vitamins and a trained collector and later confirmed by the coleopteran
pheromones, nitrogen recycling, and resistance to pathogens, taxonomist of the National Institute of Biodiversity. Once in
each of which has important implications for host fitness [8, the laboratory, the larval specimens were chilled at −20∘C for
15, 24, 25]. In return, the insect provides a stable environment 10minutes, surface sterilizedwith ethanol, and then dissected
for the microbial growth with a steady intake of nutrients. in a sterile laminar flow hood.
Furthermore, the host can evolve to where tripartite beetle-
fungi-bacteria mutualism takes place [23, 26]. This phe- 2.2. Isolation of Bacteria and Fungi. The entire gut was
nomenon seems to be widespread, although the mechanisms removed from each larva and placed on a sterile Petri dish,
that govern these interactions are still poorly understood crushed, and spread onto three differentmedia plates. For iso-
[4, 27]. lation of fungi we used Potato-Dextrose Agar (PDA, Difco)
Most of the previous studies on the microbial diversity of amendedwith chlortetracycline (120mg/L) and streptomycin
the coleopteran gut highlighted either bacterial diversity or, (120mg/L). For the isolation of bacteria we used one-third
to a lesser extent, fungal diversity, generally obtained from a strength Luria-Bertani medium (3 g/L peptone, 5 g/L yeast-
small number of economically important beetle species. In extract, 10 g/LNaCl, and 15 g/liter agar, pH 7.0) and the self-
addition, some studies used metagenomic profiling for the developed medium called LIGNO (1.5 g/L KH PO , 1.75 g/L
discovery of novel lignocellulolytic genes.The purpose of the 2 4K HPO , 0.8 g/L KNO , 0.5 g/L MgSO , 1mL/L CaCl 0.1M,
present work was to isolate and describe the composition 2 4 3 4 24 g/L sawdust, 2 g/L bagasse powder, and 17 g/L agar, pH 7.0).
of the cultivable fungi and bacteria associated with the guts Plates were incubated at 28∘C for up to three weeks and
of wood-feeding larvae of five families of Coleoptera and
to determine their lignocellulolytic activities. For this, we checked every other day regularly for visible microorganism
collected larvae in tropical wet forests of several national growths. Each emerging fungus was transferred into a fresh
parks in Costa Rica, isolated both fungi and bacteria from PDA plate amended with the antibiotics mentioned above,
their guts, performed bioinformatics analysis, and assayed while each emerging bacterial colony was replated into
for the presence of five enzymatic activities related to the Luria-Bertani medium (Difco). An initial screening, based
degradation of lignocellulosicmaterials.This work represents on the morphological traits of the fungi, was performed
an initial step toward understanding relationships existing to discard redundant isolates from the same sample (char-
among the fungi, the bacteria, and the beetle host and the acteristics such as the color, shape, border type, mycelial
surrounding environment in a country having a particularly density, presence-absence of secretions, and growth rate were
large biodiversity of Coleoptera. compared). A second screening was based on molecular tax-onomy.The resulting nonredundant isolates were included in
a database with associated information andwere preserved in
2. Material and Methods the National Biodiversity Institute’s culture collection.
2.1. Insect Sampling. This studywas conducted in tropical wet 2.3. Lignocellulolytic Assays. We screened all the isolates for
forests of 10 protected areas of Costa Rica with the respective the presence of five different pathways for lignocellulolytic
permit resolutions R-CM-INBio-40-2008-OT and R-CM- activity possibly associated with degradation of structural
INBio-48-2008-OT of the National Authority of theMinistry wood components, including cellulose, lignin, 𝛽-D-xylan, 𝛽-
of Environment. At each site, approximately 3 km of natural D-cellobiose, and 𝛽-D-glucans.These assays were performed
trails was explored, looking for decaying fallen trees within by addition of specific substrates to the medium, or directly
25m on each side of the path. The sampling area represented on the microbial culture, and hydrolysis was determined by a
nearly 150,000m2 (or 0.15 km2) per national park. Every color change. All of themicroorganismswere assessed at least
International Journal of Microbiology 3
Table 1: Description of the location and main environmental parameters of the 10 national parks of Costa Rica, where sampling was carried
out. The selected environments are classified as tropical wet forest and cover most of the natural distribution of the five coleopteran families
studied.
National park LatitudeLongitude Altitude (m) Mean temperature (
∘C) Annual precipitation (mm)
Arenal 10
∘26󸀠49󸀠󸀠 N
84∘43󸀠41 W 589 24 4000–5000󸀠󸀠
9∘58󸀠43󸀠󸀠Barbilla N83∘28󸀠23󸀠󸀠 W 460 21 3000–4000
∘ 󸀠 󸀠󸀠
Braulio Carrillo 10 9 33 N83 56 10 W 507 24 3500–4500∘ 󸀠 󸀠󸀠
Carara 9
∘46󸀠41󸀠󸀠 N
84 36 20 W 78 27 2500–3000∘ 󸀠 󸀠󸀠
∘ 󸀠 󸀠󸀠
Hitoy Cerere 9 40 18 N83 01 39 W 150 25 3000–4000∘ 󸀠 󸀠󸀠
8∘41󸀠 󸀠󸀠Piedras Blancas 56 N83 12 198 28 5000–6000∘ 󸀠29󸀠󸀠 W
∘ 󸀠 󸀠󸀠
Rincón de la Vieja 10 46 29 N85 20 41 W 782 22 2500–3000∘ 󸀠 󸀠󸀠
∘
Tapanti 9 44
󸀠40󸀠󸀠 N
83∘46󸀠56󸀠󸀠 W 1287 19 6000–7000
10∘42󸀠25󸀠󸀠Tenorio N84∘59󸀠22󸀠󸀠 W 727 22 3000–4000
∘ 󸀠 󸀠󸀠
Tortuguero 10 32 5 N83∘29󸀠56 W 0 26 5000–6000󸀠󸀠
Table 2: Distribution of the number of larvae samples according to and 15 g/L agar, pH 7.0). After this incubation period, the
the insect family and national park. At each site, an approximate area microorganism-containing agar plates were flooded with
of 150.000m2 was explored for the presence of wood-feeding larvae. 0.05% Congo red for 10min, until the dye bound CMC. The
The number of insect groups found varied according to the natural reaction was fixed with NaCl (50mM) for 5min and then
condition of each forest. Each sample was composed of one to three
individuals of the same species. rinsed with distilled water. The zones where the microor-
ganism hydrolyzed the CMC were visible as clear halos [28].
National park Cer Ela Pas Sca Ten Total The oxidative degradation of lignin was determined based
Arenal 1 3 4 on the decolorization of the dye Remazol Brilliant Blue R
Barbilla 6 6 (RBBR, Sigma) when grown on solid media [29–31]. Plates
Braulio 2 2 1 5 with MEA-RBBR medium (20 g/L malt extract, 15 g/L agar,
Carara 1 3 1 5 and 0.02%wt/vol RBBR, pH 7.0) were inoculated with the
Hitoy Cerere 7 7 bacterial and fungal isolates and incubated at 28
∘C. At daily
Piedras Blancas 1 2 1 4 intervals for a period of 14 days, the plates were checked
Rincón de la Vieja 1 2 3 for the presence of a decolorized area around the colony or
Tapanti 2 1 1 1 5 mycelia. Determination of the𝛽-glucosidase,𝛽-xylanase, and
Tenorio 1 1 1 3 cellobiose hydrolase activities was performed using as sub-strates 10mM 4-nitrophenyl 𝛽-D-glucopyranoside (Sigma),
Tortuguero 1 2 2 1 6 4-nitrophenyl𝛽-D-xylopyranoside (Sigma), or 4-nitrophenyl
Total 8 5 16 16 3 48
𝛽-D-cellobioside (Sigma) dissolved in 50mM ammonium
Cer: Cerambycidae, Ela: Elateridae, Pas: Passalidae, Sca: Scarabaeidae, and acetate buffer, pH 5.0, amended with 0.7% of agar, and kept
Ten: Tenebrionidae. at 55∘C. A drop of these solutions was placed directly on the
bacterial colonies or fungal mycelia followed by incubation
two times for each enzymatic screening. The degradation for 30min at room temperature. The catalytic action of the
of cellulose was determined using carboxymethylcellulose microbial enzymes on the substrate was detected by the
(CMC, Sigma) as the sole carbon source in the medium development of a yellow coloration, produced by the release
followed by staining with Congo red. Briefly, the bacte- of the p-nitrophenol group [30, 32].
rial isolates were grown for 48 h and fungi for 72 h, at
28∘C on CMC medium (0.94 g/L KH PO , 1.9 g/L K HPO , 2.4. Molecular Analyses. All the isolates were grown in Petri
2 4 2 4
1.6 g/L KCl, 1.43 g/L NaCl, 0.15 g/L NH Cl, 0.037 g/L MgSO - dishes containing the same media used for preservation.
4 4
7H O, 0.017 g/L CaCl , 0.1 g/L yeast-extract, 7.5 g/L CMC, For the fungal DNA extraction, 400mg of mycelia was
2 2
4 International Journal of Microbiology
ground with mortar and pestle in liquid nitrogen and fur- Bray-Curtis distances between the insect fungal communities
ther extracted using the DNeasy Plant kit (Qiagen, USA), with the function vegdist. The advantage of this approach is
including a pretreatment step consisting of the incubation that Bray-Curtis measures avoid the double zero problem:
at 60∘C for one hour with 400 𝜇L of lysis buffer and accounting for absences that are not indicators of similarities
30 𝜇L of Proteinase K (20mg/mL, Sigma Aldrich, USA). between sample units [37]. The generated distance matrix
The bacterial DNA was extracted following the instructions was used to cluster similarities between the microbial com-
of the NucleoSpin Tissue DNA Extraction kit (Macherey- positions of the larval families with the hclust function. The
Nagel, Germany). The fungal ITS1-5.8S-ITS2 regions were function tabasco was used to display compact integrated
amplified by PCR from the total DNA using as forward community information, plotting the fungal taxonomical
primer the ITS1 5󸀠-TCCGTAGGTGAACCTGCGG-3󸀠 and relationships in the rows, the similarities between the insects’
the reverse primer ITS4 5󸀠-TCCTCCGCTTATTGATATGC- microbial compositions in the columns, and a heatmap with
3󸀠 [33] with the following reaction program: 95∘C for 10min, the respective abundance distribution [36]. The function cca
35 cycles at 94∘C for 1min, 54∘C for 1min, 72∘C for 1min, was used to perform canonical correspondence analysis of
and additional extension at 72∘C for 10min. The 16S rRNA the communities associated with the coleopteran families. A
gene was amplified using primers 27f and 1492r [34] with the similar procedure was performed to analyze the community
following program: 95∘C for 10min and 35 cycles of 94∘C composition of bacteria and also for detecting differences
for 1min, 52∘C for 1min and 72∘C for 1min, and 10min among national parks.
extension at 72∘C. The PCR products were purified using
the NucleoSpin Extract II kit (Macherey-Nagel, Germany)
according to manufacturer’s protocol. Sanger sequencing of 3. Results
the samples was performed at the sequencing facility of
the Dana Farber Cancer Institute at the Harvard University, 3.1. Taxonomic Composition of the Fungi and Bacteria Isolated.
Boston, Massachusetts, using the abovementioned forward In this study, we isolated 92 fungal strains and 135 bacterial
and reverse primers for fungi and primers 27f and 785r for strains from larvae of five families of Coleoptera that were
bacteria. Sequences were assembled using Seqman program feeding ondecayingwood in tropicalwet forests of 10 national
ofDNASTARLasergene 8.0 (GenBank accession: GU827479- parks of Costa Rica. The 92 fungal isolates were assigned to
GU827553, HM770962-HM771112). three phyla, 16 orders, 24 families, and 40 genera (one differ-
ent genus every 2.3 isolates).Within the phylumZygomycota,
2.5. Taxonomy. The taxonomic assignment of the bacterial we isolated members of the order Mucorales and within the
sequences was performed by comparing the database against phylum Basidiomycota members of the orders Agaricales,
the 16S rRNA reference set 10 implemented in the Classifier Polyporales, and Trichosporonales. Most of the fungi isolated
tool of the Ribosomal Database Project, which assigned the from the gastrointestinal tracts of the larvae belonged to the
16S rRNA sequences to corresponding taxonomical hierarchy phylum Ascomycota (89% of total). They were distributed
based on a naı̈ve Bayesian rRNA classifier [35].The taxonomy in 12 orders, with Hypocreales being the dominant one; it
of the fungi was inferred by comparing the ITS1-5.8S- comprised nearly 55% of the isolates (Table 3). The genus
ITS2 sequences against the Warcup Fungal ITS trainset 1, Trichoderma was the most abundant; it was the only one
a curated reference dataset implemented in the Classifier associatedwith all five families of Coleoptera and also present
tool of the Ribosomal Database Project [35]. Every fungal in each national park sampled.
taxonomical assignment was verified against the Index Fun- Most fungal orders and genera were sparsely represented,
gorum (http://www.indexfungorum.org/) and appropriately with 68% of the orders and 55% of the genera found associ-
corrected when synonyms or current names were identified. ated with a specific coleopteran family at a particular site. All
the insect families also presented unique fungal isolates per
2.6. Ecological Analyses. The analysis of the microbial com- site, with Tenebrionidae being the only coleopteran family in
munities was performed using the Vegan package imple- which all isolates were phylogenetically distinct. Regarding
mented in the statistical programming environment and the sampling sites, Tenorio National Park showed the highestnumber of unique phylotypes, but Piedras Blancas National
language R [36]. For this, a table with the taxonomic clas- Park contained a more phylogenetically diverse array of
sifications to the levels of order, class, subphylum, phylum, isolates.
and subkingdom of the fungal isolates was converted to The 135 bacterial isolates were classified within five
taxonomic pairwise distances with the function taxa2dist phyla, 13 orders, 22 families, and 35 genera (one different
and using variable step lengths between successive categories, genus from every 3.8 isolates), including members of Acti-
proportional to the number of groups within each taxonom- nobacteria, Proteobacteria, Firmicutes, Flavobacteria, and
ical level. This distance matrix was then used to construct Fusobacteria. Approximately 82% of the bacteria belonged
a hierarchical clustering tree with the function hclust and to 𝛾-Proteobacteria and Firmicutes, accounting for 44% and
the UPGMA distance method. A second matrix with the 38% of the isolates, respectively.Within the 𝛾-Proteobacteria,
abundance distribution of the fungal isolates per insect family the genera Serratia and Pseudomonas were abundant, being
was prepared with the larval families in the rows, the fungal present in all host families studied. Within Firmicutes, the
orders in the columns, and cells containing the counts of genus Bacillus was clearly the most dominant. This single
isolates in each taxon. This matrix was used to calculate genus, which accounted for 20% of all the isolates, was
International Journal of Microbiology 5
Table 3: Taxonomic distribution of the fungal isolates identified in this study. The number of isolates at the order and genera level is shown
for each of the coleopteran families.
Order Genus Cer Ela Pas Sca Ten Total
Botryosphaeriales Botryosphaeria 1 1
Capnodiales Ramichloridium 1 1
Cladophialophora 2 2
Chaetothyriales Fonsecaea 1 1
Rhynchostoma 1 1
Diaporthales Phomopsis 1 1
Aspergillus∗ 1 1
Eurotiales Paecilomyces 1 1 1 3
Penicillium 3 1 1 5
Helotiales Scytalidium 1 1
Acremonium 1 1
Bionectria∗ 1 1 2
Cladobotryum 1 1
Cordyceps 1 1
Cosmospora 1 1
Elaphocordyceps 1 1
Fusarium 1 1 2
Hypocreales Gliocladiopsis 1 1
Isaria 1 1 2
Lanatonectria 1 1
Mariannaea∗ 1 2 3
Metacordyceps 1 1
Metarhizium 2 3 2 7
Nectria 2 2
Neonectria 1 1
Trichoderma∗ 6 4 4 8 1 23
Graphium 1 1 2
Microascales Pseudallescheria 1 1 2
Scedosporium 1 1
Ophiostomatales Sporothrix 2 2 4
Pleosporales Leptosphaerulina 1 1
Saccharomycetales Geotrichum∗ 1 1 2
Xylariales Eutypa 1 1
Pestalotiopsis 1 1 2
Agaricales Coprinellus 2 1 3
Polyporales Phlebia 1 1
Trametes∗ 1 1
Trichosporonales Trichosporon 1 1 2
∗
Mucorales Mucor 1 1 2
Rhizomucor 1 1
Total 25 12 19 29 7 92
Cer: Cerambycidae, Ela: Elateridae, Pas: Passalidae, Sca: Scarabaeidae, and Ten: Tenebrionidae.
∗Genera that presented positive enzymatic activities in more than four pathways.
a common gut inhabitant of all the insect families and was less than 6% of the isolates. When calculating the percent-
present at almost all the sites sampled (Table 4). age of isolates that were specific to a single site and host
The remaining bacterial classes obtained in this study family, results showed that 42% of the isolates exhibited
were less represented. For example, members of Actinobac- this characteristic, while the remaining 58% of the genera
teria accounted for 11% of the isolates whereas 𝛼- and 𝛽- presented a broader host-site range. A small number of the
Proteobacteria, Flavobacteria, and Fusobacteria represented genera were found in one site but in different host families
6 International Journal of Microbiology
Table 4: Taxonomic distribution of the bacterial isolates obtained in this study. The number of isolates at the phylum and genera level is
shown for each of the coleopteran families.
Class Genus Cer Ela Pas Sca Ten Total
Arthrobacter 1 1
Cellulomonas 1 1
Leifsonia 1 1
Actinobacteria Leucobacter 3 3
Microbacterium 1 1
Streptomyces 2 5 7
Tsukamurella 1 1
𝛼-Proteobacteria Novosphingobium 1 1
Rhizobium 2 2
𝛽-Proteobacteria Achromobacter 1 1 2
Chromobacterium 1 1
Acinetobacter∗ 2 4 1 3 10
Alishewanella 1 1
Azorhizophilus 1 1
Citrobacter 2 2
Dyella 1 1
Enterobacter∗ 4 4 1 3 12
-Proteobacteria Erwinia 1 1𝛾
Klebsiella 1 1 2
Kluyvera 1 1
Pseudomonas 2 1 1 4 1 9
Raoultella 3 1 4
Salmonella 2 2
Serratia 4 2 2 2 2 12
Stenotrophomonas 1 1 2
Bacillus∗ 4 4 6 10 3 27
Enterococcus 3 2 5
Firmicutes Lactococcus 1 5 2 8
Lysinibacillus 1 2 4 7
Paenibacillus 1 1 1 3
Staphylococcus 1 1 2
Flavobacteria Chryseobacterium 1 1
Fusobacteria Sebaldella 1 1
Total 18 20 41 46 10 135
Cer: Cerambycidae, Ela: Elateridae, Pas: Passalidae, Sca: Scarabaeidae, and Ten: Tenebrionidae.
∗Genera that presented positive enzymatic activities in more than four pathways.
(i.e., Rhizobium in Hitoy Cerere National Park); others were degrading lignocellulosic materials than bacteria, with gen-
associated with the same beetle family but in different sites era such as Trichoderma, Bionectria, and Trametes showing
(i.e., Leucobacter with Passalidae). Results of the analysis positive results in all the assays performed. Within bacteria,
of unique phylotypes per site and insect family showed Bacillus, Enterobacter, and Acinetobacter, some of the most
that Hitoy Cerere National Park and Elateridae, respectively, abundant genera isolated from the larval guts, tested positive
presented the highest percentages of single bacterial isolates. for four out of the five enzymatic activities assayed: cellulase,
𝛽-glucosidase, 𝛽-xylanase, and cellobiose hydrolase activi-
ties. However, neither these genera nor any other bacterial
3.2. Lignocellulolytic Activity Determination. Nearly 65% of group screened were able to degrade the Remazol Brilliant
the fungal genera and 48% of the bacterial genera presented Bluemolecules, while 30%of the fungal genera tested positive
positive results in at least one of the five lignocellulolytic for this lignin-related degradation activity.
activities evaluated, with carboxymethylcellulose degrada-
tion being the most common activity observed in both 3.3. Comparison of Gut Inhabitants between Families of
groups (Table 5). In general, fungi showedmore capability for Coleoptera. We performed community analysis with Vegan
International Journal of Microbiology 7
Table 5: Results of the screening for lignocellulolytic activities.
Fungal genera with positive results are shown in the upper group
and bacterial genera in the lower group.
Genus CMC lignin 𝛽-gluc 𝛽-xyl celob EurotialesChaetothyriales
Aspergillus + + + + Saccharomycetales
Bionectria + + + + + PleosporalesBotryosphaeriales
Botryosphaeria + CapnodialesXylariales
Coprinellus + Hypocreales
Elaphocordyceps + MicroascalesOphiostomatales
Eutypa + + DiaporthalesHelotiales
Fusarium + Trichosporonales
Geotrichum + + + + AgaricalesPolyporales
Graphium + Mucorales
Isaria + + +
Lanatonectria +
Mariannaea + + + +
Metacordyceps + + +
Mucor + + + + Figure 1: Heatmap of the abundance distribution of fungal com-
Nectria + + + munities associated with the guts of five wood-feeding families
Paecilomyces + + of Coleoptera. The taxonomic relationship of the fungal genera is
Penicillium + + shown in the rows, while the clustering of the coleopteran families,
Pestalotiopsis + determined by their composition similarities, is shown in thecolumns. Higher intensities of the color reveal higher abundances
Phlebia + + + of the isolates.
Phomopsis +
Pseudallescheria + + +
Scytalidium + + the abundance distribution of the isolates, considering also
Sporothrix + their phylogenetic relationships. The results showed that the
Trametes + + + + + fungal composition of the isolates associated with larvae
Trichoderma + + + + + of Cerambycidae, Scarabaeidae, and Passalidae clustered
Trichosporon + together; Cerambycidae and Passalidae shared one order
Acinetobacter + + + + of Basidiomycota and three orders of Ascomycota, while
Bacillus + + + + Scarabaeidae and Passalidae had in common four orders
Citrobacter + of Ascomycota. A second cluster was formed by the fungalmicrobiotas isolated fromTenebrionidae and Elateridae; they
Enterobacter + + + + shared two orders of Ascomycota and one of Basidiomycota
Enterococcus + + + (Figure 1). The analysis of the bacterial dataset showed that
Lactococcus + the microbial compositions associated with Scarabaeidae
Novosphingobium + and Passalidae formed part of the same cluster, sharing
Paenibacillus + isolates belonging to 𝛽- and 𝛾-Proteobacteria, Actinobac-
Pseudomonas + teria, and Firmicutes. The second cluster was formed by
Rhizobium + Tenebrionidae, Elateridae, andCerambycidae that shared iso-
Serratia + lates assigned to Pseudomonadales, Enterobacteriales, and
Stenotrophomonas + Bacillales (Figure 2). In addition, we performed canonical
Arthrobacter + correspondence analysis for exploring relationships betweenthe microbial communities of the coleopteran hosts. Results
Microbacterium + of this analysis where consistent with results obtained with
Streptomyces + the Bray-Curtis clustering for both the fungal and bacterial
Tsukamurella + communities (Figure S1 in Supplementary Material available
CMC: cellulase activity on carboxymethylcellulose, lignin: ligninolytic activ- online at http://dx.doi.org/10.1155/2015/285018).
ity onRemazol Brilliant Blue R,𝛽-gluc:𝛽-glucosidase,𝛽-xyl:𝛽-xylanase, and
celob: cellobiose hydrolase activity.
4. Discussion
We collected larvae of five families of wood-feeding
to gain insight into how the microbial gut composition of the Coleoptera in tropical forests of Costa Rica with the aim of
beetle families related to one another.This approach clustered estimating the species composition of cultivable fungi and
the environments according to Bray-Curtis distances of bacteria inhabiting their guts and to identify microorganisms
Cerambycidae
Passalidae
Scarabaeidae
Elateridae
Tenebrionidae
8 International Journal of Microbiology
of Hypocreales, as also observed in a similar study performed
in other locations of Costa Rica [41]. This is relevant for
bioprospecting purposes, since wood-feeding beetles might
Flavobacteriales constitute a good source of Trichoderma,Metarhizium,Meta-
Neisseriales cordyceps, Bionectria, and other fungal genera known to
Burkholderiales possess a wide array of biotechnological applications [42–
Alteromonadales 45]. The remaining orders presented a lower abundance
Pseudomonadales and, in most of the cases, were represented by a single
Enterobacteriales
Xanthomonadales genus. Nevertheless, many of the genera showed the ability
Rhizobiales to degrade lignocellulose-related hexoses and pentoses, as
Sphingomonadales also shown in other studies [46–50]. Within the phylum
Actinomycetales Basidiomycota, the genus Trametes showed positive results
Bacillales in all the lignocellulolytic assays related to the degradation
Lactobacillales
Fusobacteriales of structural wood components. This white-rot fungus is a
knownmodel for studying degradation of lignin in free-living
conditions and in this work reported in its association with
the gut microbiota of wood-feeding insects [51, 52].
It is difficult to know whether these fungal isolates are
truly endosymbionts of the intestinal tracts of the coleopteran
Figure 2: Heatmap of the abundance distribution of bacterial larvae or are transitory inhabitants associated with host
communities associated with the guts of five wood-feeding families feeding habits. Hence, it is also possible that some of
of Coleoptera. The taxonomic relationship of the bacterial genera is these microorganisms could be commensals, parasites, and
shown in the rows, while the clustering of the coleopteran families, facultative endosymbionts. They might even be using the
determined by their composition similarities, is shown in the insect as a dispersal mechanism [15, 53]. It is clear, however,
columns. Higher intensities of the color reveal higher abundances that the overall taxonomic composition of the gut-inhabiting
of the isolates. microbes and the proportion of lignocellulolytic-positive
fungi seem to be particular to the larval microenvironment.
The structure of this endosymbiotic community is distin-
with relevant lignocellulolytic activities. The main limitation guished from the fungal composition observed in other
of this study is that the cultivation-dependent approach, wood-related microhabitats such as the fungal populations
based on artificial media, covers only a small proportion of in living plant tissues. They are also dominated by members
the total microbial diversity present in this particular niche. of Ascomycetes, but they present a different abundance
The positive trade-off of this approach was the identification distribution of fungal families [54, 55]; decaying logs are
of several isolates with lignocellulose-degrading capabilities, dominated mainly by Basidiomycetes [56–58].
which can be further used for the respective enzyme The analysis of the taxonomic composition of the bacte-
characterization, for direct degradation assays on residues rial isolates showed the presence of seven major phylogenetic
from agriculture and forestry, for the treatment of industrial classes, codominated by 𝛾-Proteobacteria and Firmicutes.
effluents, and for bioprospecting novel metabolites with This finding is consistent with results obtained in similar
other biotechnological applications. Despite the inherent studies [6, 13, 14, 44, 59]. Within the 𝛾-Proteobacteria, the
bias of the isolation method, our results suggest that gut most abundant genera were Enterobacter, Serratia, Acineto-
microbiota of wood-feeding tropical beetles presents a bacter, and Pseudomonas. Interestingly, Serratia and Pseu-
relatively high diversity in terms of microbial richness, domonas were isolated from all five coleopteran families
phylogenetic composition, and lignocellulolytic activities. studied; Enterobacter and Acinetobacter were present in four
The order Hypocreales represented about 60% of the out of the five insect families, and they exhibited positive
total number of fungal isolates. Within this group, the genus results in the lignocellulolytic assays, except for lignin degra-
Trichoderma was the most abundant, comprising nearly a dation. Similar characteristics related to the degradation of
quarter of the fungal collection. This genus was a common lignocellulose and to fermentativemetabolismwere observed
gut inhabitant of beetle larvae regardless of the host family in Bacillus, the most abundant genus within Firmicutes [11,
or the geographic location. The reason for this dominance 60]. Together, these results support the notion that some
is not entirely clear; however, one possible explanation is species of fungi and bacteria, such as Trichoderma, Serratia,
that several species belonging to this fungal genus contain Pseudomonas, and Bacillus, can be common gut inhabitants
a number of glycoside hydrolases, peroxidases, laccases, of wood-feeding larvae in tropical forests, suggesting that
and phenol oxidases, among other enzymes related to the certain affinities might have developed between the beetle
degradation of lignocellulose materials. This feature might host and its microbiota [41, 61–64].
provide some advantages for using the recalcitrant polymeric When comparing the fungal and bacterial species compo-
materials passing through the gastrointestinal tract [16, 38– sition among the beetle families, the plots of the Bray-Curtis
40]. distances and canonical correspondence analyses produced
In addition, our data indicate that guts of wood-feeding biologically meaningful clusters to group the environments
larvae were from environments having a high representation that share similar microbial compositions. The first fungal
Cerambycidae
Elateridae
Tenebrionidae
Scarabaeidae
Passalidae 
International Journal of Microbiology 9
cluster relates the microbiota associated with the guts of Acknowledgments
Cerambycidae, Passalidae, and Scarabaeidae. This is con-
sistent with the observation of a high diversity of isolates The authors acknowledge the contribution of the follow-
from Cerambycidae that shared members of the fungal ing people during the development of this project: Luis
phyla Basidiomycota and Ascomycota with Passalidae and GuillermoAcosta for the field sampling and early insect iden-
members of Zygomycota and Ascomycota with Scarabaeidae. tification; Angel Solis, Carlos Hernández, and Elena Ulate for
The cluster formed by Tenebrionidae-Elateridae shared, in the identification of some adult specimens; Jorge Blanco for
a lower proportion, members of the Basidiomycota and the fungal isolation; Angelica Acuña and Beatriz Rivera for
Ascomycota.The bacterial microbiota associated with Passal- theDNA extraction and enzymatic assays;Manuel Ferrer and
idae and Scarabaeidae also formed a cluster, sharingmembers Cesar Mateo for their advices on the lignocellulolytic activity
of five major bacterial clades; microbiota of Cerambycidae, determination; Ana Lorena Guevara and Giselle Tamayo
Elateridae, and Tenebrionidae shared members only of - for the overall support; the editor and reviewers of this𝛾
Proteobacteria and Firmicutes. journal for critical comments on the paper.This research was
The clustering analyses revealed that Cerambycidae pre- funded by the support of the National Council of Science and
sented a high diversity of fungi but not of bacteria, while Technology (CONICIT, FV-027-2007), the CSIC and CRUSA
Passalidae and Elateridae exhibited a high diversity of bac- Foundation (2007 CR0034), and Florida Ice & FarmCo.They
teria and moderate diversity of fungi. Scarabaeidae and thank ACLAC, ACOPAC, ACOSA, ACTo, ACLAP, ACCVC,
Tenebrionidae contained a similar composition of both. ACAT, ACAHN, ACG, and ACLAC National Conservation
These results suggest that the nature of the beetle host Areas and CONAGEBIO for granting the sample collecting
has an important effect on the phylogenetic diversity of its permits (R-CM-INBio-40-2008-OT, R-CM-INBio-48-2008-
associated microbiota and that many factors can influence OT).
its configuration. These factors may include the biology of
the host, the physical and chemical characteristics of the References
gut compartments, the feeding habits of the insects, and the
microbial diversity associated with the environment in which [1] M. Tien and C.-P. D. Tu, “Cloning and sequencing of a cDNA
the insect is living [23, 26, 65, 66]. for a ligninase from Phanerochaete chrysosporium,” Nature, vol.
326, no. 6112, pp. 520–523, 1987.
Our results consistently showed that both the fungal
and bacterial populations associated with the guts of beetle [2] P. Béguin, “Molecular biology of cellulose degradation,” AnnualReview of Microbiology, vol. 44, pp. 219–248, 1990.
larvae are highly diverse in terms of the number of species
obtained and in their phylogenetic composition. These [3] J. Pérez, J. Muñoz-Dorado, T. de la Rubia, and J. Mart́ınez,
microbial inhabitants could be forming complex consortia “Biodegradation and biological treatments of cellulose, hemi-cellulose and lignin: an overview,” International Microbiology,
that would be acting synergistically to provide many of the vol. 5, no. 2, pp. 53–63, 2002.
nutritional needs of the beetle host. Some of these functions [4] M. R. Berenbaum and T. Eisner, “Ecology. Bugs’ bugs,” Science,
include the degradation and fermentation of lignocellulosic vol. 322, no. 5898, pp. 52–53, 2008.
materials, as shown by the high percentage of fungal and
bacterial genera that presented positive activities or by the [5] Z. Zhang, “Phylum Arthropoda von Siebold, 1948. In: animalbiodiversity: an outline of higher-level classification and survey
production of proteins and other metabolites necessary for of taxonomic richness,” Zootaxa, vol. 3148, pp. 99–103, 1948.
the development of the insect [25, 44, 67–69]. Furthermore, [6] J. Morales-Jiménez, G. Zúñiga, L. Villa-Tanaca, and C.
certain affinities for substrates can be expected according to Hernández-Rodŕıguez, “Bacterial community and nitrogen
the nature of the gut inhabitant. For example, members of fixation in the red turpentine beetle, Dendroctonus valens
the Basidiomycota could possibly degrade larger polymeric LeConte (Coleoptera: Curculionidae: Scolytinae),” Microbial
molecules, the Ascomycota deplete diverse lignocellulosic Ecology, vol. 58, no. 4, pp. 879–891, 2009.
constituents, while the bacteria degrade and ferment the [7] S. M. Geib, M. del Mar Jimenez-Gasco, J. E. Carlson, M. Tien,
smaller monomeric and dimeric hexoses and pentoses pro- R. Jabbour, and K. Hoover, “Microbial community profiling
duced by the fungal counterparts. The bacteria also likely to investigate transmission of bacteria between life stages of
use these sugars to produce other nutrients and metabo- the wood-boring beetle, Anoplophora glabripennis,” Microbial
lites. Consequently, the present work raises new lines of Ecology, vol. 58, no. 1, pp. 199–211, 2009.
investigation concerning the existence of microbial consortia [8] P. Engel and N. A. Moran, “The gut microbiota of insects—
acting synergistically to provide the nutritional needs of the diversity in structure and function,” FEMS Microbiology
hosts, the nature of the ecological and evolutionary processes Reviews, vol. 37, no. 5, pp. 699–735, 2013.
that contribute to ensure the fitness of the insect, and the [9] W. Shi, S. Xie, X. Chen et al., “Comparative genomic anal-
mechanisms that rule the interactions among the fungi, the ysis of the microbiome of herbivorous insects reveals eco-
bacteria, and the beetle host. environmental adaptations: biotechnology applications,” PLoS
Genetics, vol. 9, no. 1, Article ID e1003131, 2013.
[10] I. Hanski and Y. Cambefort, Dung Beetle Ecology, Princeton
Conflict of Interests University Press, Princeton, NJ, USA, 1991.
[11] M. Egert, B. Wagner, T. Lemke, A. Brune, and M. W. Friedrich,
The authors declared that there is no conflict of interests “Microbial community structure in midgut and hindgut of
regarding this paper. the humus-feeding larva of Pachnoda ephippiata (Coleoptera:
10 International Journal of Microbiology
Scarabaeidae),” Applied and Environmental Microbiology, vol. [29] B. R.M.Vyas andH. P.Molitoris, “Involvement of an extracellu-
69, no. 11, pp. 6659–6668, 2003. lar H O -dependent ligninolytic activity of the white rot fungus
2 2
[12] R. N. Coulson, “Population dynamics of bark beetles,” Annual Pleurotus ostreatus in the decolorization of Remazol brilliant
Review of Entomology, vol. 24, no. 1, pp. 417–447, 1979. blue R,” Applied and Environmental Microbiology, vol. 61, no. 11,
[13] P. D. Schloss, I. Delalibera Jr., J. Handelsman, and K. F. Raffa, pp. 3919–3927, 1995.
“Bacteria associated with the guts of two wood-boring beetles: [30] J.-D. Bok, D. A. Yernool, and D. E. Eveleigh, “Purification,
anoplophora glabripennis and Saperda vestita (Cerambycidae),” characterization, and molecular analysis of thermostable cellu-
Environmental Entomology, vol. 35, no. 3, pp. 625–629, 2006. lases CelA andCelB fromThermotoga neapolitana,”Applied and
[14] A. Vasanthakumar, J. O. Handelsman, P. D. Schloss, L. S. Bauer, EnvironmentalMicrobiology, vol. 64, no. 12, pp. 4774–4781, 1998.
and K. F. Raffa, “Gut microbiota of an invasive subcortical [31] K. M. G. Machado, D. R. Matheus, and V. L. R. Bononi,
beetle, Agrilus planipennis Fairmaire, across various life stages,” “Ligninolytic enzymes production and Remazol Brilliant Blue
Environmental Entomology, vol. 37, no. 5, pp. 1344–1353, 2008. R decolorization by tropical Brazilian basidiomycetes fungi,”
[15] J. Morales-Jiménez, G. Zúñiga, H. C. Ramı́rez-Saad, and C. Brazilian Journal of Microbiology, vol. 36, no. 3, pp. 246–252,
Hernández-Rodŕıguez, “Gut-associated bacteria throughout 2005.
the life cycle of the bark beetle Dendroctonus rhizophagus [32] G. T. Howard and B. A. White, “Molecular cloning and expres-
Thomas and Bright (Curculionidae: Scolytinae) and their cellu- sion of cellulase genes fromRuminococcus albus 8 in Escherichia
lolytic activities,”Microbial Ecology, vol. 64, no. 1, pp. 268–278, coli bacteriophage 𝜆,” Applied and Environmental Microbiology,
2012. vol. 54, no. 7, pp. 1752–1755, 1988.
[16] R. Kumar, S. Singh, and O. V. Singh, “Bioconversion of lig- [33] N. L. Glass and G. C. Donaldson, “Development of primer
nocellulosic biomass: biochemical and molecular perspectives,” sets designed for use with the PCR to amplify conserved genes
Journal of IndustrialMicrobiology and Biotechnology, vol. 35, no. from filamentous ascomycetes,” Applied and Environmental
5, pp. 377–391, 2008. Microbiology, vol. 61, no. 4, pp. 1323–1330, 1995.
[17] C. Sánchez, “Lignocellulosic residues: biodegradation and bio- [34] D. Lane, “16S/23S rRNA sequencing,” inNucleic Acid Techniques
conversion by fungi,” Biotechnology Advances, vol. 27, no. 2, pp. in Bacterial Systematics, E. Stachebrandt and M. Goodfellow,
185–194, 2009. Eds., Wiley, Chichester, UK, 1991.
[18] E. D. Scully, S. M. Geib, K. Hoover et al., “Metagenomic [35] Q. Wang, G. M. Garrity, J. M. Tiedje, and J. R. Cole, “Näıve
profiling reveals lignocellulose degrading system in a microbial Bayesian classifier for rapid assignment of rRNA sequences
community associated with a wood-feeding beetle,” PLoS ONE, into the new bacterial taxonomy,” Applied and Environmental
vol. 8, no. 9, Article ID e73827, 2013. Microbiology, vol. 73, no. 16, pp. 5261–5267, 2007.
[19] T. L. Erwin, “Tropical forest: their richness in Coleoptera and [36] J. Oksanen, G. Blanchet, R. Kindt et al., Vegan: Community
other arthropod species,”The Coleopterists Bulletin, vol. 36, no. Ecology Package, 2014.
1, pp. 74–75, 1982. [37] P. L. Buttigieg and A. Ramette, “A guide to statistical analysis
[20] Y. Basset, L. Cizek, P. Cuénoud et al., “Arthropod diversity in a in microbial ecology: a community-focused, living review of
tropical forest,” Science, vol. 338, no. 6113, pp. 1481–1484, 2012. multivariate data analyses,” FEMSMicrobiology Ecology, vol. 90,
[21] A. Solis, Escarabajos de Costa Rica: Las Familias Mas Comunes, no. 3, pp. 543–550, 2014.
Heredia, Costa Rica, Editorial Inbio, 1999. [38] D.Martinez, R.M. Berka, B. Henrissat et al., “Genome sequenc-
[22] D. Borror and R. White, A Field Guide to the Insects, Houghton ing and analysis of the biomass-degrading fungus Trichoderma
Mifflin Company, New York, NY, USA, 1987. reesei (syn. Hypocrea jecorina),” Nature Biotechnology, vol. 26,
[23] M. Egert, U. Stingl, L. D. Bruun, B. Pommerenke, A. Brune, no. 5, pp. 553–560, 2008.
and M. W. Friedrich, “Structure and topology of microbial [39] E. D. Scully, K. Hoover, J. E. Carlson, M. Tien, and S. M. Geib,
communities in the major gut compartments of Melolontha “Midgut transcriptome profiling of Anoplophora glabripennis,
melolontha larvae (Coleoptera: Scarabaeidae),” Applied and a lignocellulose degrading cerambycid beetle,” BMC Genomics,
EnvironmentalMicrobiology, vol. 71, no. 8, pp. 4556–4566, 2005. vol. 14, article 850, 2013.
[24] S.-O. Suh, J. V. McHugh, D. D. Pollock, and M. Blackwell, “The [40] P. K. Foreman, D. Brown, L. Dankmeyer et al., “Transcriptional
beetle gut: a hyperdiverse source of novel yeasts,” Mycological regulation of biomass-degrading enzymes in the filamentous
Research, vol. 109, no. 3, pp. 261–265, 2005. fungus Trichoderma reesei,”The Journal of Biological Chemistry,
[25] J. J. Scott, D.-C. Oh, M. C. Yuceer, K. D. Klepzig, J. Clardy, and vol. 278, no. 34, pp. 31988–31997, 2003.
C. R. Currie, “Bacterial protection of beetle-fungusmutualism,” [41] G. Vargas-Asensio, A. Pinto-Tomas, B. Rivera et al., “Uncover-
Science, vol. 322, no. 5898, p. 63, 2008. ing the cultivable microbial diversity of costa rican beetles and
[26] J. A. Ceja-Navarro, N. H. Nguyen, U. Karaoz et al., “Com- its ability to break down plant cell wall components,”PLoSONE,
partmentalized microbial composition, oxygen gradients and vol. 9, no. 11, Article ID e113303, 2014.
nitrogen fixation in the gut of Odontotaenius disjunctus,” The [42] M. P. Coughlan, “The properties of fungal and bacterial cel-
ISME Journal, vol. 8, no. 1, pp. 6–18, 2014. lulases with comment on their production and application,”
[27] J. B. Nardi, C. M. Bee, L. A. Miller, N. H. Nguyen, S.-O. Suh, Biotechnology and Genetic Engineering Reviews, vol. 3, no. 1, pp.
and M. Blackwell, “Communities of microbes that inhabit the 39–110, 1985.
changing hindgut landscape of a subsocial beetle,” Arthropod [43] L. R. Lynd, P. J. Weimer, W. H. Van Zyl, and I. S. Pretorius,
Structure and Development, vol. 35, no. 1, pp. 57–68, 2006. “Microbial cellulose utilization: fundamentals and biotechnol-
[28] R.M. Teather and P. J.Wood, “Use of Congo red-polysaccharide ogy,”Microbiology andMolecular Biology Reviews, vol. 66, no. 3,
interactions in enumeration and characterization of cellulolytic pp. 506–577, 2002.
bacteria from the bovine rumen,” Applied and Environmental [44] S. M. Geib, T. R. Filley, P. G. Hatcher et al., “Lignin degradation
Microbiology, vol. 43, no. 4, pp. 777–780, 1982. in wood-feeding insects,” Proceedings of the National Academy
International Journal of Microbiology 11
of Sciences of the United States of America, vol. 105, no. 35, pp. [60] C.C. Lee, R. E. Kibblewhite-Accinelli,M. R. Smith, K.Wagschal,
12932–12937, 2008. W. J. Orts, and D.W. S.Wong, “Cloning of Bacillus licheniformis
[45] H. Alper and G. Stephanopoulos, “Engineering for biofuels: xylanase gene and characterization of recombinant enzyme,”
exploiting innate microbial capacity or importing biosynthetic Current Microbiology, vol. 57, no. 4, pp. 301–305, 2008.
potential?” Nature Reviews Microbiology, vol. 7, no. 10, pp. 715– [61] T. L. Rhoads, A. T. Mikell Jr., and M. H. Eley, “Investigation of
723, 2009. the lignin-degrading activity of Serratia marcescens: biochem-
[46] K. Yaoi and Y. Mitsuishi, “Purification, characterization, ical screening and ultrastructural evidence,” Canadian Journal
cloning, and expression of a novel xyloglucan-specific glycosi- of Microbiology, vol. 41, no. 7, pp. 592–600, 1995.
dase, oligoxyloglucan reducing end-specific cellobiohydrolase,” [62] T. Nagy, K. Emami, C. M. G. A. Fontes, L. M. A. Ferreira,
The Journal of Biological Chemistry, vol. 277, no. 50, pp. 48276– D. R. Humphry, and H. J. Gilbert, “The membrane-bound
48281, 2002. 𝛼-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-O-
[47] L. Ayed, N. Assas, S. Sayadi, and M. Hamdi, “Involvement methyl-D-glucuronoxylooligosaccharides but not 4-O-methyl-
of lignin peroxidase in the decolourization of black olive D-glucuronoxylan,” Journal of Bacteriology, vol. 184, no. 17, pp.
mill wastewaters by Geotrichum candidum,” Letters in Applied 4925–4929, 2002.
Microbiology, vol. 40, no. 1, pp. 7–11, 2005. [63] J. Weslien, L. B. Djupström, M. Schroeder, and O. Widenfalk,
[48] Y. Baba, A. Shimonaka, J. Koga, H. Kubota, and T. Kono, “Long-term priority effects among insects and fungi colonizing
“Alternative splicing produces two endoglucanases with one decaying wood,” The Journal of Animal Ecology, vol. 80, no. 6,
or two carbohydrate-binding modules in Mucor circinelloides,” pp. 1155–1162, 2011.
Journal of Bacteriology, vol. 187, no. 9, pp. 3045–3051, 2005. [64] R. T. Jones, L. G. Sanchez, and N. Fierer, “A cross-taxon analysis
[49] M. Dashtban, H. Schraft, andW. Qin, “Fungal bioconversion of of insect-associated bacterial diversity,” PLoS ONE, vol. 8, no. 4,
lignocellulosic residues; opportunities & perspectives,” Interna- Article ID e61218, 2013.
tional Journal of Biological Sciences, vol. 5, no. 6, pp. 578–595, [65] T. Lemke, U. Stingl, M. Egert, M. W. Friedrich, and A. Brune,
2009. “Physicochemical conditions and microbial activities in the
[50] D. A. Ribeiro, J. Cota, T. M. Alvarez et al., “The Penicillium highly alkaline gut of the humus-feeding larva of Pachnoda
echinulatum secretome on sugar cane bagasse,” PLoS ONE, vol. ephippiata (Coleoptera: Scarabaeidae),” Applied and Environ-
7, no. 12, Article ID e50571, 2012. mental Microbiology, vol. 69, no. 11, pp. 6650–6658, 2003.
[51] O. Borokhov and S. Rothenburger, “Rapid dye decolorization [66] S. M. Geib, M. D. M. Jimenez-Gasco, J. E. Carlson, M. Tien,
method for screening potential wood preservatives,” Applied and K. Hoover, “Effect of host tree species on cellulase activity
and Environmental Microbiology, vol. 66, no. 12, pp. 5457–5459, and bacterial community composition in the gut of larval Asian
2000. longhorned beetle,” Environmental Entomology, vol. 38, no. 3,
[52] E. Abadulla, T. Tzanov, S. Costa, K.-H. Robra, A. Cavaco- pp. 686–699, 2009.
Paulo, and G. M. Gubitz, “Decolorization and detoxification of [67] A. E. Cazemier, J. C. Verdoes, F. A. G. Reubsaet, J. H. P.
textile dyes with a laccase from Trametes hirsuta,” Applied and Hackstein, C. van der Drift, and H. J. M. Op den Camp,
Environmental Microbiology, vol. 66, no. 8, pp. 3357–3362, 2000. “Promicromonospora pachnodae sp. nov., a member of the
[53] T. J. Dreaden, J. M. Davis, Z. W. de Beer et al., “Phylogeny (hemi)cellulolytic hindgut flora of larvae of the scarab beetle
of ambrosia beetle symbionts in the genus Raffaelea,” Fungal Pachnoda marginata,” Antonie van Leeuwenhoek, vol. 83, no. 2,
Biology, vol. 118, no. 12, pp. 970–978, 2014. pp. 135–148, 2003.
[54] T. S. Suryanarayanan, T. S. Murali, and G. Venkatesan, “Occur- [68] F. A. Genta, R. J. Dillon, W. R. Terra, and C. Ferreira, “Potential
rence and distribution of fungal endophytes in tropical forests role for gut microbiota in cell wall digestion and glucoside
across a rainfall gradient,” Canadian Journal of Botany, vol. 80, detoxification in Tenebrio molitor larvae,” Journal of Insect
no. 8, pp. 818–826, 2002. Physiology, vol. 52, no. 6, pp. 593–601, 2006.
[55] A. E. Arnold and F. Lutzoni, “Diversity and host range of foliar [69] D.-C. Oh, J. J. Scott, C. R. Currie, and J. Clardy, “Mycangimycin,
fungal endophytes: are tropical leaves biodiversity hotspots?” a polyene peroxide from a mutualist Streptomyces sp.,” Organic
Ecology, vol. 88, no. 3, pp. 541–549, 2007. Letters, vol. 11, no. 3, pp. 633–636, 2009.
[56] J. Heilmann-Clausen and L. Boddy, “Inhibition and stimulation
effects in communities of wood decay fungi: exudates from
colonized wood influence growth by other species,” Microbial
Ecology, vol. 49, no. 3, pp. 399–406, 2005.
[57] A. Kubartová, E. Ottosson, A. Dahlberg, and J. Stenlid, “Pat-
terns of fungal communities among and within decaying logs,
revealed by 454 sequencing,” Molecular Ecology, vol. 21, no. 18,
pp. 4514–4532, 2012.
[58] L. Prewitt, Y. Kang, M. L. Kakumanu, andM.Williams, “Fungal
and bacterial community succession differs for three wood
types during decay in a forest soil,” Microbial Ecology, vol. 68,
no. 2, pp. 212–221, 2014.
[59] N. M. Reid, S. L. Addison, L. J. Macdonald, and G. Lloyd-Jones,
“Biodiversity of active and inactive bacteria in the gut flora
of wood-feeding Huhu beetle larvae (Prionoplus reticularis),”
Applied and Environmental Microbiology, vol. 77, no. 19, pp.
7000–7006, 2011.