Research ArticleIsolation of Fungi and Bacteria Associated withthe Guts of Tropical Wood-Feeding Coleoptera andDetermination of Their Lignocellulolytic Activities
Keilor Rojas-Jimeacutenez12 and Myriam Hernaacutendez1
1 Instituto Nacional de Biodiversidad Apartado Postal 22-3100 Santo Domingo Heredia Costa Rica2Universidad Latina de Costa Rica Campus San Pedro Apartado Postal 10138-1000 San Jose Costa Rica
Correspondence should be addressed to Keilor Rojas-Jimenez keilorrojasgmailcom
Received 23 May 2015 Accepted 12 August 2015
Academic Editor Karl Drlica
Copyright copy 2015 K Rojas-Jimenez and M Hernandez This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited
The guts of beetle larvae constitute a complex system where relationships among fungi bacteria and the insect host occur In thisstudy we collected larvae of five families of wood-feeding Coleoptera in tropical forests of Costa Rica isolated fungi and bacteriafrom their intestinal tracts and determined the presence of five different pathways for lignocellulolytic activity The fungal isolateswere assigned to three phyla 16 orders 24 families and 40 genera Trichoderma was the most abundant genus detected in allinsect families and at all sites The bacterial isolates were assigned to five phyla 13 orders 22 families and 35 genera BacillusSerratia and Pseudomonas were the dominant genera present in all the Coleopteran families Positive results for activities relatedto degradation of wood components were determined in 65 and 48 of the fungal and bacterial genera respectively Our resultsshowed that both the fungal and bacterial populations were highly diverse in terms of number of species and their phylogeneticcomposition although the structure of themicrobial communities variedwith insect host family and the surrounding environmentThe recurrent identification of some lignocellulolytic-positive inhabitants suggests that particular microbial groups play importantroles in providing nutritional needs for the Coleopteran host
1 Introduction
Plant cell walls are predominantly composed of lignin cellu-lose and hemicellulose Together these three polymers repre-sent one of themost abundant sources of renewable energy onEarth [1ndash3]These polymers also constitute the basic nutritionsource for a large number of terrestrial insects of whichthe order Coleoptera is perhaps the most representative [45] The adaptation of the coleopteran insects to nutrient-limited diets such as wood constituents is attributed to theestablishment of relationships with microorganisms Thesemicroorganisms highly prominent in the digestive tracts ofthe host perform essential functions including digestion oflignocellulosic biomass nutrient production and compounddetoxification [6ndash9]
Recently there has been an increasing interest in thegut microorganisms of wood-feeding Coleoptera since this
microbial-host interaction is highly relevant from severalperspectives For example in natural ecosystems beetlesand their associated microorganisms perform importantfunctions as prime contributors to the degradation of organicmatter [10 11]Moreover some species of beetles have becomesignificant forest pests that cause large-scale economic lossesTherefore a better understanding of their feeding capabilitiesis relevant for establishing novel management strategies [12ndash15] From the biotechnological point of view the coleopterangut inhabitants represent a novel source for bioprospectingof enzymes related to the conversion of plant biomass intobiofuels production of industrial value-added products andbioremediation of pollutants [16ndash18]
Most of the 300000 beetle species described to date occurin tropical rainforests [19 20] Costa Rican rainforests forexample are known to harbor approximately 10 of thespecies and 60 of the families of Coleoptera including
Hindawi Publishing CorporationInternational Journal of MicrobiologyVolume 2015 Article ID 285018 11 pageshttpdxdoiorg1011552015285018
2 International Journal of Microbiology
a number of wood-feeding beetles from the ScarabaeidaePassalidae Cerambycidae Elateridae and Tenebrionidaefamilies [21] The life cycles of these insects are highlyseasonal with most of the developmental stages occurringduring the rainy season The feeding sources of the adultsinclude dung carrion and various plant parts such as rootsstems foliage flowers pollen fruits and seeds Converselythe diets of the larvae are more restricted to roots decom-posing organic matter and decaying wood [11 20ndash22]
The substrates on which the insects feed are majordeterminants for the gut microbial diversity However itis also possible that certain microbes have adapted to theendointestinal lifestyle and have developed mutualistic rela-tionships necessary for the host survival Hence a fraction ofthe endosymbiont microbial community could be verticallytransmitted [6 7 23] Among the microbial groups fungaland bacterial endosymbionts form complex communitiesthat besides the basic digestive functions also performnonconventional roles that include synthesis of vitamins andpheromones nitrogen recycling and resistance to pathogenseach of which has important implications for host fitness [815 24 25] In return the insect provides a stable environmentfor the microbial growth with a steady intake of nutrientsFurthermore the host can evolve to where tripartite beetle-fungi-bacteria mutualism takes place [23 26] This phe-nomenon seems to be widespread although the mechanismsthat govern these interactions are still poorly understood[4 27]
Most of the previous studies on the microbial diversity ofthe coleopteran gut highlighted either bacterial diversity orto a lesser extent fungal diversity generally obtained from asmall number of economically important beetle species Inaddition some studies used metagenomic profiling for thediscovery of novel lignocellulolytic genesThe purpose of thepresent work was to isolate and describe the compositionof the cultivable fungi and bacteria associated with the gutsof wood-feeding larvae of five families of Coleoptera andto determine their lignocellulolytic activities For this wecollected larvae in tropical wet forests of several nationalparks in Costa Rica isolated both fungi and bacteria fromtheir guts performed bioinformatics analysis and assayedfor the presence of five enzymatic activities related to thedegradation of lignocellulosicmaterialsThis work representsan initial step toward understanding relationships existingamong the fungi the bacteria and the beetle host and thesurrounding environment in a country having a particularlylarge biodiversity of Coleoptera
2 Material and Methods
21 Insect Sampling This studywas conducted in tropical wetforests of 10 protected areas of Costa Rica with the respectivepermit resolutions R-CM-INBio-40-2008-OT and R-CM-INBio-48-2008-OT of the National Authority of theMinistryof Environment At each site approximately 3 km of naturaltrails was explored looking for decaying fallen trees within25m on each side of the path The sampling area representednearly 150000m2 (or 015 km2) per national park Every
fallen tree found was exhaustively examined for the presenceof galleries of wood-feeding beetle larvae particularly fromthe Scarabaeidae Passalidae Elateridae Cerambycidae andTenebrionidae families The selected national parks coveredmost of the natural distribution of the five coleopteranfamilies studied wet tropical forest ranging from zero to1300m altitude (Table 1)
The observed frequency of fallen trees and the presenceof insect galleries within them varied according to theconditions of each siteTherefore sample collection and studyresults were normalized to unit area Most of the insectgalleries contained insects of only one coleopteran familybut in few cases two or three different families were presentThe insect larvae in the late second or third larval instarswere collected placed in polyethylene boxes together withpieces of their feedingwood and kept at ambient temperatureuntil being transported to the laboratory (Table 2)The initialidentification of the larval families was performed on site bya trained collector and later confirmed by the coleopterantaxonomist of the National Institute of Biodiversity Once inthe laboratory the larval specimens were chilled at minus20∘C for10minutes surface sterilizedwith ethanol and then dissectedin a sterile laminar flow hood
22 Isolation of Bacteria and Fungi The entire gut wasremoved from each larva and placed on a sterile Petri dishcrushed and spread onto three differentmedia plates For iso-lation of fungi we used Potato-Dextrose Agar (PDA Difco)amendedwith chlortetracycline (120mgL) and streptomycin(120mgL) For the isolation of bacteria we used one-thirdstrength Luria-Bertani medium (3 gL peptone 5 gL yeast-extract 10 gLNaCl and 15 gliter agar pH 70) and the self-developed medium called LIGNO (15 gL KH
2PO4 175 gL
K2HPO4 08 gL KNO
3 05 gL MgSO
4 1 mLL CaCl
201M
4 gL sawdust 2 gL bagasse powder and 17 gL agar pH 70)Plates were incubated at 28∘C for up to three weeks andchecked every other day regularly for visible microorganismgrowths Each emerging fungus was transferred into a freshPDA plate amended with the antibiotics mentioned abovewhile each emerging bacterial colony was replated intoLuria-Bertani medium (Difco) An initial screening basedon the morphological traits of the fungi was performedto discard redundant isolates from the same sample (char-acteristics such as the color shape border type mycelialdensity presence-absence of secretions and growth rate werecompared) A second screening was based on molecular tax-onomyThe resulting nonredundant isolates were included ina database with associated information andwere preserved inthe National Biodiversity Institutersquos culture collection
23 Lignocellulolytic Assays We screened all the isolates forthe presence of five different pathways for lignocellulolyticactivity possibly associated with degradation of structuralwood components including cellulose lignin 120573-D-xylan 120573-D-cellobiose and 120573-D-glucansThese assays were performedby addition of specific substrates to the medium or directlyon the microbial culture and hydrolysis was determined by acolor 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 carriedout The selected environments are classified as tropical wet forest and cover most of the natural distribution of the five coleopteran familiesstudied
National park LatitudeLongitude Altitude (m) Mean temperature (∘C) Annual precipitation (mm)
Table 2 Distribution of the number of larvae samples according tothe insect family and national park At each site an approximate areaof 150000m2 was explored for the presence of wood-feeding larvaeThe number of insect groups found varied according to the naturalcondition of each forest Each sample was composed of one to threeindividuals of the same species
National park Cer Ela Pas Sca Ten TotalArenal 1 3 4Barbilla 6 6Braulio 2 2 1 5Carara 1 3 1 5Hitoy Cerere 7 7Piedras Blancas 1 2 1 4Rincon de la Vieja 1 2 3Tapanti 2 1 1 1 5Tenorio 1 1 1 3Tortuguero 1 2 2 1 6Total 8 5 16 16 3 48Cer Cerambycidae Ela Elateridae Pas Passalidae Sca Scarabaeidae andTen Tenebrionidae
two times for each enzymatic screening The degradationof cellulose was determined using carboxymethylcellulose(CMC Sigma) as the sole carbon source in the mediumfollowed by staining with Congo red Briefly the bacte-rial isolates were grown for 48 h and fungi for 72 h at28∘C on CMC medium (094 gL KH
2PO4 19 gL K
2HPO4
16 gL KCl 143 gL NaCl 015 gL NH4Cl 0037 gL MgSO
4-
7H2O 0017 gL CaCl
2 01 gL yeast-extract 75 gL CMC
and 15 gL agar pH 70) After this incubation period themicroorganism-containing agar plates were flooded with005 Congo red for 10min until the dye bound CMC Thereaction was fixed with NaCl (50mM) for 5min and thenrinsed with distilled water The zones where the microor-ganism hydrolyzed the CMC were visible as clear halos [28]The oxidative degradation of lignin was determined basedon the decolorization of the dye Remazol Brilliant Blue R(RBBR Sigma) when grown on solid media [29ndash31] Plateswith MEA-RBBR medium (20 gL malt extract 15 gL agarand 002wtvol RBBR pH 70) were inoculated with thebacterial and fungal isolates and incubated at 28∘C At dailyintervals for a period of 14 days the plates were checkedfor the presence of a decolorized area around the colony ormycelia Determination of the120573-glucosidase120573-xylanase andcellobiose hydrolase activities was performed using as sub-strates 10mM 4-nitrophenyl 120573-D-glucopyranoside (Sigma)4-nitrophenyl120573-D-xylopyranoside (Sigma) or 4-nitrophenyl120573-D-cellobioside (Sigma) dissolved in 50mM ammoniumacetate buffer pH 50 amended with 07 of agar and keptat 55∘C A drop of these solutions was placed directly on thebacterial colonies or fungal mycelia followed by incubationfor 30min at room temperature The catalytic action of themicrobial enzymes on the substrate was detected by thedevelopment of a yellow coloration produced by the releaseof the p-nitrophenol group [30 32]
24 Molecular Analyses All the isolates were grown in Petridishes containing the same media used for preservationFor the fungal DNA extraction 400mg of mycelia was
4 International Journal of Microbiology
ground with mortar and pestle in liquid nitrogen and fur-ther extracted using the DNeasy Plant kit (Qiagen USA)including a pretreatment step consisting of the incubationat 60∘C for one hour with 400 120583L of lysis buffer and30 120583L of Proteinase K (20mgmL Sigma Aldrich USA)The bacterial DNA was extracted following the instructionsof the NucleoSpin Tissue DNA Extraction kit (Macherey-Nagel Germany) The fungal ITS1-58S-ITS2 regions wereamplified by PCR from the total DNA using as forwardprimer the ITS1 51015840-TCCGTAGGTGAACCTGCGG-31015840 andthe reverse primer ITS4 51015840-TCCTCCGCTTATTGATATGC-31015840 [33] with the following reaction program 95∘C for 10min35 cycles at 94∘C for 1min 54∘C for 1min 72∘C for 1minand additional extension at 72∘C for 10min The 16S rRNAgene was amplified using primers 27f and 1492r [34] with thefollowing program 95∘C for 10min and 35 cycles of 94∘Cfor 1min 52∘C for 1min and 72∘C for 1min and 10minextension at 72∘C The PCR products were purified usingthe NucleoSpin Extract II kit (Macherey-Nagel Germany)according to manufacturerrsquos protocol Sanger sequencing ofthe samples was performed at the sequencing facility ofthe Dana Farber Cancer Institute at the Harvard UniversityBoston Massachusetts using the abovementioned forwardand reverse primers for fungi and primers 27f and 785r forbacteria Sequences were assembled using Seqman programofDNASTARLasergene 80 (GenBank accession GU827479-GU827553 HM770962-HM771112)
25 Taxonomy The taxonomic assignment of the bacterialsequences was performed by comparing the database againstthe 16S rRNA reference set 10 implemented in the Classifiertool of the Ribosomal Database Project which assigned the16S rRNA sequences to corresponding taxonomical hierarchybased on a naıve Bayesian rRNA classifier [35]The taxonomyof the fungi was inferred by comparing the ITS1-58S-ITS2 sequences against the Warcup Fungal ITS trainset 1a curated reference dataset implemented in the Classifiertool of the Ribosomal Database Project [35] Every fungaltaxonomical assignment was verified against the Index Fun-gorum (httpwwwindexfungorumorg) and appropriatelycorrected when synonyms or current names were identified
26 Ecological Analyses The analysis of the microbial com-munities was performed using the Vegan package imple-mented in the statistical programming environment andlanguage R [36] For this a table with the taxonomic clas-sifications to the levels of order class subphylum phylumand subkingdom of the fungal isolates was converted totaxonomic pairwise distances with the function taxa2distand using variable step lengths between successive categoriesproportional to the number of groups within each taxonom-ical level This distance matrix was then used to constructa hierarchical clustering tree with the function hclust andthe UPGMA distance method A second matrix with theabundance distribution of the fungal isolates per insect familywas prepared with the larval families in the rows the fungalorders in the columns and cells containing the counts ofisolates in each taxon This matrix was used to calculate
Bray-Curtis distances between the insect fungal communitieswith the function vegdist The advantage of this approach isthat Bray-Curtis measures avoid the double zero problemaccounting for absences that are not indicators of similaritiesbetween sample units [37] The generated distance matrixwas used to cluster similarities between the microbial com-positions of the larval families with the hclust function Thefunction tabasco was used to display compact integratedcommunity information plotting the fungal taxonomicalrelationships in the rows the similarities between the insectsrsquomicrobial compositions in the columns and a heatmap withthe respective abundance distribution [36] The function ccawas used to perform canonical correspondence analysis ofthe communities associated with the coleopteran families Asimilar procedure was performed to analyze the communitycomposition of bacteria and also for detecting differencesamong national parks
3 Results
31 Taxonomic Composition of the Fungi and Bacteria IsolatedIn this study we isolated 92 fungal strains and 135 bacterialstrains from larvae of five families of Coleoptera that werefeeding ondecayingwood in tropicalwet forests of 10 nationalparks of Costa Rica The 92 fungal isolates were assigned tothree phyla 16 orders 24 families and 40 genera (one differ-ent genus every 23 isolates)Within the phylumZygomycotawe isolated members of the order Mucorales and within thephylum Basidiomycota members of the orders AgaricalesPolyporales and Trichosporonales Most of the fungi isolatedfrom the gastrointestinal tracts of the larvae belonged to thephylum Ascomycota (89 of total) They were distributedin 12 orders with Hypocreales being the dominant one itcomprised nearly 55 of the isolates (Table 3) The genusTrichoderma was the most abundant it was the only oneassociatedwith all five families of Coleoptera and also presentin each national park sampled
Most fungal orders and genera were sparsely representedwith 68 of the orders and 55 of the genera found associ-ated with a specific coleopteran family at a particular site Allthe insect families also presented unique fungal isolates persite with Tenebrionidae being the only coleopteran family inwhich all isolates were phylogenetically distinct Regardingthe sampling sites Tenorio National Park showed the highestnumber of unique phylotypes but Piedras Blancas NationalPark contained a more phylogenetically diverse array ofisolates
The 135 bacterial isolates were classified within fivephyla 13 orders 22 families and 35 genera (one differentgenus from every 38 isolates) including members of Acti-nobacteria Proteobacteria Firmicutes Flavobacteria andFusobacteria Approximately 82 of the bacteria belongedto 120574-Proteobacteria and Firmicutes accounting for 44 and38 of the isolates respectivelyWithin the 120574-Proteobacteriathe genera Serratia and Pseudomonas were abundant beingpresent in all host families studied Within Firmicutes thegenus Bacillus was clearly the most dominant This singlegenus 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 shownfor each of the coleopteran families
Order Genus Cer Ela Pas Sca Ten TotalBotryosphaeriales Botryosphaeria 1 1Capnodiales Ramichloridium 1 1
Total 25 12 19 29 7 92Cer Cerambycidae Ela Elateridae Pas Passalidae Sca Scarabaeidae and Ten TenebrionidaelowastGenera that presented positive enzymatic activities in more than four pathways
a common gut inhabitant of all the insect families and waspresent at almost all the sites sampled (Table 4)
The remaining bacterial classes obtained in this studywere less represented For example members of Actinobac-teria accounted for 11 of the isolates whereas 120572- and 120573-Proteobacteria Flavobacteria and Fusobacteria represented
less than 6 of the isolates When calculating the percent-age of isolates that were specific to a single site and hostfamily results showed that 42 of the isolates exhibitedthis characteristic while the remaining 58 of the generapresented a broader host-site range A small number of thegenera 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 isshown for each of the coleopteran families
Flavobacteria Chryseobacterium 1 1Fusobacteria Sebaldella 1 1Total 18 20 41 46 10 135Cer Cerambycidae Ela Elateridae Pas Passalidae Sca Scarabaeidae and Ten TenebrionidaelowastGenera that presented positive enzymatic activities in more than four pathways
(ie Rhizobium in Hitoy Cerere National Park) others wereassociated with the same beetle family but in different sites(ie Leucobacter with Passalidae) Results of the analysisof unique phylotypes per site and insect family showedthat Hitoy Cerere National Park and Elateridae respectivelypresented the highest percentages of single bacterial isolates
32 Lignocellulolytic Activity Determination Nearly 65 ofthe fungal genera and 48 of the bacterial genera presentedpositive results in at least one of the five lignocellulolyticactivities evaluated with carboxymethylcellulose degrada-tion being the most common activity observed in bothgroups (Table 5) In general fungi showedmore capability for
degrading lignocellulosic materials than bacteria with gen-era such as Trichoderma Bionectria and Trametes showingpositive results in all the assays performed Within bacteriaBacillus Enterobacter and Acinetobacter some of the mostabundant genera isolated from the larval guts tested positivefor four out of the five enzymatic activities assayed cellulase120573-glucosidase 120573-xylanase and cellobiose hydrolase activi-ties However neither these genera nor any other bacterialgroup screened were able to degrade the Remazol BrilliantBluemolecules while 30of the fungal genera tested positivefor this lignin-related degradation activity
33 Comparison of Gut Inhabitants between Families ofColeoptera We performed community analysis with Vegan
International Journal of Microbiology 7
Table 5 Results of the screening for lignocellulolytic activitiesFungal genera with positive results are shown in the upper groupand bacterial genera in the lower group
to gain insight into how the microbial gut composition of thebeetle families related to one anotherThis approach clusteredthe environments according to Bray-Curtis distances of
Figure 1 Heatmap of the abundance distribution of fungal com-munities associated with the guts of five wood-feeding familiesof Coleoptera The taxonomic relationship of the fungal genera isshown in the rows while the clustering of the coleopteran familiesdetermined by their composition similarities is shown in thecolumns Higher intensities of the color reveal higher abundancesof the isolates
the abundance distribution of the isolates considering alsotheir phylogenetic relationships The results showed that thefungal composition of the isolates associated with larvaeof Cerambycidae Scarabaeidae and Passalidae clusteredtogether Cerambycidae and Passalidae shared one orderof Basidiomycota and three orders of Ascomycota whileScarabaeidae and Passalidae had in common four ordersof Ascomycota A second cluster was formed by the fungalmicrobiotas isolated fromTenebrionidae and Elateridae theyshared two orders of Ascomycota and one of Basidiomycota(Figure 1) The analysis of the bacterial dataset showed thatthe microbial compositions associated with Scarabaeidaeand Passalidae formed part of the same cluster sharingisolates belonging to 120573- and 120574-Proteobacteria Actinobac-teria and Firmicutes The second cluster was formed byTenebrionidae Elateridae andCerambycidae that shared iso-lates assigned to Pseudomonadales Enterobacteriales andBacillales (Figure 2) In addition we performed canonicalcorrespondence analysis for exploring relationships betweenthe microbial communities of the coleopteran hosts Resultsof this analysis where consistent with results obtained withthe Bray-Curtis clustering for both the fungal and bacterialcommunities (Figure S1 in Supplementary Material availableonline at httpdxdoiorg1011552015285018)
4 Discussion
We collected larvae of five families of wood-feedingColeoptera in tropical forests of Costa Rica with the aim ofestimating the species composition of cultivable fungi andbacteria inhabiting their guts and to identify microorganisms
Figure 2 Heatmap of the abundance distribution of bacterialcommunities associated with the guts of five wood-feeding familiesof Coleoptera The taxonomic relationship of the bacterial genera isshown in the rows while the clustering of the coleopteran familiesdetermined by their composition similarities is shown in thecolumns Higher intensities of the color reveal higher abundancesof the isolates
with relevant lignocellulolytic activities The main limitationof this study is that the cultivation-dependent approachbased on artificial media covers only a small proportion ofthe total microbial diversity present in this particular nicheThe positive trade-off of this approach was the identificationof several isolates with lignocellulose-degrading capabilitieswhich can be further used for the respective enzymecharacterization for direct degradation assays on residuesfrom agriculture and forestry for the treatment of industrialeffluents and for bioprospecting novel metabolites withother biotechnological applications Despite the inherentbias of the isolation method our results suggest that gutmicrobiota of wood-feeding tropical beetles presents arelatively high diversity in terms of microbial richnessphylogenetic composition and lignocellulolytic activities
The order Hypocreales represented about 60 of thetotal number of fungal isolates Within this group the genusTrichoderma was the most abundant comprising nearly aquarter of the fungal collection This genus was a commongut inhabitant of beetle larvae regardless of the host familyor the geographic location The reason for this dominanceis not entirely clear however one possible explanation isthat several species belonging to this fungal genus containa number of glycoside hydrolases peroxidases laccasesand phenol oxidases among other enzymes related to thedegradation of lignocellulose materials This feature mightprovide some advantages for using the recalcitrant polymericmaterials passing through the gastrointestinal tract [16 38ndash40]
In addition our data indicate that guts of wood-feedinglarvae were from environments having a high representation
of Hypocreales as also observed in a similar study performedin other locations of Costa Rica [41] This is relevant forbioprospecting purposes since wood-feeding beetles mightconstitute a good source of TrichodermaMetarhiziumMeta-cordyceps Bionectria and other fungal genera known topossess a wide array of biotechnological applications [42ndash45] The remaining orders presented a lower abundanceand in most of the cases were represented by a singlegenus Nevertheless many of the genera showed the abilityto degrade lignocellulose-related hexoses and pentoses asalso shown in other studies [46ndash50] Within the phylumBasidiomycota the genus Trametes showed positive resultsin all the lignocellulolytic assays related to the degradationof structural wood components This white-rot fungus is aknownmodel for studying degradation of lignin in free-livingconditions and in this work reported in its association withthe gut microbiota of wood-feeding insects [51 52]
It is difficult to know whether these fungal isolates aretruly endosymbionts of the intestinal tracts of the coleopteranlarvae or are transitory inhabitants associated with hostfeeding habits Hence it is also possible that some ofthese microorganisms could be commensals parasites andfacultative endosymbionts They might even be using theinsect as a dispersal mechanism [15 53] It is clear howeverthat the overall taxonomic composition of the gut-inhabitingmicrobes and the proportion of lignocellulolytic-positivefungi seem to be particular to the larval microenvironmentThe structure of this endosymbiotic community is distin-guished from the fungal composition observed in otherwood-related microhabitats such as the fungal populationsin living plant tissues They are also dominated by membersof Ascomycetes but they present a different abundancedistribution of fungal families [54 55] decaying logs aredominated mainly by Basidiomycetes [56ndash58]
The analysis of the taxonomic composition of the bacte-rial isolates showed the presence of seven major phylogeneticclasses codominated by 120574-Proteobacteria and FirmicutesThis finding is consistent with results obtained in similarstudies [6 13 14 44 59] Within the 120574-Proteobacteria themost abundant genera were Enterobacter Serratia Acineto-bacter and Pseudomonas Interestingly Serratia and Pseu-domonas were isolated from all five coleopteran familiesstudied Enterobacter and Acinetobacter were present in fourout of the five insect families and they exhibited positiveresults in the lignocellulolytic assays except for lignin degra-dation Similar characteristics related to the degradation oflignocellulose and to fermentativemetabolismwere observedin Bacillus the most abundant genus within Firmicutes [1160] Together these results support the notion that somespecies of fungi and bacteria such as Trichoderma SerratiaPseudomonas and Bacillus can be common gut inhabitantsof wood-feeding larvae in tropical forests suggesting thatcertain affinities might have developed between the beetlehost and its microbiota [41 61ndash64]
When comparing the fungal and bacterial species compo-sition among the beetle families the plots of the Bray-Curtisdistances and canonical correspondence analyses producedbiologically meaningful clusters to group the environmentsthat share similar microbial compositions The first fungal
International Journal of Microbiology 9
cluster relates the microbiota associated with the guts ofCerambycidae Passalidae and Scarabaeidae This is con-sistent with the observation of a high diversity of isolatesfrom Cerambycidae that shared members of the fungalphyla Basidiomycota and Ascomycota with Passalidae andmembers of Zygomycota and Ascomycota with ScarabaeidaeThe cluster formed by Tenebrionidae-Elateridae shared ina lower proportion members of the Basidiomycota andAscomycotaThe bacterial microbiota associated with Passal-idae and Scarabaeidae also formed a cluster sharingmembersof five major bacterial clades microbiota of CerambycidaeElateridae and Tenebrionidae shared members only of 120574-Proteobacteria and Firmicutes
The clustering analyses revealed that Cerambycidae pre-sented a high diversity of fungi but not of bacteria whilePassalidae and Elateridae exhibited a high diversity of bac-teria and moderate diversity of fungi Scarabaeidae andTenebrionidae contained a similar composition of bothThese results suggest that the nature of the beetle hosthas an important effect on the phylogenetic diversity of itsassociated microbiota and that many factors can influenceits configuration These factors may include the biology ofthe host the physical and chemical characteristics of thegut compartments the feeding habits of the insects and themicrobial diversity associated with the environment in whichthe insect is living [23 26 65 66]
Our results consistently showed that both the fungaland bacterial populations associated with the guts of beetlelarvae are highly diverse in terms of the number of speciesobtained and in their phylogenetic composition Thesemicrobial inhabitants could be forming complex consortiathat would be acting synergistically to provide many of thenutritional needs of the beetle host Some of these functionsinclude the degradation and fermentation of lignocellulosicmaterials as shown by the high percentage of fungal andbacterial genera that presented positive activities or by theproduction of proteins and other metabolites necessary forthe development of the insect [25 44 67ndash69] Furthermorecertain affinities for substrates can be expected according tothe nature of the gut inhabitant For example members ofthe Basidiomycota could possibly degrade larger polymericmolecules the Ascomycota deplete diverse lignocellulosicconstituents while the bacteria degrade and ferment thesmaller monomeric and dimeric hexoses and pentoses pro-duced by the fungal counterparts The bacteria also likelyuse these sugars to produce other nutrients and metabo-lites Consequently the present work raises new lines ofinvestigation concerning the existence of microbial consortiaacting synergistically to provide the nutritional needs of thehosts the nature of the ecological and evolutionary processesthat contribute to ensure the fitness of the insect and themechanisms that rule the interactions among the fungi thebacteria and the beetle host
Conflict of Interests
The authors declared that there is no conflict of interestsregarding this paper
Acknowledgments
The authors acknowledge the contribution of the follow-ing people during the development of this project LuisGuillermoAcosta for the field sampling and early insect iden-tification Angel Solis Carlos Hernandez and Elena Ulate forthe identification of some adult specimens Jorge Blanco forthe fungal isolation Angelica Acuna and Beatriz Rivera fortheDNA extraction and enzymatic assaysManuel Ferrer andCesar Mateo for their advices on the lignocellulolytic activitydetermination Ana Lorena Guevara and Giselle Tamayofor the overall support the editor and reviewers of thisjournal for critical comments on the paperThis research wasfunded by the support of the National Council of Science andTechnology (CONICIT FV-027-2007) the CSIC and CRUSAFoundation (2007 CR0034) and Florida Ice amp FarmCoTheythank ACLAC ACOPAC ACOSA ACTo ACLAP ACCVCACAT ACAHN ACG and ACLAC National ConservationAreas and CONAGEBIO for granting the sample collectingpermits (R-CM-INBio-40-2008-OT R-CM-INBio-48-2008-OT)
References
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[2] P Beguin ldquoMolecular biology of cellulose degradationrdquo AnnualReview of Microbiology vol 44 pp 219ndash248 1990
[3] J Perez J Munoz-Dorado T de la Rubia and J MartınezldquoBiodegradation and biological treatments of cellulose hemi-cellulose and lignin an overviewrdquo International Microbiologyvol 5 no 2 pp 53ndash63 2002
[4] M R Berenbaum and T Eisner ldquoEcology Bugsrsquo bugsrdquo Sciencevol 322 no 5898 pp 52ndash53 2008
[5] Z Zhang ldquoPhylum Arthropoda von Siebold 1948 In animalbiodiversity an outline of higher-level classification and surveyof taxonomic richnessrdquo Zootaxa vol 3148 pp 99ndash103 1948
[6] J Morales-Jimenez G Zuniga L Villa-Tanaca and CHernandez-Rodrıguez ldquoBacterial community and nitrogenfixation in the red turpentine beetle Dendroctonus valensLeConte (Coleoptera Curculionidae Scolytinae)rdquo MicrobialEcology vol 58 no 4 pp 879ndash891 2009
[7] S M Geib M del Mar Jimenez-Gasco J E Carlson M TienR Jabbour and K Hoover ldquoMicrobial community profilingto investigate transmission of bacteria between life stages ofthe wood-boring beetle Anoplophora glabripennisrdquo MicrobialEcology vol 58 no 1 pp 199ndash211 2009
[8] P Engel and N A Moran ldquoThe gut microbiota of insectsmdashdiversity in structure and functionrdquo FEMS MicrobiologyReviews vol 37 no 5 pp 699ndash735 2013
[9] W Shi S Xie X Chen et al ldquoComparative genomic anal-ysis of the microbiome of herbivorous insects reveals eco-environmental adaptations biotechnology applicationsrdquo PLoSGenetics vol 9 no 1 Article ID e1003131 2013
[10] I Hanski and Y Cambefort Dung Beetle Ecology PrincetonUniversity Press Princeton NJ USA 1991
[11] M Egert B Wagner T Lemke A Brune and M W FriedrichldquoMicrobial community structure in midgut and hindgut ofthe humus-feeding larva of Pachnoda ephippiata (Coleoptera
10 International Journal of Microbiology
Scarabaeidae)rdquo Applied and Environmental Microbiology vol69 no 11 pp 6659ndash6668 2003
[12] R N Coulson ldquoPopulation dynamics of bark beetlesrdquo AnnualReview of Entomology vol 24 no 1 pp 417ndash447 1979
[13] P D Schloss I Delalibera Jr J Handelsman and K F RaffaldquoBacteria associated with the guts of two wood-boring beetlesanoplophora glabripennis and Saperda vestita (Cerambycidae)rdquoEnvironmental Entomology vol 35 no 3 pp 625ndash629 2006
[14] A Vasanthakumar J O Handelsman P D Schloss L S Bauerand K F Raffa ldquoGut microbiota of an invasive subcorticalbeetle Agrilus planipennis Fairmaire across various life stagesrdquoEnvironmental Entomology vol 37 no 5 pp 1344ndash1353 2008
[15] J Morales-Jimenez G Zuniga H C Ramırez-Saad and CHernandez-Rodrıguez ldquoGut-associated bacteria throughoutthe life cycle of the bark beetle Dendroctonus rhizophagusThomas and Bright (Curculionidae Scolytinae) and their cellu-lolytic activitiesrdquoMicrobial Ecology vol 64 no 1 pp 268ndash2782012
[16] R Kumar S Singh and O V Singh ldquoBioconversion of lig-nocellulosic biomass biochemical and molecular perspectivesrdquoJournal of IndustrialMicrobiology and Biotechnology vol 35 no5 pp 377ndash391 2008
[17] C Sanchez ldquoLignocellulosic residues biodegradation and bio-conversion by fungirdquo Biotechnology Advances vol 27 no 2 pp185ndash194 2009
[18] E D Scully S M Geib K Hoover et al ldquoMetagenomicprofiling reveals lignocellulose degrading system in a microbialcommunity associated with a wood-feeding beetlerdquo PLoS ONEvol 8 no 9 Article ID e73827 2013
[19] T L Erwin ldquoTropical forest their richness in Coleoptera andother arthropod speciesrdquoThe Coleopterists Bulletin vol 36 no1 pp 74ndash75 1982
[20] Y Basset L Cizek P Cuenoud et al ldquoArthropod diversity in atropical forestrdquo Science vol 338 no 6113 pp 1481ndash1484 2012
[21] A Solis Escarabajos de Costa Rica Las Familias Mas ComunesHeredia Costa Rica Editorial Inbio 1999
[22] D Borror and R White A Field Guide to the Insects HoughtonMifflin Company New York NY USA 1987
[23] M Egert U Stingl L D Bruun B Pommerenke A Bruneand M W Friedrich ldquoStructure and topology of microbialcommunities in the major gut compartments of Melolonthamelolontha larvae (Coleoptera Scarabaeidae)rdquo Applied andEnvironmentalMicrobiology vol 71 no 8 pp 4556ndash4566 2005
[24] S-O Suh J V McHugh D D Pollock and M Blackwell ldquoThebeetle gut a hyperdiverse source of novel yeastsrdquo MycologicalResearch vol 109 no 3 pp 261ndash265 2005
[25] J J Scott D-C Oh M C Yuceer K D Klepzig J Clardy andC R Currie ldquoBacterial protection of beetle-fungusmutualismrdquoScience vol 322 no 5898 p 63 2008
[26] J A Ceja-Navarro N H Nguyen U Karaoz et al ldquoCom-partmentalized microbial composition oxygen gradients andnitrogen fixation in the gut of Odontotaenius disjunctusrdquo TheISME Journal vol 8 no 1 pp 6ndash18 2014
[27] J B Nardi C M Bee L A Miller N H Nguyen S-O Suhand M Blackwell ldquoCommunities of microbes that inhabit thechanging hindgut landscape of a subsocial beetlerdquo ArthropodStructure and Development vol 35 no 1 pp 57ndash68 2006
[28] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982
[29] B RMVyas andH PMolitoris ldquoInvolvement of an extracellu-lar H2O2-dependent ligninolytic activity of the white rot fungus
Pleurotus ostreatus in the decolorization of Remazol brilliantblue Rrdquo Applied and Environmental Microbiology vol 61 no 11pp 3919ndash3927 1995
[30] J-D Bok D A Yernool and D E Eveleigh ldquoPurificationcharacterization and molecular analysis of thermostable cellu-lases CelA andCelB fromThermotoga neapolitanardquoApplied andEnvironmentalMicrobiology vol 64 no 12 pp 4774ndash4781 1998
[31] K M G Machado D R Matheus and V L R BononildquoLigninolytic enzymes production and Remazol Brilliant BlueR decolorization by tropical Brazilian basidiomycetes fungirdquoBrazilian Journal of Microbiology vol 36 no 3 pp 246ndash2522005
[32] G T Howard and B A White ldquoMolecular cloning and expres-sion of cellulase genes fromRuminococcus albus 8 in Escherichiacoli bacteriophage 120582rdquo Applied and Environmental Microbiologyvol 54 no 7 pp 1752ndash1755 1988
[33] N L Glass and G C Donaldson ldquoDevelopment of primersets designed for use with the PCR to amplify conserved genesfrom filamentous ascomycetesrdquo Applied and EnvironmentalMicrobiology vol 61 no 4 pp 1323ndash1330 1995
[34] D Lane ldquo16S23S rRNA sequencingrdquo inNucleic Acid Techniquesin Bacterial Systematics E Stachebrandt and M GoodfellowEds Wiley Chichester UK 1991
[35] Q Wang G M Garrity J M Tiedje and J R Cole ldquoNaıveBayesian classifier for rapid assignment of rRNA sequencesinto the new bacterial taxonomyrdquo Applied and EnvironmentalMicrobiology vol 73 no 16 pp 5261ndash5267 2007
[36] J Oksanen G Blanchet R Kindt et al Vegan CommunityEcology Package 2014
[37] P L Buttigieg and A Ramette ldquoA guide to statistical analysisin microbial ecology a community-focused living review ofmultivariate data analysesrdquo FEMSMicrobiology Ecology vol 90no 3 pp 543ndash550 2014
[38] DMartinez RM Berka B Henrissat et al ldquoGenome sequenc-ing and analysis of the biomass-degrading fungus Trichodermareesei (syn Hypocrea jecorina)rdquo Nature Biotechnology vol 26no 5 pp 553ndash560 2008
[39] E D Scully K Hoover J E Carlson M Tien and S M GeibldquoMidgut transcriptome profiling of Anoplophora glabripennisa lignocellulose degrading cerambycid beetlerdquo BMC Genomicsvol 14 article 850 2013
[40] P K Foreman D Brown L Dankmeyer et al ldquoTranscriptionalregulation of biomass-degrading enzymes in the filamentousfungus Trichoderma reeseirdquoThe Journal of Biological Chemistryvol 278 no 34 pp 31988ndash31997 2003
[41] G Vargas-Asensio A Pinto-Tomas B Rivera et al ldquoUncover-ing the cultivable microbial diversity of costa rican beetles andits ability to break down plant cell wall componentsrdquoPLoSONEvol 9 no 11 Article ID e113303 2014
[42] M P Coughlan ldquoThe properties of fungal and bacterial cel-lulases with comment on their production and applicationrdquoBiotechnology and Genetic Engineering Reviews vol 3 no 1 pp39ndash110 1985
[43] L R Lynd P J Weimer W H Van Zyl and I S PretoriusldquoMicrobial cellulose utilization fundamentals and biotechnol-ogyrdquoMicrobiology andMolecular Biology Reviews vol 66 no 3pp 506ndash577 2002
[44] S M Geib T R Filley P G Hatcher et al ldquoLignin degradationin wood-feeding insectsrdquo Proceedings of the National Academy
International Journal of Microbiology 11
of Sciences of the United States of America vol 105 no 35 pp12932ndash12937 2008
[45] H Alper and G Stephanopoulos ldquoEngineering for biofuelsexploiting innate microbial capacity or importing biosyntheticpotentialrdquo Nature Reviews Microbiology vol 7 no 10 pp 715ndash723 2009
[46] K Yaoi and Y Mitsuishi ldquoPurification characterizationcloning and expression of a novel xyloglucan-specific glycosi-dase oligoxyloglucan reducing end-specific cellobiohydrolaserdquoThe Journal of Biological Chemistry vol 277 no 50 pp 48276ndash48281 2002
[47] L Ayed N Assas S Sayadi and M Hamdi ldquoInvolvementof lignin peroxidase in the decolourization of black olivemill wastewaters by Geotrichum candidumrdquo Letters in AppliedMicrobiology vol 40 no 1 pp 7ndash11 2005
[48] Y Baba A Shimonaka J Koga H Kubota and T KonoldquoAlternative splicing produces two endoglucanases with oneor two carbohydrate-binding modules in Mucor circinelloidesrdquoJournal of Bacteriology vol 187 no 9 pp 3045ndash3051 2005
[49] M Dashtban H Schraft andW Qin ldquoFungal bioconversion oflignocellulosic residues opportunities amp perspectivesrdquo Interna-tional Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009
[50] D A Ribeiro J Cota T M Alvarez et al ldquoThe Penicilliumechinulatum secretome on sugar cane bagasserdquo PLoS ONE vol7 no 12 Article ID e50571 2012
[51] O Borokhov and S Rothenburger ldquoRapid dye decolorizationmethod for screening potential wood preservativesrdquo Appliedand Environmental Microbiology vol 66 no 12 pp 5457ndash54592000
[52] E Abadulla T Tzanov S Costa K-H Robra A Cavaco-Paulo and G M Gubitz ldquoDecolorization and detoxification oftextile dyes with a laccase from Trametes hirsutardquo Applied andEnvironmental Microbiology vol 66 no 8 pp 3357ndash3362 2000
[53] T J Dreaden J M Davis Z W de Beer et al ldquoPhylogenyof ambrosia beetle symbionts in the genus Raffaeleardquo FungalBiology vol 118 no 12 pp 970ndash978 2014
[54] T S Suryanarayanan T S Murali and G Venkatesan ldquoOccur-rence and distribution of fungal endophytes in tropical forestsacross a rainfall gradientrdquo Canadian Journal of Botany vol 80no 8 pp 818ndash826 2002
[55] A E Arnold and F Lutzoni ldquoDiversity and host range of foliarfungal endophytes are tropical leaves biodiversity hotspotsrdquoEcology vol 88 no 3 pp 541ndash549 2007
[56] J Heilmann-Clausen and L Boddy ldquoInhibition and stimulationeffects in communities of wood decay fungi exudates fromcolonized wood influence growth by other speciesrdquo MicrobialEcology vol 49 no 3 pp 399ndash406 2005
[57] A Kubartova E Ottosson A Dahlberg and J Stenlid ldquoPat-terns of fungal communities among and within decaying logsrevealed by 454 sequencingrdquo Molecular Ecology vol 21 no 18pp 4514ndash4532 2012
[58] L Prewitt Y Kang M L Kakumanu andMWilliams ldquoFungaland bacterial community succession differs for three woodtypes during decay in a forest soilrdquo Microbial Ecology vol 68no 2 pp 212ndash221 2014
[59] N M Reid S L Addison L J Macdonald and G Lloyd-JonesldquoBiodiversity of active and inactive bacteria in the gut floraof wood-feeding Huhu beetle larvae (Prionoplus reticularis)rdquoApplied and Environmental Microbiology vol 77 no 19 pp7000ndash7006 2011
[60] CC Lee R E Kibblewhite-AccinelliM R Smith KWagschalW J Orts and DW SWong ldquoCloning of Bacillus licheniformisxylanase gene and characterization of recombinant enzymerdquoCurrent Microbiology vol 57 no 4 pp 301ndash305 2008
[61] T L Rhoads A T Mikell Jr and M H Eley ldquoInvestigation ofthe lignin-degrading activity of Serratia marcescens biochem-ical screening and ultrastructural evidencerdquo Canadian Journalof Microbiology vol 41 no 7 pp 592ndash600 1995
[62] T Nagy K Emami C M G A Fontes L M A FerreiraD R Humphry and H J Gilbert ldquoThe membrane-bound120572-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-O-methyl-D-glucuronoxylooligosaccharides but not 4-O-methyl-D-glucuronoxylanrdquo Journal of Bacteriology vol 184 no 17 pp4925ndash4929 2002
[63] J Weslien L B Djupstrom M Schroeder and O WidenfalkldquoLong-term priority effects among insects and fungi colonizingdecaying woodrdquo The Journal of Animal Ecology vol 80 no 6pp 1155ndash1162 2011
[64] R T Jones L G Sanchez and N Fierer ldquoA cross-taxon analysisof insect-associated bacterial diversityrdquo PLoS ONE vol 8 no 4Article ID e61218 2013
[65] T Lemke U Stingl M Egert M W Friedrich and A BruneldquoPhysicochemical conditions and microbial activities in thehighly alkaline gut of the humus-feeding larva of Pachnodaephippiata (Coleoptera Scarabaeidae)rdquo Applied and Environ-mental Microbiology vol 69 no 11 pp 6650ndash6658 2003
[66] S M Geib M D M Jimenez-Gasco J E Carlson M Tienand K Hoover ldquoEffect of host tree species on cellulase activityand bacterial community composition in the gut of larval Asianlonghorned beetlerdquo Environmental Entomology vol 38 no 3pp 686ndash699 2009
[67] A E Cazemier J C Verdoes F A G Reubsaet J H PHackstein C van der Drift and H J M Op den CampldquoPromicromonospora pachnodae sp nov a member of the(hemi)cellulolytic hindgut flora of larvae of the scarab beetlePachnoda marginatardquo Antonie van Leeuwenhoek vol 83 no 2pp 135ndash148 2003
[68] F A Genta R J Dillon W R Terra and C Ferreira ldquoPotentialrole for gut microbiota in cell wall digestion and glucosidedetoxification in Tenebrio molitor larvaerdquo Journal of InsectPhysiology vol 52 no 6 pp 593ndash601 2006
[69] D-C Oh J J Scott C R Currie and J Clardy ldquoMycangimycina polyene peroxide from a mutualist Streptomyces sprdquo OrganicLetters vol 11 no 3 pp 633ndash636 2009
a number of wood-feeding beetles from the ScarabaeidaePassalidae Cerambycidae Elateridae and Tenebrionidaefamilies [21] The life cycles of these insects are highlyseasonal with most of the developmental stages occurringduring the rainy season The feeding sources of the adultsinclude dung carrion and various plant parts such as rootsstems foliage flowers pollen fruits and seeds Converselythe diets of the larvae are more restricted to roots decom-posing organic matter and decaying wood [11 20ndash22]
The substrates on which the insects feed are majordeterminants for the gut microbial diversity However itis also possible that certain microbes have adapted to theendointestinal lifestyle and have developed mutualistic rela-tionships necessary for the host survival Hence a fraction ofthe endosymbiont microbial community could be verticallytransmitted [6 7 23] Among the microbial groups fungaland bacterial endosymbionts form complex communitiesthat besides the basic digestive functions also performnonconventional roles that include synthesis of vitamins andpheromones nitrogen recycling and resistance to pathogenseach of which has important implications for host fitness [815 24 25] In return the insect provides a stable environmentfor the microbial growth with a steady intake of nutrientsFurthermore the host can evolve to where tripartite beetle-fungi-bacteria mutualism takes place [23 26] This phe-nomenon seems to be widespread although the mechanismsthat govern these interactions are still poorly understood[4 27]
Most of the previous studies on the microbial diversity ofthe coleopteran gut highlighted either bacterial diversity orto a lesser extent fungal diversity generally obtained from asmall number of economically important beetle species Inaddition some studies used metagenomic profiling for thediscovery of novel lignocellulolytic genesThe purpose of thepresent work was to isolate and describe the compositionof the cultivable fungi and bacteria associated with the gutsof wood-feeding larvae of five families of Coleoptera andto determine their lignocellulolytic activities For this wecollected larvae in tropical wet forests of several nationalparks in Costa Rica isolated both fungi and bacteria fromtheir guts performed bioinformatics analysis and assayedfor the presence of five enzymatic activities related to thedegradation of lignocellulosicmaterialsThis work representsan initial step toward understanding relationships existingamong the fungi the bacteria and the beetle host and thesurrounding environment in a country having a particularlylarge biodiversity of Coleoptera
2 Material and Methods
21 Insect Sampling This studywas conducted in tropical wetforests of 10 protected areas of Costa Rica with the respectivepermit resolutions R-CM-INBio-40-2008-OT and R-CM-INBio-48-2008-OT of the National Authority of theMinistryof Environment At each site approximately 3 km of naturaltrails was explored looking for decaying fallen trees within25m on each side of the path The sampling area representednearly 150000m2 (or 015 km2) per national park Every
fallen tree found was exhaustively examined for the presenceof galleries of wood-feeding beetle larvae particularly fromthe Scarabaeidae Passalidae Elateridae Cerambycidae andTenebrionidae families The selected national parks coveredmost of the natural distribution of the five coleopteranfamilies studied wet tropical forest ranging from zero to1300m altitude (Table 1)
The observed frequency of fallen trees and the presenceof insect galleries within them varied according to theconditions of each siteTherefore sample collection and studyresults were normalized to unit area Most of the insectgalleries contained insects of only one coleopteran familybut in few cases two or three different families were presentThe insect larvae in the late second or third larval instarswere collected placed in polyethylene boxes together withpieces of their feedingwood and kept at ambient temperatureuntil being transported to the laboratory (Table 2)The initialidentification of the larval families was performed on site bya trained collector and later confirmed by the coleopterantaxonomist of the National Institute of Biodiversity Once inthe laboratory the larval specimens were chilled at minus20∘C for10minutes surface sterilizedwith ethanol and then dissectedin a sterile laminar flow hood
22 Isolation of Bacteria and Fungi The entire gut wasremoved from each larva and placed on a sterile Petri dishcrushed and spread onto three differentmedia plates For iso-lation of fungi we used Potato-Dextrose Agar (PDA Difco)amendedwith chlortetracycline (120mgL) and streptomycin(120mgL) For the isolation of bacteria we used one-thirdstrength Luria-Bertani medium (3 gL peptone 5 gL yeast-extract 10 gLNaCl and 15 gliter agar pH 70) and the self-developed medium called LIGNO (15 gL KH
2PO4 175 gL
K2HPO4 08 gL KNO
3 05 gL MgSO
4 1 mLL CaCl
201M
4 gL sawdust 2 gL bagasse powder and 17 gL agar pH 70)Plates were incubated at 28∘C for up to three weeks andchecked every other day regularly for visible microorganismgrowths Each emerging fungus was transferred into a freshPDA plate amended with the antibiotics mentioned abovewhile each emerging bacterial colony was replated intoLuria-Bertani medium (Difco) An initial screening basedon the morphological traits of the fungi was performedto discard redundant isolates from the same sample (char-acteristics such as the color shape border type mycelialdensity presence-absence of secretions and growth rate werecompared) A second screening was based on molecular tax-onomyThe resulting nonredundant isolates were included ina database with associated information andwere preserved inthe National Biodiversity Institutersquos culture collection
23 Lignocellulolytic Assays We screened all the isolates forthe presence of five different pathways for lignocellulolyticactivity possibly associated with degradation of structuralwood components including cellulose lignin 120573-D-xylan 120573-D-cellobiose and 120573-D-glucansThese assays were performedby addition of specific substrates to the medium or directlyon the microbial culture and hydrolysis was determined by acolor 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 carriedout The selected environments are classified as tropical wet forest and cover most of the natural distribution of the five coleopteran familiesstudied
National park LatitudeLongitude Altitude (m) Mean temperature (∘C) Annual precipitation (mm)
Table 2 Distribution of the number of larvae samples according tothe insect family and national park At each site an approximate areaof 150000m2 was explored for the presence of wood-feeding larvaeThe number of insect groups found varied according to the naturalcondition of each forest Each sample was composed of one to threeindividuals of the same species
National park Cer Ela Pas Sca Ten TotalArenal 1 3 4Barbilla 6 6Braulio 2 2 1 5Carara 1 3 1 5Hitoy Cerere 7 7Piedras Blancas 1 2 1 4Rincon de la Vieja 1 2 3Tapanti 2 1 1 1 5Tenorio 1 1 1 3Tortuguero 1 2 2 1 6Total 8 5 16 16 3 48Cer Cerambycidae Ela Elateridae Pas Passalidae Sca Scarabaeidae andTen Tenebrionidae
two times for each enzymatic screening The degradationof cellulose was determined using carboxymethylcellulose(CMC Sigma) as the sole carbon source in the mediumfollowed by staining with Congo red Briefly the bacte-rial isolates were grown for 48 h and fungi for 72 h at28∘C on CMC medium (094 gL KH
2PO4 19 gL K
2HPO4
16 gL KCl 143 gL NaCl 015 gL NH4Cl 0037 gL MgSO
4-
7H2O 0017 gL CaCl
2 01 gL yeast-extract 75 gL CMC
and 15 gL agar pH 70) After this incubation period themicroorganism-containing agar plates were flooded with005 Congo red for 10min until the dye bound CMC Thereaction was fixed with NaCl (50mM) for 5min and thenrinsed with distilled water The zones where the microor-ganism hydrolyzed the CMC were visible as clear halos [28]The oxidative degradation of lignin was determined basedon the decolorization of the dye Remazol Brilliant Blue R(RBBR Sigma) when grown on solid media [29ndash31] Plateswith MEA-RBBR medium (20 gL malt extract 15 gL agarand 002wtvol RBBR pH 70) were inoculated with thebacterial and fungal isolates and incubated at 28∘C At dailyintervals for a period of 14 days the plates were checkedfor the presence of a decolorized area around the colony ormycelia Determination of the120573-glucosidase120573-xylanase andcellobiose hydrolase activities was performed using as sub-strates 10mM 4-nitrophenyl 120573-D-glucopyranoside (Sigma)4-nitrophenyl120573-D-xylopyranoside (Sigma) or 4-nitrophenyl120573-D-cellobioside (Sigma) dissolved in 50mM ammoniumacetate buffer pH 50 amended with 07 of agar and keptat 55∘C A drop of these solutions was placed directly on thebacterial colonies or fungal mycelia followed by incubationfor 30min at room temperature The catalytic action of themicrobial enzymes on the substrate was detected by thedevelopment of a yellow coloration produced by the releaseof the p-nitrophenol group [30 32]
24 Molecular Analyses All the isolates were grown in Petridishes containing the same media used for preservationFor the fungal DNA extraction 400mg of mycelia was
4 International Journal of Microbiology
ground with mortar and pestle in liquid nitrogen and fur-ther extracted using the DNeasy Plant kit (Qiagen USA)including a pretreatment step consisting of the incubationat 60∘C for one hour with 400 120583L of lysis buffer and30 120583L of Proteinase K (20mgmL Sigma Aldrich USA)The bacterial DNA was extracted following the instructionsof the NucleoSpin Tissue DNA Extraction kit (Macherey-Nagel Germany) The fungal ITS1-58S-ITS2 regions wereamplified by PCR from the total DNA using as forwardprimer the ITS1 51015840-TCCGTAGGTGAACCTGCGG-31015840 andthe reverse primer ITS4 51015840-TCCTCCGCTTATTGATATGC-31015840 [33] with the following reaction program 95∘C for 10min35 cycles at 94∘C for 1min 54∘C for 1min 72∘C for 1minand additional extension at 72∘C for 10min The 16S rRNAgene was amplified using primers 27f and 1492r [34] with thefollowing program 95∘C for 10min and 35 cycles of 94∘Cfor 1min 52∘C for 1min and 72∘C for 1min and 10minextension at 72∘C The PCR products were purified usingthe NucleoSpin Extract II kit (Macherey-Nagel Germany)according to manufacturerrsquos protocol Sanger sequencing ofthe samples was performed at the sequencing facility ofthe Dana Farber Cancer Institute at the Harvard UniversityBoston Massachusetts using the abovementioned forwardand reverse primers for fungi and primers 27f and 785r forbacteria Sequences were assembled using Seqman programofDNASTARLasergene 80 (GenBank accession GU827479-GU827553 HM770962-HM771112)
25 Taxonomy The taxonomic assignment of the bacterialsequences was performed by comparing the database againstthe 16S rRNA reference set 10 implemented in the Classifiertool of the Ribosomal Database Project which assigned the16S rRNA sequences to corresponding taxonomical hierarchybased on a naıve Bayesian rRNA classifier [35]The taxonomyof the fungi was inferred by comparing the ITS1-58S-ITS2 sequences against the Warcup Fungal ITS trainset 1a curated reference dataset implemented in the Classifiertool of the Ribosomal Database Project [35] Every fungaltaxonomical assignment was verified against the Index Fun-gorum (httpwwwindexfungorumorg) and appropriatelycorrected when synonyms or current names were identified
26 Ecological Analyses The analysis of the microbial com-munities was performed using the Vegan package imple-mented in the statistical programming environment andlanguage R [36] For this a table with the taxonomic clas-sifications to the levels of order class subphylum phylumand subkingdom of the fungal isolates was converted totaxonomic pairwise distances with the function taxa2distand using variable step lengths between successive categoriesproportional to the number of groups within each taxonom-ical level This distance matrix was then used to constructa hierarchical clustering tree with the function hclust andthe UPGMA distance method A second matrix with theabundance distribution of the fungal isolates per insect familywas prepared with the larval families in the rows the fungalorders in the columns and cells containing the counts ofisolates in each taxon This matrix was used to calculate
Bray-Curtis distances between the insect fungal communitieswith the function vegdist The advantage of this approach isthat Bray-Curtis measures avoid the double zero problemaccounting for absences that are not indicators of similaritiesbetween sample units [37] The generated distance matrixwas used to cluster similarities between the microbial com-positions of the larval families with the hclust function Thefunction tabasco was used to display compact integratedcommunity information plotting the fungal taxonomicalrelationships in the rows the similarities between the insectsrsquomicrobial compositions in the columns and a heatmap withthe respective abundance distribution [36] The function ccawas used to perform canonical correspondence analysis ofthe communities associated with the coleopteran families Asimilar procedure was performed to analyze the communitycomposition of bacteria and also for detecting differencesamong national parks
3 Results
31 Taxonomic Composition of the Fungi and Bacteria IsolatedIn this study we isolated 92 fungal strains and 135 bacterialstrains from larvae of five families of Coleoptera that werefeeding ondecayingwood in tropicalwet forests of 10 nationalparks of Costa Rica The 92 fungal isolates were assigned tothree phyla 16 orders 24 families and 40 genera (one differ-ent genus every 23 isolates)Within the phylumZygomycotawe isolated members of the order Mucorales and within thephylum Basidiomycota members of the orders AgaricalesPolyporales and Trichosporonales Most of the fungi isolatedfrom the gastrointestinal tracts of the larvae belonged to thephylum Ascomycota (89 of total) They were distributedin 12 orders with Hypocreales being the dominant one itcomprised nearly 55 of the isolates (Table 3) The genusTrichoderma was the most abundant it was the only oneassociatedwith all five families of Coleoptera and also presentin each national park sampled
Most fungal orders and genera were sparsely representedwith 68 of the orders and 55 of the genera found associ-ated with a specific coleopteran family at a particular site Allthe insect families also presented unique fungal isolates persite with Tenebrionidae being the only coleopteran family inwhich all isolates were phylogenetically distinct Regardingthe sampling sites Tenorio National Park showed the highestnumber of unique phylotypes but Piedras Blancas NationalPark contained a more phylogenetically diverse array ofisolates
The 135 bacterial isolates were classified within fivephyla 13 orders 22 families and 35 genera (one differentgenus from every 38 isolates) including members of Acti-nobacteria Proteobacteria Firmicutes Flavobacteria andFusobacteria Approximately 82 of the bacteria belongedto 120574-Proteobacteria and Firmicutes accounting for 44 and38 of the isolates respectivelyWithin the 120574-Proteobacteriathe genera Serratia and Pseudomonas were abundant beingpresent in all host families studied Within Firmicutes thegenus Bacillus was clearly the most dominant This singlegenus 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 shownfor each of the coleopteran families
Order Genus Cer Ela Pas Sca Ten TotalBotryosphaeriales Botryosphaeria 1 1Capnodiales Ramichloridium 1 1
Total 25 12 19 29 7 92Cer Cerambycidae Ela Elateridae Pas Passalidae Sca Scarabaeidae and Ten TenebrionidaelowastGenera that presented positive enzymatic activities in more than four pathways
a common gut inhabitant of all the insect families and waspresent at almost all the sites sampled (Table 4)
The remaining bacterial classes obtained in this studywere less represented For example members of Actinobac-teria accounted for 11 of the isolates whereas 120572- and 120573-Proteobacteria Flavobacteria and Fusobacteria represented
less than 6 of the isolates When calculating the percent-age of isolates that were specific to a single site and hostfamily results showed that 42 of the isolates exhibitedthis characteristic while the remaining 58 of the generapresented a broader host-site range A small number of thegenera 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 isshown for each of the coleopteran families
Flavobacteria Chryseobacterium 1 1Fusobacteria Sebaldella 1 1Total 18 20 41 46 10 135Cer Cerambycidae Ela Elateridae Pas Passalidae Sca Scarabaeidae and Ten TenebrionidaelowastGenera that presented positive enzymatic activities in more than four pathways
(ie Rhizobium in Hitoy Cerere National Park) others wereassociated with the same beetle family but in different sites(ie Leucobacter with Passalidae) Results of the analysisof unique phylotypes per site and insect family showedthat Hitoy Cerere National Park and Elateridae respectivelypresented the highest percentages of single bacterial isolates
32 Lignocellulolytic Activity Determination Nearly 65 ofthe fungal genera and 48 of the bacterial genera presentedpositive results in at least one of the five lignocellulolyticactivities evaluated with carboxymethylcellulose degrada-tion being the most common activity observed in bothgroups (Table 5) In general fungi showedmore capability for
degrading lignocellulosic materials than bacteria with gen-era such as Trichoderma Bionectria and Trametes showingpositive results in all the assays performed Within bacteriaBacillus Enterobacter and Acinetobacter some of the mostabundant genera isolated from the larval guts tested positivefor four out of the five enzymatic activities assayed cellulase120573-glucosidase 120573-xylanase and cellobiose hydrolase activi-ties However neither these genera nor any other bacterialgroup screened were able to degrade the Remazol BrilliantBluemolecules while 30of the fungal genera tested positivefor this lignin-related degradation activity
33 Comparison of Gut Inhabitants between Families ofColeoptera We performed community analysis with Vegan
International Journal of Microbiology 7
Table 5 Results of the screening for lignocellulolytic activitiesFungal genera with positive results are shown in the upper groupand bacterial genera in the lower group
to gain insight into how the microbial gut composition of thebeetle families related to one anotherThis approach clusteredthe environments according to Bray-Curtis distances of
Figure 1 Heatmap of the abundance distribution of fungal com-munities associated with the guts of five wood-feeding familiesof Coleoptera The taxonomic relationship of the fungal genera isshown in the rows while the clustering of the coleopteran familiesdetermined by their composition similarities is shown in thecolumns Higher intensities of the color reveal higher abundancesof the isolates
the abundance distribution of the isolates considering alsotheir phylogenetic relationships The results showed that thefungal composition of the isolates associated with larvaeof Cerambycidae Scarabaeidae and Passalidae clusteredtogether Cerambycidae and Passalidae shared one orderof Basidiomycota and three orders of Ascomycota whileScarabaeidae and Passalidae had in common four ordersof Ascomycota A second cluster was formed by the fungalmicrobiotas isolated fromTenebrionidae and Elateridae theyshared two orders of Ascomycota and one of Basidiomycota(Figure 1) The analysis of the bacterial dataset showed thatthe microbial compositions associated with Scarabaeidaeand Passalidae formed part of the same cluster sharingisolates belonging to 120573- and 120574-Proteobacteria Actinobac-teria and Firmicutes The second cluster was formed byTenebrionidae Elateridae andCerambycidae that shared iso-lates assigned to Pseudomonadales Enterobacteriales andBacillales (Figure 2) In addition we performed canonicalcorrespondence analysis for exploring relationships betweenthe microbial communities of the coleopteran hosts Resultsof this analysis where consistent with results obtained withthe Bray-Curtis clustering for both the fungal and bacterialcommunities (Figure S1 in Supplementary Material availableonline at httpdxdoiorg1011552015285018)
4 Discussion
We collected larvae of five families of wood-feedingColeoptera in tropical forests of Costa Rica with the aim ofestimating the species composition of cultivable fungi andbacteria inhabiting their guts and to identify microorganisms
Figure 2 Heatmap of the abundance distribution of bacterialcommunities associated with the guts of five wood-feeding familiesof Coleoptera The taxonomic relationship of the bacterial genera isshown in the rows while the clustering of the coleopteran familiesdetermined by their composition similarities is shown in thecolumns Higher intensities of the color reveal higher abundancesof the isolates
with relevant lignocellulolytic activities The main limitationof this study is that the cultivation-dependent approachbased on artificial media covers only a small proportion ofthe total microbial diversity present in this particular nicheThe positive trade-off of this approach was the identificationof several isolates with lignocellulose-degrading capabilitieswhich can be further used for the respective enzymecharacterization for direct degradation assays on residuesfrom agriculture and forestry for the treatment of industrialeffluents and for bioprospecting novel metabolites withother biotechnological applications Despite the inherentbias of the isolation method our results suggest that gutmicrobiota of wood-feeding tropical beetles presents arelatively high diversity in terms of microbial richnessphylogenetic composition and lignocellulolytic activities
The order Hypocreales represented about 60 of thetotal number of fungal isolates Within this group the genusTrichoderma was the most abundant comprising nearly aquarter of the fungal collection This genus was a commongut inhabitant of beetle larvae regardless of the host familyor the geographic location The reason for this dominanceis not entirely clear however one possible explanation isthat several species belonging to this fungal genus containa number of glycoside hydrolases peroxidases laccasesand phenol oxidases among other enzymes related to thedegradation of lignocellulose materials This feature mightprovide some advantages for using the recalcitrant polymericmaterials passing through the gastrointestinal tract [16 38ndash40]
In addition our data indicate that guts of wood-feedinglarvae were from environments having a high representation
of Hypocreales as also observed in a similar study performedin other locations of Costa Rica [41] This is relevant forbioprospecting purposes since wood-feeding beetles mightconstitute a good source of TrichodermaMetarhiziumMeta-cordyceps Bionectria and other fungal genera known topossess a wide array of biotechnological applications [42ndash45] The remaining orders presented a lower abundanceand in most of the cases were represented by a singlegenus Nevertheless many of the genera showed the abilityto degrade lignocellulose-related hexoses and pentoses asalso shown in other studies [46ndash50] Within the phylumBasidiomycota the genus Trametes showed positive resultsin all the lignocellulolytic assays related to the degradationof structural wood components This white-rot fungus is aknownmodel for studying degradation of lignin in free-livingconditions and in this work reported in its association withthe gut microbiota of wood-feeding insects [51 52]
It is difficult to know whether these fungal isolates aretruly endosymbionts of the intestinal tracts of the coleopteranlarvae or are transitory inhabitants associated with hostfeeding habits Hence it is also possible that some ofthese microorganisms could be commensals parasites andfacultative endosymbionts They might even be using theinsect as a dispersal mechanism [15 53] It is clear howeverthat the overall taxonomic composition of the gut-inhabitingmicrobes and the proportion of lignocellulolytic-positivefungi seem to be particular to the larval microenvironmentThe structure of this endosymbiotic community is distin-guished from the fungal composition observed in otherwood-related microhabitats such as the fungal populationsin living plant tissues They are also dominated by membersof Ascomycetes but they present a different abundancedistribution of fungal families [54 55] decaying logs aredominated mainly by Basidiomycetes [56ndash58]
The analysis of the taxonomic composition of the bacte-rial isolates showed the presence of seven major phylogeneticclasses codominated by 120574-Proteobacteria and FirmicutesThis finding is consistent with results obtained in similarstudies [6 13 14 44 59] Within the 120574-Proteobacteria themost abundant genera were Enterobacter Serratia Acineto-bacter and Pseudomonas Interestingly Serratia and Pseu-domonas were isolated from all five coleopteran familiesstudied Enterobacter and Acinetobacter were present in fourout of the five insect families and they exhibited positiveresults in the lignocellulolytic assays except for lignin degra-dation Similar characteristics related to the degradation oflignocellulose and to fermentativemetabolismwere observedin Bacillus the most abundant genus within Firmicutes [1160] Together these results support the notion that somespecies of fungi and bacteria such as Trichoderma SerratiaPseudomonas and Bacillus can be common gut inhabitantsof wood-feeding larvae in tropical forests suggesting thatcertain affinities might have developed between the beetlehost and its microbiota [41 61ndash64]
When comparing the fungal and bacterial species compo-sition among the beetle families the plots of the Bray-Curtisdistances and canonical correspondence analyses producedbiologically meaningful clusters to group the environmentsthat share similar microbial compositions The first fungal
International Journal of Microbiology 9
cluster relates the microbiota associated with the guts ofCerambycidae Passalidae and Scarabaeidae This is con-sistent with the observation of a high diversity of isolatesfrom Cerambycidae that shared members of the fungalphyla Basidiomycota and Ascomycota with Passalidae andmembers of Zygomycota and Ascomycota with ScarabaeidaeThe cluster formed by Tenebrionidae-Elateridae shared ina lower proportion members of the Basidiomycota andAscomycotaThe bacterial microbiota associated with Passal-idae and Scarabaeidae also formed a cluster sharingmembersof five major bacterial clades microbiota of CerambycidaeElateridae and Tenebrionidae shared members only of 120574-Proteobacteria and Firmicutes
The clustering analyses revealed that Cerambycidae pre-sented a high diversity of fungi but not of bacteria whilePassalidae and Elateridae exhibited a high diversity of bac-teria and moderate diversity of fungi Scarabaeidae andTenebrionidae contained a similar composition of bothThese results suggest that the nature of the beetle hosthas an important effect on the phylogenetic diversity of itsassociated microbiota and that many factors can influenceits configuration These factors may include the biology ofthe host the physical and chemical characteristics of thegut compartments the feeding habits of the insects and themicrobial diversity associated with the environment in whichthe insect is living [23 26 65 66]
Our results consistently showed that both the fungaland bacterial populations associated with the guts of beetlelarvae are highly diverse in terms of the number of speciesobtained and in their phylogenetic composition Thesemicrobial inhabitants could be forming complex consortiathat would be acting synergistically to provide many of thenutritional needs of the beetle host Some of these functionsinclude the degradation and fermentation of lignocellulosicmaterials as shown by the high percentage of fungal andbacterial genera that presented positive activities or by theproduction of proteins and other metabolites necessary forthe development of the insect [25 44 67ndash69] Furthermorecertain affinities for substrates can be expected according tothe nature of the gut inhabitant For example members ofthe Basidiomycota could possibly degrade larger polymericmolecules the Ascomycota deplete diverse lignocellulosicconstituents while the bacteria degrade and ferment thesmaller monomeric and dimeric hexoses and pentoses pro-duced by the fungal counterparts The bacteria also likelyuse these sugars to produce other nutrients and metabo-lites Consequently the present work raises new lines ofinvestigation concerning the existence of microbial consortiaacting synergistically to provide the nutritional needs of thehosts the nature of the ecological and evolutionary processesthat contribute to ensure the fitness of the insect and themechanisms that rule the interactions among the fungi thebacteria and the beetle host
Conflict of Interests
The authors declared that there is no conflict of interestsregarding this paper
Acknowledgments
The authors acknowledge the contribution of the follow-ing people during the development of this project LuisGuillermoAcosta for the field sampling and early insect iden-tification Angel Solis Carlos Hernandez and Elena Ulate forthe identification of some adult specimens Jorge Blanco forthe fungal isolation Angelica Acuna and Beatriz Rivera fortheDNA extraction and enzymatic assaysManuel Ferrer andCesar Mateo for their advices on the lignocellulolytic activitydetermination Ana Lorena Guevara and Giselle Tamayofor the overall support the editor and reviewers of thisjournal for critical comments on the paperThis research wasfunded by the support of the National Council of Science andTechnology (CONICIT FV-027-2007) the CSIC and CRUSAFoundation (2007 CR0034) and Florida Ice amp FarmCoTheythank ACLAC ACOPAC ACOSA ACTo ACLAP ACCVCACAT ACAHN ACG and ACLAC National ConservationAreas and CONAGEBIO for granting the sample collectingpermits (R-CM-INBio-40-2008-OT R-CM-INBio-48-2008-OT)
References
[1] M Tien and C-P D Tu ldquoCloning and sequencing of a cDNAfor a ligninase from Phanerochaete chrysosporiumrdquo Nature vol326 no 6112 pp 520ndash523 1987
[2] P Beguin ldquoMolecular biology of cellulose degradationrdquo AnnualReview of Microbiology vol 44 pp 219ndash248 1990
[3] J Perez J Munoz-Dorado T de la Rubia and J MartınezldquoBiodegradation and biological treatments of cellulose hemi-cellulose and lignin an overviewrdquo International Microbiologyvol 5 no 2 pp 53ndash63 2002
[4] M R Berenbaum and T Eisner ldquoEcology Bugsrsquo bugsrdquo Sciencevol 322 no 5898 pp 52ndash53 2008
[5] Z Zhang ldquoPhylum Arthropoda von Siebold 1948 In animalbiodiversity an outline of higher-level classification and surveyof taxonomic richnessrdquo Zootaxa vol 3148 pp 99ndash103 1948
[6] J Morales-Jimenez G Zuniga L Villa-Tanaca and CHernandez-Rodrıguez ldquoBacterial community and nitrogenfixation in the red turpentine beetle Dendroctonus valensLeConte (Coleoptera Curculionidae Scolytinae)rdquo MicrobialEcology vol 58 no 4 pp 879ndash891 2009
[7] S M Geib M del Mar Jimenez-Gasco J E Carlson M TienR Jabbour and K Hoover ldquoMicrobial community profilingto investigate transmission of bacteria between life stages ofthe wood-boring beetle Anoplophora glabripennisrdquo MicrobialEcology vol 58 no 1 pp 199ndash211 2009
[8] P Engel and N A Moran ldquoThe gut microbiota of insectsmdashdiversity in structure and functionrdquo FEMS MicrobiologyReviews vol 37 no 5 pp 699ndash735 2013
[9] W Shi S Xie X Chen et al ldquoComparative genomic anal-ysis of the microbiome of herbivorous insects reveals eco-environmental adaptations biotechnology applicationsrdquo PLoSGenetics vol 9 no 1 Article ID e1003131 2013
[10] I Hanski and Y Cambefort Dung Beetle Ecology PrincetonUniversity Press Princeton NJ USA 1991
[11] M Egert B Wagner T Lemke A Brune and M W FriedrichldquoMicrobial community structure in midgut and hindgut ofthe humus-feeding larva of Pachnoda ephippiata (Coleoptera
10 International Journal of Microbiology
Scarabaeidae)rdquo Applied and Environmental Microbiology vol69 no 11 pp 6659ndash6668 2003
[12] R N Coulson ldquoPopulation dynamics of bark beetlesrdquo AnnualReview of Entomology vol 24 no 1 pp 417ndash447 1979
[13] P D Schloss I Delalibera Jr J Handelsman and K F RaffaldquoBacteria associated with the guts of two wood-boring beetlesanoplophora glabripennis and Saperda vestita (Cerambycidae)rdquoEnvironmental Entomology vol 35 no 3 pp 625ndash629 2006
[14] A Vasanthakumar J O Handelsman P D Schloss L S Bauerand K F Raffa ldquoGut microbiota of an invasive subcorticalbeetle Agrilus planipennis Fairmaire across various life stagesrdquoEnvironmental Entomology vol 37 no 5 pp 1344ndash1353 2008
[15] J Morales-Jimenez G Zuniga H C Ramırez-Saad and CHernandez-Rodrıguez ldquoGut-associated bacteria throughoutthe life cycle of the bark beetle Dendroctonus rhizophagusThomas and Bright (Curculionidae Scolytinae) and their cellu-lolytic activitiesrdquoMicrobial Ecology vol 64 no 1 pp 268ndash2782012
[16] R Kumar S Singh and O V Singh ldquoBioconversion of lig-nocellulosic biomass biochemical and molecular perspectivesrdquoJournal of IndustrialMicrobiology and Biotechnology vol 35 no5 pp 377ndash391 2008
[17] C Sanchez ldquoLignocellulosic residues biodegradation and bio-conversion by fungirdquo Biotechnology Advances vol 27 no 2 pp185ndash194 2009
[18] E D Scully S M Geib K Hoover et al ldquoMetagenomicprofiling reveals lignocellulose degrading system in a microbialcommunity associated with a wood-feeding beetlerdquo PLoS ONEvol 8 no 9 Article ID e73827 2013
[19] T L Erwin ldquoTropical forest their richness in Coleoptera andother arthropod speciesrdquoThe Coleopterists Bulletin vol 36 no1 pp 74ndash75 1982
[20] Y Basset L Cizek P Cuenoud et al ldquoArthropod diversity in atropical forestrdquo Science vol 338 no 6113 pp 1481ndash1484 2012
[21] A Solis Escarabajos de Costa Rica Las Familias Mas ComunesHeredia Costa Rica Editorial Inbio 1999
[22] D Borror and R White A Field Guide to the Insects HoughtonMifflin Company New York NY USA 1987
[23] M Egert U Stingl L D Bruun B Pommerenke A Bruneand M W Friedrich ldquoStructure and topology of microbialcommunities in the major gut compartments of Melolonthamelolontha larvae (Coleoptera Scarabaeidae)rdquo Applied andEnvironmentalMicrobiology vol 71 no 8 pp 4556ndash4566 2005
[24] S-O Suh J V McHugh D D Pollock and M Blackwell ldquoThebeetle gut a hyperdiverse source of novel yeastsrdquo MycologicalResearch vol 109 no 3 pp 261ndash265 2005
[25] J J Scott D-C Oh M C Yuceer K D Klepzig J Clardy andC R Currie ldquoBacterial protection of beetle-fungusmutualismrdquoScience vol 322 no 5898 p 63 2008
[26] J A Ceja-Navarro N H Nguyen U Karaoz et al ldquoCom-partmentalized microbial composition oxygen gradients andnitrogen fixation in the gut of Odontotaenius disjunctusrdquo TheISME Journal vol 8 no 1 pp 6ndash18 2014
[27] J B Nardi C M Bee L A Miller N H Nguyen S-O Suhand M Blackwell ldquoCommunities of microbes that inhabit thechanging hindgut landscape of a subsocial beetlerdquo ArthropodStructure and Development vol 35 no 1 pp 57ndash68 2006
[28] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982
[29] B RMVyas andH PMolitoris ldquoInvolvement of an extracellu-lar H2O2-dependent ligninolytic activity of the white rot fungus
Pleurotus ostreatus in the decolorization of Remazol brilliantblue Rrdquo Applied and Environmental Microbiology vol 61 no 11pp 3919ndash3927 1995
[30] J-D Bok D A Yernool and D E Eveleigh ldquoPurificationcharacterization and molecular analysis of thermostable cellu-lases CelA andCelB fromThermotoga neapolitanardquoApplied andEnvironmentalMicrobiology vol 64 no 12 pp 4774ndash4781 1998
[31] K M G Machado D R Matheus and V L R BononildquoLigninolytic enzymes production and Remazol Brilliant BlueR decolorization by tropical Brazilian basidiomycetes fungirdquoBrazilian Journal of Microbiology vol 36 no 3 pp 246ndash2522005
[32] G T Howard and B A White ldquoMolecular cloning and expres-sion of cellulase genes fromRuminococcus albus 8 in Escherichiacoli bacteriophage 120582rdquo Applied and Environmental Microbiologyvol 54 no 7 pp 1752ndash1755 1988
[33] N L Glass and G C Donaldson ldquoDevelopment of primersets designed for use with the PCR to amplify conserved genesfrom filamentous ascomycetesrdquo Applied and EnvironmentalMicrobiology vol 61 no 4 pp 1323ndash1330 1995
[34] D Lane ldquo16S23S rRNA sequencingrdquo inNucleic Acid Techniquesin Bacterial Systematics E Stachebrandt and M GoodfellowEds Wiley Chichester UK 1991
[35] Q Wang G M Garrity J M Tiedje and J R Cole ldquoNaıveBayesian classifier for rapid assignment of rRNA sequencesinto the new bacterial taxonomyrdquo Applied and EnvironmentalMicrobiology vol 73 no 16 pp 5261ndash5267 2007
[36] J Oksanen G Blanchet R Kindt et al Vegan CommunityEcology Package 2014
[37] P L Buttigieg and A Ramette ldquoA guide to statistical analysisin microbial ecology a community-focused living review ofmultivariate data analysesrdquo FEMSMicrobiology Ecology vol 90no 3 pp 543ndash550 2014
[38] DMartinez RM Berka B Henrissat et al ldquoGenome sequenc-ing and analysis of the biomass-degrading fungus Trichodermareesei (syn Hypocrea jecorina)rdquo Nature Biotechnology vol 26no 5 pp 553ndash560 2008
[39] E D Scully K Hoover J E Carlson M Tien and S M GeibldquoMidgut transcriptome profiling of Anoplophora glabripennisa lignocellulose degrading cerambycid beetlerdquo BMC Genomicsvol 14 article 850 2013
[40] P K Foreman D Brown L Dankmeyer et al ldquoTranscriptionalregulation of biomass-degrading enzymes in the filamentousfungus Trichoderma reeseirdquoThe Journal of Biological Chemistryvol 278 no 34 pp 31988ndash31997 2003
[41] G Vargas-Asensio A Pinto-Tomas B Rivera et al ldquoUncover-ing the cultivable microbial diversity of costa rican beetles andits ability to break down plant cell wall componentsrdquoPLoSONEvol 9 no 11 Article ID e113303 2014
[42] M P Coughlan ldquoThe properties of fungal and bacterial cel-lulases with comment on their production and applicationrdquoBiotechnology and Genetic Engineering Reviews vol 3 no 1 pp39ndash110 1985
[43] L R Lynd P J Weimer W H Van Zyl and I S PretoriusldquoMicrobial cellulose utilization fundamentals and biotechnol-ogyrdquoMicrobiology andMolecular Biology Reviews vol 66 no 3pp 506ndash577 2002
[44] S M Geib T R Filley P G Hatcher et al ldquoLignin degradationin wood-feeding insectsrdquo Proceedings of the National Academy
International Journal of Microbiology 11
of Sciences of the United States of America vol 105 no 35 pp12932ndash12937 2008
[45] H Alper and G Stephanopoulos ldquoEngineering for biofuelsexploiting innate microbial capacity or importing biosyntheticpotentialrdquo Nature Reviews Microbiology vol 7 no 10 pp 715ndash723 2009
[46] K Yaoi and Y Mitsuishi ldquoPurification characterizationcloning and expression of a novel xyloglucan-specific glycosi-dase oligoxyloglucan reducing end-specific cellobiohydrolaserdquoThe Journal of Biological Chemistry vol 277 no 50 pp 48276ndash48281 2002
[47] L Ayed N Assas S Sayadi and M Hamdi ldquoInvolvementof lignin peroxidase in the decolourization of black olivemill wastewaters by Geotrichum candidumrdquo Letters in AppliedMicrobiology vol 40 no 1 pp 7ndash11 2005
[48] Y Baba A Shimonaka J Koga H Kubota and T KonoldquoAlternative splicing produces two endoglucanases with oneor two carbohydrate-binding modules in Mucor circinelloidesrdquoJournal of Bacteriology vol 187 no 9 pp 3045ndash3051 2005
[49] M Dashtban H Schraft andW Qin ldquoFungal bioconversion oflignocellulosic residues opportunities amp perspectivesrdquo Interna-tional Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009
[50] D A Ribeiro J Cota T M Alvarez et al ldquoThe Penicilliumechinulatum secretome on sugar cane bagasserdquo PLoS ONE vol7 no 12 Article ID e50571 2012
[51] O Borokhov and S Rothenburger ldquoRapid dye decolorizationmethod for screening potential wood preservativesrdquo Appliedand Environmental Microbiology vol 66 no 12 pp 5457ndash54592000
[52] E Abadulla T Tzanov S Costa K-H Robra A Cavaco-Paulo and G M Gubitz ldquoDecolorization and detoxification oftextile dyes with a laccase from Trametes hirsutardquo Applied andEnvironmental Microbiology vol 66 no 8 pp 3357ndash3362 2000
[53] T J Dreaden J M Davis Z W de Beer et al ldquoPhylogenyof ambrosia beetle symbionts in the genus Raffaeleardquo FungalBiology vol 118 no 12 pp 970ndash978 2014
[54] T S Suryanarayanan T S Murali and G Venkatesan ldquoOccur-rence and distribution of fungal endophytes in tropical forestsacross a rainfall gradientrdquo Canadian Journal of Botany vol 80no 8 pp 818ndash826 2002
[55] A E Arnold and F Lutzoni ldquoDiversity and host range of foliarfungal endophytes are tropical leaves biodiversity hotspotsrdquoEcology vol 88 no 3 pp 541ndash549 2007
[56] J Heilmann-Clausen and L Boddy ldquoInhibition and stimulationeffects in communities of wood decay fungi exudates fromcolonized wood influence growth by other speciesrdquo MicrobialEcology vol 49 no 3 pp 399ndash406 2005
[57] A Kubartova E Ottosson A Dahlberg and J Stenlid ldquoPat-terns of fungal communities among and within decaying logsrevealed by 454 sequencingrdquo Molecular Ecology vol 21 no 18pp 4514ndash4532 2012
[58] L Prewitt Y Kang M L Kakumanu andMWilliams ldquoFungaland bacterial community succession differs for three woodtypes during decay in a forest soilrdquo Microbial Ecology vol 68no 2 pp 212ndash221 2014
[59] N M Reid S L Addison L J Macdonald and G Lloyd-JonesldquoBiodiversity of active and inactive bacteria in the gut floraof wood-feeding Huhu beetle larvae (Prionoplus reticularis)rdquoApplied and Environmental Microbiology vol 77 no 19 pp7000ndash7006 2011
[60] CC Lee R E Kibblewhite-AccinelliM R Smith KWagschalW J Orts and DW SWong ldquoCloning of Bacillus licheniformisxylanase gene and characterization of recombinant enzymerdquoCurrent Microbiology vol 57 no 4 pp 301ndash305 2008
[61] T L Rhoads A T Mikell Jr and M H Eley ldquoInvestigation ofthe lignin-degrading activity of Serratia marcescens biochem-ical screening and ultrastructural evidencerdquo Canadian Journalof Microbiology vol 41 no 7 pp 592ndash600 1995
[62] T Nagy K Emami C M G A Fontes L M A FerreiraD R Humphry and H J Gilbert ldquoThe membrane-bound120572-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-O-methyl-D-glucuronoxylooligosaccharides but not 4-O-methyl-D-glucuronoxylanrdquo Journal of Bacteriology vol 184 no 17 pp4925ndash4929 2002
[63] J Weslien L B Djupstrom M Schroeder and O WidenfalkldquoLong-term priority effects among insects and fungi colonizingdecaying woodrdquo The Journal of Animal Ecology vol 80 no 6pp 1155ndash1162 2011
[64] R T Jones L G Sanchez and N Fierer ldquoA cross-taxon analysisof insect-associated bacterial diversityrdquo PLoS ONE vol 8 no 4Article ID e61218 2013
[65] T Lemke U Stingl M Egert M W Friedrich and A BruneldquoPhysicochemical conditions and microbial activities in thehighly alkaline gut of the humus-feeding larva of Pachnodaephippiata (Coleoptera Scarabaeidae)rdquo Applied and Environ-mental Microbiology vol 69 no 11 pp 6650ndash6658 2003
[66] S M Geib M D M Jimenez-Gasco J E Carlson M Tienand K Hoover ldquoEffect of host tree species on cellulase activityand bacterial community composition in the gut of larval Asianlonghorned beetlerdquo Environmental Entomology vol 38 no 3pp 686ndash699 2009
[67] A E Cazemier J C Verdoes F A G Reubsaet J H PHackstein C van der Drift and H J M Op den CampldquoPromicromonospora pachnodae sp nov a member of the(hemi)cellulolytic hindgut flora of larvae of the scarab beetlePachnoda marginatardquo Antonie van Leeuwenhoek vol 83 no 2pp 135ndash148 2003
[68] F A Genta R J Dillon W R Terra and C Ferreira ldquoPotentialrole for gut microbiota in cell wall digestion and glucosidedetoxification in Tenebrio molitor larvaerdquo Journal of InsectPhysiology vol 52 no 6 pp 593ndash601 2006
[69] D-C Oh J J Scott C R Currie and J Clardy ldquoMycangimycina polyene peroxide from a mutualist Streptomyces sprdquo OrganicLetters vol 11 no 3 pp 633ndash636 2009
Table 1 Description of the location and main environmental parameters of the 10 national parks of Costa Rica where sampling was carriedout The selected environments are classified as tropical wet forest and cover most of the natural distribution of the five coleopteran familiesstudied
National park LatitudeLongitude Altitude (m) Mean temperature (∘C) Annual precipitation (mm)
Table 2 Distribution of the number of larvae samples according tothe insect family and national park At each site an approximate areaof 150000m2 was explored for the presence of wood-feeding larvaeThe number of insect groups found varied according to the naturalcondition of each forest Each sample was composed of one to threeindividuals of the same species
National park Cer Ela Pas Sca Ten TotalArenal 1 3 4Barbilla 6 6Braulio 2 2 1 5Carara 1 3 1 5Hitoy Cerere 7 7Piedras Blancas 1 2 1 4Rincon de la Vieja 1 2 3Tapanti 2 1 1 1 5Tenorio 1 1 1 3Tortuguero 1 2 2 1 6Total 8 5 16 16 3 48Cer Cerambycidae Ela Elateridae Pas Passalidae Sca Scarabaeidae andTen Tenebrionidae
two times for each enzymatic screening The degradationof cellulose was determined using carboxymethylcellulose(CMC Sigma) as the sole carbon source in the mediumfollowed by staining with Congo red Briefly the bacte-rial isolates were grown for 48 h and fungi for 72 h at28∘C on CMC medium (094 gL KH
2PO4 19 gL K
2HPO4
16 gL KCl 143 gL NaCl 015 gL NH4Cl 0037 gL MgSO
4-
7H2O 0017 gL CaCl
2 01 gL yeast-extract 75 gL CMC
and 15 gL agar pH 70) After this incubation period themicroorganism-containing agar plates were flooded with005 Congo red for 10min until the dye bound CMC Thereaction was fixed with NaCl (50mM) for 5min and thenrinsed with distilled water The zones where the microor-ganism hydrolyzed the CMC were visible as clear halos [28]The oxidative degradation of lignin was determined basedon the decolorization of the dye Remazol Brilliant Blue R(RBBR Sigma) when grown on solid media [29ndash31] Plateswith MEA-RBBR medium (20 gL malt extract 15 gL agarand 002wtvol RBBR pH 70) were inoculated with thebacterial and fungal isolates and incubated at 28∘C At dailyintervals for a period of 14 days the plates were checkedfor the presence of a decolorized area around the colony ormycelia Determination of the120573-glucosidase120573-xylanase andcellobiose hydrolase activities was performed using as sub-strates 10mM 4-nitrophenyl 120573-D-glucopyranoside (Sigma)4-nitrophenyl120573-D-xylopyranoside (Sigma) or 4-nitrophenyl120573-D-cellobioside (Sigma) dissolved in 50mM ammoniumacetate buffer pH 50 amended with 07 of agar and keptat 55∘C A drop of these solutions was placed directly on thebacterial colonies or fungal mycelia followed by incubationfor 30min at room temperature The catalytic action of themicrobial enzymes on the substrate was detected by thedevelopment of a yellow coloration produced by the releaseof the p-nitrophenol group [30 32]
24 Molecular Analyses All the isolates were grown in Petridishes containing the same media used for preservationFor the fungal DNA extraction 400mg of mycelia was
4 International Journal of Microbiology
ground with mortar and pestle in liquid nitrogen and fur-ther extracted using the DNeasy Plant kit (Qiagen USA)including a pretreatment step consisting of the incubationat 60∘C for one hour with 400 120583L of lysis buffer and30 120583L of Proteinase K (20mgmL Sigma Aldrich USA)The bacterial DNA was extracted following the instructionsof the NucleoSpin Tissue DNA Extraction kit (Macherey-Nagel Germany) The fungal ITS1-58S-ITS2 regions wereamplified by PCR from the total DNA using as forwardprimer the ITS1 51015840-TCCGTAGGTGAACCTGCGG-31015840 andthe reverse primer ITS4 51015840-TCCTCCGCTTATTGATATGC-31015840 [33] with the following reaction program 95∘C for 10min35 cycles at 94∘C for 1min 54∘C for 1min 72∘C for 1minand additional extension at 72∘C for 10min The 16S rRNAgene was amplified using primers 27f and 1492r [34] with thefollowing program 95∘C for 10min and 35 cycles of 94∘Cfor 1min 52∘C for 1min and 72∘C for 1min and 10minextension at 72∘C The PCR products were purified usingthe NucleoSpin Extract II kit (Macherey-Nagel Germany)according to manufacturerrsquos protocol Sanger sequencing ofthe samples was performed at the sequencing facility ofthe Dana Farber Cancer Institute at the Harvard UniversityBoston Massachusetts using the abovementioned forwardand reverse primers for fungi and primers 27f and 785r forbacteria Sequences were assembled using Seqman programofDNASTARLasergene 80 (GenBank accession GU827479-GU827553 HM770962-HM771112)
25 Taxonomy The taxonomic assignment of the bacterialsequences was performed by comparing the database againstthe 16S rRNA reference set 10 implemented in the Classifiertool of the Ribosomal Database Project which assigned the16S rRNA sequences to corresponding taxonomical hierarchybased on a naıve Bayesian rRNA classifier [35]The taxonomyof the fungi was inferred by comparing the ITS1-58S-ITS2 sequences against the Warcup Fungal ITS trainset 1a curated reference dataset implemented in the Classifiertool of the Ribosomal Database Project [35] Every fungaltaxonomical assignment was verified against the Index Fun-gorum (httpwwwindexfungorumorg) and appropriatelycorrected when synonyms or current names were identified
26 Ecological Analyses The analysis of the microbial com-munities was performed using the Vegan package imple-mented in the statistical programming environment andlanguage R [36] For this a table with the taxonomic clas-sifications to the levels of order class subphylum phylumand subkingdom of the fungal isolates was converted totaxonomic pairwise distances with the function taxa2distand using variable step lengths between successive categoriesproportional to the number of groups within each taxonom-ical level This distance matrix was then used to constructa hierarchical clustering tree with the function hclust andthe UPGMA distance method A second matrix with theabundance distribution of the fungal isolates per insect familywas prepared with the larval families in the rows the fungalorders in the columns and cells containing the counts ofisolates in each taxon This matrix was used to calculate
Bray-Curtis distances between the insect fungal communitieswith the function vegdist The advantage of this approach isthat Bray-Curtis measures avoid the double zero problemaccounting for absences that are not indicators of similaritiesbetween sample units [37] The generated distance matrixwas used to cluster similarities between the microbial com-positions of the larval families with the hclust function Thefunction tabasco was used to display compact integratedcommunity information plotting the fungal taxonomicalrelationships in the rows the similarities between the insectsrsquomicrobial compositions in the columns and a heatmap withthe respective abundance distribution [36] The function ccawas used to perform canonical correspondence analysis ofthe communities associated with the coleopteran families Asimilar procedure was performed to analyze the communitycomposition of bacteria and also for detecting differencesamong national parks
3 Results
31 Taxonomic Composition of the Fungi and Bacteria IsolatedIn this study we isolated 92 fungal strains and 135 bacterialstrains from larvae of five families of Coleoptera that werefeeding ondecayingwood in tropicalwet forests of 10 nationalparks of Costa Rica The 92 fungal isolates were assigned tothree phyla 16 orders 24 families and 40 genera (one differ-ent genus every 23 isolates)Within the phylumZygomycotawe isolated members of the order Mucorales and within thephylum Basidiomycota members of the orders AgaricalesPolyporales and Trichosporonales Most of the fungi isolatedfrom the gastrointestinal tracts of the larvae belonged to thephylum Ascomycota (89 of total) They were distributedin 12 orders with Hypocreales being the dominant one itcomprised nearly 55 of the isolates (Table 3) The genusTrichoderma was the most abundant it was the only oneassociatedwith all five families of Coleoptera and also presentin each national park sampled
Most fungal orders and genera were sparsely representedwith 68 of the orders and 55 of the genera found associ-ated with a specific coleopteran family at a particular site Allthe insect families also presented unique fungal isolates persite with Tenebrionidae being the only coleopteran family inwhich all isolates were phylogenetically distinct Regardingthe sampling sites Tenorio National Park showed the highestnumber of unique phylotypes but Piedras Blancas NationalPark contained a more phylogenetically diverse array ofisolates
The 135 bacterial isolates were classified within fivephyla 13 orders 22 families and 35 genera (one differentgenus from every 38 isolates) including members of Acti-nobacteria Proteobacteria Firmicutes Flavobacteria andFusobacteria Approximately 82 of the bacteria belongedto 120574-Proteobacteria and Firmicutes accounting for 44 and38 of the isolates respectivelyWithin the 120574-Proteobacteriathe genera Serratia and Pseudomonas were abundant beingpresent in all host families studied Within Firmicutes thegenus Bacillus was clearly the most dominant This singlegenus 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 shownfor each of the coleopteran families
Order Genus Cer Ela Pas Sca Ten TotalBotryosphaeriales Botryosphaeria 1 1Capnodiales Ramichloridium 1 1
Total 25 12 19 29 7 92Cer Cerambycidae Ela Elateridae Pas Passalidae Sca Scarabaeidae and Ten TenebrionidaelowastGenera that presented positive enzymatic activities in more than four pathways
a common gut inhabitant of all the insect families and waspresent at almost all the sites sampled (Table 4)
The remaining bacterial classes obtained in this studywere less represented For example members of Actinobac-teria accounted for 11 of the isolates whereas 120572- and 120573-Proteobacteria Flavobacteria and Fusobacteria represented
less than 6 of the isolates When calculating the percent-age of isolates that were specific to a single site and hostfamily results showed that 42 of the isolates exhibitedthis characteristic while the remaining 58 of the generapresented a broader host-site range A small number of thegenera 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 isshown for each of the coleopteran families
Flavobacteria Chryseobacterium 1 1Fusobacteria Sebaldella 1 1Total 18 20 41 46 10 135Cer Cerambycidae Ela Elateridae Pas Passalidae Sca Scarabaeidae and Ten TenebrionidaelowastGenera that presented positive enzymatic activities in more than four pathways
(ie Rhizobium in Hitoy Cerere National Park) others wereassociated with the same beetle family but in different sites(ie Leucobacter with Passalidae) Results of the analysisof unique phylotypes per site and insect family showedthat Hitoy Cerere National Park and Elateridae respectivelypresented the highest percentages of single bacterial isolates
32 Lignocellulolytic Activity Determination Nearly 65 ofthe fungal genera and 48 of the bacterial genera presentedpositive results in at least one of the five lignocellulolyticactivities evaluated with carboxymethylcellulose degrada-tion being the most common activity observed in bothgroups (Table 5) In general fungi showedmore capability for
degrading lignocellulosic materials than bacteria with gen-era such as Trichoderma Bionectria and Trametes showingpositive results in all the assays performed Within bacteriaBacillus Enterobacter and Acinetobacter some of the mostabundant genera isolated from the larval guts tested positivefor four out of the five enzymatic activities assayed cellulase120573-glucosidase 120573-xylanase and cellobiose hydrolase activi-ties However neither these genera nor any other bacterialgroup screened were able to degrade the Remazol BrilliantBluemolecules while 30of the fungal genera tested positivefor this lignin-related degradation activity
33 Comparison of Gut Inhabitants between Families ofColeoptera We performed community analysis with Vegan
International Journal of Microbiology 7
Table 5 Results of the screening for lignocellulolytic activitiesFungal genera with positive results are shown in the upper groupand bacterial genera in the lower group
to gain insight into how the microbial gut composition of thebeetle families related to one anotherThis approach clusteredthe environments according to Bray-Curtis distances of
Figure 1 Heatmap of the abundance distribution of fungal com-munities associated with the guts of five wood-feeding familiesof Coleoptera The taxonomic relationship of the fungal genera isshown in the rows while the clustering of the coleopteran familiesdetermined by their composition similarities is shown in thecolumns Higher intensities of the color reveal higher abundancesof the isolates
the abundance distribution of the isolates considering alsotheir phylogenetic relationships The results showed that thefungal composition of the isolates associated with larvaeof Cerambycidae Scarabaeidae and Passalidae clusteredtogether Cerambycidae and Passalidae shared one orderof Basidiomycota and three orders of Ascomycota whileScarabaeidae and Passalidae had in common four ordersof Ascomycota A second cluster was formed by the fungalmicrobiotas isolated fromTenebrionidae and Elateridae theyshared two orders of Ascomycota and one of Basidiomycota(Figure 1) The analysis of the bacterial dataset showed thatthe microbial compositions associated with Scarabaeidaeand Passalidae formed part of the same cluster sharingisolates belonging to 120573- and 120574-Proteobacteria Actinobac-teria and Firmicutes The second cluster was formed byTenebrionidae Elateridae andCerambycidae that shared iso-lates assigned to Pseudomonadales Enterobacteriales andBacillales (Figure 2) In addition we performed canonicalcorrespondence analysis for exploring relationships betweenthe microbial communities of the coleopteran hosts Resultsof this analysis where consistent with results obtained withthe Bray-Curtis clustering for both the fungal and bacterialcommunities (Figure S1 in Supplementary Material availableonline at httpdxdoiorg1011552015285018)
4 Discussion
We collected larvae of five families of wood-feedingColeoptera in tropical forests of Costa Rica with the aim ofestimating the species composition of cultivable fungi andbacteria inhabiting their guts and to identify microorganisms
Figure 2 Heatmap of the abundance distribution of bacterialcommunities associated with the guts of five wood-feeding familiesof Coleoptera The taxonomic relationship of the bacterial genera isshown in the rows while the clustering of the coleopteran familiesdetermined by their composition similarities is shown in thecolumns Higher intensities of the color reveal higher abundancesof the isolates
with relevant lignocellulolytic activities The main limitationof this study is that the cultivation-dependent approachbased on artificial media covers only a small proportion ofthe total microbial diversity present in this particular nicheThe positive trade-off of this approach was the identificationof several isolates with lignocellulose-degrading capabilitieswhich can be further used for the respective enzymecharacterization for direct degradation assays on residuesfrom agriculture and forestry for the treatment of industrialeffluents and for bioprospecting novel metabolites withother biotechnological applications Despite the inherentbias of the isolation method our results suggest that gutmicrobiota of wood-feeding tropical beetles presents arelatively high diversity in terms of microbial richnessphylogenetic composition and lignocellulolytic activities
The order Hypocreales represented about 60 of thetotal number of fungal isolates Within this group the genusTrichoderma was the most abundant comprising nearly aquarter of the fungal collection This genus was a commongut inhabitant of beetle larvae regardless of the host familyor the geographic location The reason for this dominanceis not entirely clear however one possible explanation isthat several species belonging to this fungal genus containa number of glycoside hydrolases peroxidases laccasesand phenol oxidases among other enzymes related to thedegradation of lignocellulose materials This feature mightprovide some advantages for using the recalcitrant polymericmaterials passing through the gastrointestinal tract [16 38ndash40]
In addition our data indicate that guts of wood-feedinglarvae were from environments having a high representation
of Hypocreales as also observed in a similar study performedin other locations of Costa Rica [41] This is relevant forbioprospecting purposes since wood-feeding beetles mightconstitute a good source of TrichodermaMetarhiziumMeta-cordyceps Bionectria and other fungal genera known topossess a wide array of biotechnological applications [42ndash45] The remaining orders presented a lower abundanceand in most of the cases were represented by a singlegenus Nevertheless many of the genera showed the abilityto degrade lignocellulose-related hexoses and pentoses asalso shown in other studies [46ndash50] Within the phylumBasidiomycota the genus Trametes showed positive resultsin all the lignocellulolytic assays related to the degradationof structural wood components This white-rot fungus is aknownmodel for studying degradation of lignin in free-livingconditions and in this work reported in its association withthe gut microbiota of wood-feeding insects [51 52]
It is difficult to know whether these fungal isolates aretruly endosymbionts of the intestinal tracts of the coleopteranlarvae or are transitory inhabitants associated with hostfeeding habits Hence it is also possible that some ofthese microorganisms could be commensals parasites andfacultative endosymbionts They might even be using theinsect as a dispersal mechanism [15 53] It is clear howeverthat the overall taxonomic composition of the gut-inhabitingmicrobes and the proportion of lignocellulolytic-positivefungi seem to be particular to the larval microenvironmentThe structure of this endosymbiotic community is distin-guished from the fungal composition observed in otherwood-related microhabitats such as the fungal populationsin living plant tissues They are also dominated by membersof Ascomycetes but they present a different abundancedistribution of fungal families [54 55] decaying logs aredominated mainly by Basidiomycetes [56ndash58]
The analysis of the taxonomic composition of the bacte-rial isolates showed the presence of seven major phylogeneticclasses codominated by 120574-Proteobacteria and FirmicutesThis finding is consistent with results obtained in similarstudies [6 13 14 44 59] Within the 120574-Proteobacteria themost abundant genera were Enterobacter Serratia Acineto-bacter and Pseudomonas Interestingly Serratia and Pseu-domonas were isolated from all five coleopteran familiesstudied Enterobacter and Acinetobacter were present in fourout of the five insect families and they exhibited positiveresults in the lignocellulolytic assays except for lignin degra-dation Similar characteristics related to the degradation oflignocellulose and to fermentativemetabolismwere observedin Bacillus the most abundant genus within Firmicutes [1160] Together these results support the notion that somespecies of fungi and bacteria such as Trichoderma SerratiaPseudomonas and Bacillus can be common gut inhabitantsof wood-feeding larvae in tropical forests suggesting thatcertain affinities might have developed between the beetlehost and its microbiota [41 61ndash64]
When comparing the fungal and bacterial species compo-sition among the beetle families the plots of the Bray-Curtisdistances and canonical correspondence analyses producedbiologically meaningful clusters to group the environmentsthat share similar microbial compositions The first fungal
International Journal of Microbiology 9
cluster relates the microbiota associated with the guts ofCerambycidae Passalidae and Scarabaeidae This is con-sistent with the observation of a high diversity of isolatesfrom Cerambycidae that shared members of the fungalphyla Basidiomycota and Ascomycota with Passalidae andmembers of Zygomycota and Ascomycota with ScarabaeidaeThe cluster formed by Tenebrionidae-Elateridae shared ina lower proportion members of the Basidiomycota andAscomycotaThe bacterial microbiota associated with Passal-idae and Scarabaeidae also formed a cluster sharingmembersof five major bacterial clades microbiota of CerambycidaeElateridae and Tenebrionidae shared members only of 120574-Proteobacteria and Firmicutes
The clustering analyses revealed that Cerambycidae pre-sented a high diversity of fungi but not of bacteria whilePassalidae and Elateridae exhibited a high diversity of bac-teria and moderate diversity of fungi Scarabaeidae andTenebrionidae contained a similar composition of bothThese results suggest that the nature of the beetle hosthas an important effect on the phylogenetic diversity of itsassociated microbiota and that many factors can influenceits configuration These factors may include the biology ofthe host the physical and chemical characteristics of thegut compartments the feeding habits of the insects and themicrobial diversity associated with the environment in whichthe insect is living [23 26 65 66]
Our results consistently showed that both the fungaland bacterial populations associated with the guts of beetlelarvae are highly diverse in terms of the number of speciesobtained and in their phylogenetic composition Thesemicrobial inhabitants could be forming complex consortiathat would be acting synergistically to provide many of thenutritional needs of the beetle host Some of these functionsinclude the degradation and fermentation of lignocellulosicmaterials as shown by the high percentage of fungal andbacterial genera that presented positive activities or by theproduction of proteins and other metabolites necessary forthe development of the insect [25 44 67ndash69] Furthermorecertain affinities for substrates can be expected according tothe nature of the gut inhabitant For example members ofthe Basidiomycota could possibly degrade larger polymericmolecules the Ascomycota deplete diverse lignocellulosicconstituents while the bacteria degrade and ferment thesmaller monomeric and dimeric hexoses and pentoses pro-duced by the fungal counterparts The bacteria also likelyuse these sugars to produce other nutrients and metabo-lites Consequently the present work raises new lines ofinvestigation concerning the existence of microbial consortiaacting synergistically to provide the nutritional needs of thehosts the nature of the ecological and evolutionary processesthat contribute to ensure the fitness of the insect and themechanisms that rule the interactions among the fungi thebacteria and the beetle host
Conflict of Interests
The authors declared that there is no conflict of interestsregarding this paper
Acknowledgments
The authors acknowledge the contribution of the follow-ing people during the development of this project LuisGuillermoAcosta for the field sampling and early insect iden-tification Angel Solis Carlos Hernandez and Elena Ulate forthe identification of some adult specimens Jorge Blanco forthe fungal isolation Angelica Acuna and Beatriz Rivera fortheDNA extraction and enzymatic assaysManuel Ferrer andCesar Mateo for their advices on the lignocellulolytic activitydetermination Ana Lorena Guevara and Giselle Tamayofor the overall support the editor and reviewers of thisjournal for critical comments on the paperThis research wasfunded by the support of the National Council of Science andTechnology (CONICIT FV-027-2007) the CSIC and CRUSAFoundation (2007 CR0034) and Florida Ice amp FarmCoTheythank ACLAC ACOPAC ACOSA ACTo ACLAP ACCVCACAT ACAHN ACG and ACLAC National ConservationAreas and CONAGEBIO for granting the sample collectingpermits (R-CM-INBio-40-2008-OT R-CM-INBio-48-2008-OT)
References
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[2] P Beguin ldquoMolecular biology of cellulose degradationrdquo AnnualReview of Microbiology vol 44 pp 219ndash248 1990
[3] J Perez J Munoz-Dorado T de la Rubia and J MartınezldquoBiodegradation and biological treatments of cellulose hemi-cellulose and lignin an overviewrdquo International Microbiologyvol 5 no 2 pp 53ndash63 2002
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[7] S M Geib M del Mar Jimenez-Gasco J E Carlson M TienR Jabbour and K Hoover ldquoMicrobial community profilingto investigate transmission of bacteria between life stages ofthe wood-boring beetle Anoplophora glabripennisrdquo MicrobialEcology vol 58 no 1 pp 199ndash211 2009
[8] P Engel and N A Moran ldquoThe gut microbiota of insectsmdashdiversity in structure and functionrdquo FEMS MicrobiologyReviews vol 37 no 5 pp 699ndash735 2013
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10 International Journal of Microbiology
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[39] E D Scully K Hoover J E Carlson M Tien and S M GeibldquoMidgut transcriptome profiling of Anoplophora glabripennisa lignocellulose degrading cerambycid beetlerdquo BMC Genomicsvol 14 article 850 2013
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[47] L Ayed N Assas S Sayadi and M Hamdi ldquoInvolvementof lignin peroxidase in the decolourization of black olivemill wastewaters by Geotrichum candidumrdquo Letters in AppliedMicrobiology vol 40 no 1 pp 7ndash11 2005
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[49] M Dashtban H Schraft andW Qin ldquoFungal bioconversion oflignocellulosic residues opportunities amp perspectivesrdquo Interna-tional Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009
[50] D A Ribeiro J Cota T M Alvarez et al ldquoThe Penicilliumechinulatum secretome on sugar cane bagasserdquo PLoS ONE vol7 no 12 Article ID e50571 2012
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[52] E Abadulla T Tzanov S Costa K-H Robra A Cavaco-Paulo and G M Gubitz ldquoDecolorization and detoxification oftextile dyes with a laccase from Trametes hirsutardquo Applied andEnvironmental Microbiology vol 66 no 8 pp 3357ndash3362 2000
[53] T J Dreaden J M Davis Z W de Beer et al ldquoPhylogenyof ambrosia beetle symbionts in the genus Raffaeleardquo FungalBiology vol 118 no 12 pp 970ndash978 2014
[54] T S Suryanarayanan T S Murali and G Venkatesan ldquoOccur-rence and distribution of fungal endophytes in tropical forestsacross a rainfall gradientrdquo Canadian Journal of Botany vol 80no 8 pp 818ndash826 2002
[55] A E Arnold and F Lutzoni ldquoDiversity and host range of foliarfungal endophytes are tropical leaves biodiversity hotspotsrdquoEcology vol 88 no 3 pp 541ndash549 2007
[56] J Heilmann-Clausen and L Boddy ldquoInhibition and stimulationeffects in communities of wood decay fungi exudates fromcolonized wood influence growth by other speciesrdquo MicrobialEcology vol 49 no 3 pp 399ndash406 2005
[57] A Kubartova E Ottosson A Dahlberg and J Stenlid ldquoPat-terns of fungal communities among and within decaying logsrevealed by 454 sequencingrdquo Molecular Ecology vol 21 no 18pp 4514ndash4532 2012
[58] L Prewitt Y Kang M L Kakumanu andMWilliams ldquoFungaland bacterial community succession differs for three woodtypes during decay in a forest soilrdquo Microbial Ecology vol 68no 2 pp 212ndash221 2014
[59] N M Reid S L Addison L J Macdonald and G Lloyd-JonesldquoBiodiversity of active and inactive bacteria in the gut floraof wood-feeding Huhu beetle larvae (Prionoplus reticularis)rdquoApplied and Environmental Microbiology vol 77 no 19 pp7000ndash7006 2011
[60] CC Lee R E Kibblewhite-AccinelliM R Smith KWagschalW J Orts and DW SWong ldquoCloning of Bacillus licheniformisxylanase gene and characterization of recombinant enzymerdquoCurrent Microbiology vol 57 no 4 pp 301ndash305 2008
[61] T L Rhoads A T Mikell Jr and M H Eley ldquoInvestigation ofthe lignin-degrading activity of Serratia marcescens biochem-ical screening and ultrastructural evidencerdquo Canadian Journalof Microbiology vol 41 no 7 pp 592ndash600 1995
[62] T Nagy K Emami C M G A Fontes L M A FerreiraD R Humphry and H J Gilbert ldquoThe membrane-bound120572-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-O-methyl-D-glucuronoxylooligosaccharides but not 4-O-methyl-D-glucuronoxylanrdquo Journal of Bacteriology vol 184 no 17 pp4925ndash4929 2002
[63] J Weslien L B Djupstrom M Schroeder and O WidenfalkldquoLong-term priority effects among insects and fungi colonizingdecaying woodrdquo The Journal of Animal Ecology vol 80 no 6pp 1155ndash1162 2011
[64] R T Jones L G Sanchez and N Fierer ldquoA cross-taxon analysisof insect-associated bacterial diversityrdquo PLoS ONE vol 8 no 4Article ID e61218 2013
[65] T Lemke U Stingl M Egert M W Friedrich and A BruneldquoPhysicochemical conditions and microbial activities in thehighly alkaline gut of the humus-feeding larva of Pachnodaephippiata (Coleoptera Scarabaeidae)rdquo Applied and Environ-mental Microbiology vol 69 no 11 pp 6650ndash6658 2003
[66] S M Geib M D M Jimenez-Gasco J E Carlson M Tienand K Hoover ldquoEffect of host tree species on cellulase activityand bacterial community composition in the gut of larval Asianlonghorned beetlerdquo Environmental Entomology vol 38 no 3pp 686ndash699 2009
[67] A E Cazemier J C Verdoes F A G Reubsaet J H PHackstein C van der Drift and H J M Op den CampldquoPromicromonospora pachnodae sp nov a member of the(hemi)cellulolytic hindgut flora of larvae of the scarab beetlePachnoda marginatardquo Antonie van Leeuwenhoek vol 83 no 2pp 135ndash148 2003
[68] F A Genta R J Dillon W R Terra and C Ferreira ldquoPotentialrole for gut microbiota in cell wall digestion and glucosidedetoxification in Tenebrio molitor larvaerdquo Journal of InsectPhysiology vol 52 no 6 pp 593ndash601 2006
[69] D-C Oh J J Scott C R Currie and J Clardy ldquoMycangimycina polyene peroxide from a mutualist Streptomyces sprdquo OrganicLetters vol 11 no 3 pp 633ndash636 2009
ground with mortar and pestle in liquid nitrogen and fur-ther extracted using the DNeasy Plant kit (Qiagen USA)including a pretreatment step consisting of the incubationat 60∘C for one hour with 400 120583L of lysis buffer and30 120583L of Proteinase K (20mgmL Sigma Aldrich USA)The bacterial DNA was extracted following the instructionsof the NucleoSpin Tissue DNA Extraction kit (Macherey-Nagel Germany) The fungal ITS1-58S-ITS2 regions wereamplified by PCR from the total DNA using as forwardprimer the ITS1 51015840-TCCGTAGGTGAACCTGCGG-31015840 andthe reverse primer ITS4 51015840-TCCTCCGCTTATTGATATGC-31015840 [33] with the following reaction program 95∘C for 10min35 cycles at 94∘C for 1min 54∘C for 1min 72∘C for 1minand additional extension at 72∘C for 10min The 16S rRNAgene was amplified using primers 27f and 1492r [34] with thefollowing program 95∘C for 10min and 35 cycles of 94∘Cfor 1min 52∘C for 1min and 72∘C for 1min and 10minextension at 72∘C The PCR products were purified usingthe NucleoSpin Extract II kit (Macherey-Nagel Germany)according to manufacturerrsquos protocol Sanger sequencing ofthe samples was performed at the sequencing facility ofthe Dana Farber Cancer Institute at the Harvard UniversityBoston Massachusetts using the abovementioned forwardand reverse primers for fungi and primers 27f and 785r forbacteria Sequences were assembled using Seqman programofDNASTARLasergene 80 (GenBank accession GU827479-GU827553 HM770962-HM771112)
25 Taxonomy The taxonomic assignment of the bacterialsequences was performed by comparing the database againstthe 16S rRNA reference set 10 implemented in the Classifiertool of the Ribosomal Database Project which assigned the16S rRNA sequences to corresponding taxonomical hierarchybased on a naıve Bayesian rRNA classifier [35]The taxonomyof the fungi was inferred by comparing the ITS1-58S-ITS2 sequences against the Warcup Fungal ITS trainset 1a curated reference dataset implemented in the Classifiertool of the Ribosomal Database Project [35] Every fungaltaxonomical assignment was verified against the Index Fun-gorum (httpwwwindexfungorumorg) and appropriatelycorrected when synonyms or current names were identified
26 Ecological Analyses The analysis of the microbial com-munities was performed using the Vegan package imple-mented in the statistical programming environment andlanguage R [36] For this a table with the taxonomic clas-sifications to the levels of order class subphylum phylumand subkingdom of the fungal isolates was converted totaxonomic pairwise distances with the function taxa2distand using variable step lengths between successive categoriesproportional to the number of groups within each taxonom-ical level This distance matrix was then used to constructa hierarchical clustering tree with the function hclust andthe UPGMA distance method A second matrix with theabundance distribution of the fungal isolates per insect familywas prepared with the larval families in the rows the fungalorders in the columns and cells containing the counts ofisolates in each taxon This matrix was used to calculate
Bray-Curtis distances between the insect fungal communitieswith the function vegdist The advantage of this approach isthat Bray-Curtis measures avoid the double zero problemaccounting for absences that are not indicators of similaritiesbetween sample units [37] The generated distance matrixwas used to cluster similarities between the microbial com-positions of the larval families with the hclust function Thefunction tabasco was used to display compact integratedcommunity information plotting the fungal taxonomicalrelationships in the rows the similarities between the insectsrsquomicrobial compositions in the columns and a heatmap withthe respective abundance distribution [36] The function ccawas used to perform canonical correspondence analysis ofthe communities associated with the coleopteran families Asimilar procedure was performed to analyze the communitycomposition of bacteria and also for detecting differencesamong national parks
3 Results
31 Taxonomic Composition of the Fungi and Bacteria IsolatedIn this study we isolated 92 fungal strains and 135 bacterialstrains from larvae of five families of Coleoptera that werefeeding ondecayingwood in tropicalwet forests of 10 nationalparks of Costa Rica The 92 fungal isolates were assigned tothree phyla 16 orders 24 families and 40 genera (one differ-ent genus every 23 isolates)Within the phylumZygomycotawe isolated members of the order Mucorales and within thephylum Basidiomycota members of the orders AgaricalesPolyporales and Trichosporonales Most of the fungi isolatedfrom the gastrointestinal tracts of the larvae belonged to thephylum Ascomycota (89 of total) They were distributedin 12 orders with Hypocreales being the dominant one itcomprised nearly 55 of the isolates (Table 3) The genusTrichoderma was the most abundant it was the only oneassociatedwith all five families of Coleoptera and also presentin each national park sampled
Most fungal orders and genera were sparsely representedwith 68 of the orders and 55 of the genera found associ-ated with a specific coleopteran family at a particular site Allthe insect families also presented unique fungal isolates persite with Tenebrionidae being the only coleopteran family inwhich all isolates were phylogenetically distinct Regardingthe sampling sites Tenorio National Park showed the highestnumber of unique phylotypes but Piedras Blancas NationalPark contained a more phylogenetically diverse array ofisolates
The 135 bacterial isolates were classified within fivephyla 13 orders 22 families and 35 genera (one differentgenus from every 38 isolates) including members of Acti-nobacteria Proteobacteria Firmicutes Flavobacteria andFusobacteria Approximately 82 of the bacteria belongedto 120574-Proteobacteria and Firmicutes accounting for 44 and38 of the isolates respectivelyWithin the 120574-Proteobacteriathe genera Serratia and Pseudomonas were abundant beingpresent in all host families studied Within Firmicutes thegenus Bacillus was clearly the most dominant This singlegenus 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 shownfor each of the coleopteran families
Order Genus Cer Ela Pas Sca Ten TotalBotryosphaeriales Botryosphaeria 1 1Capnodiales Ramichloridium 1 1
Total 25 12 19 29 7 92Cer Cerambycidae Ela Elateridae Pas Passalidae Sca Scarabaeidae and Ten TenebrionidaelowastGenera that presented positive enzymatic activities in more than four pathways
a common gut inhabitant of all the insect families and waspresent at almost all the sites sampled (Table 4)
The remaining bacterial classes obtained in this studywere less represented For example members of Actinobac-teria accounted for 11 of the isolates whereas 120572- and 120573-Proteobacteria Flavobacteria and Fusobacteria represented
less than 6 of the isolates When calculating the percent-age of isolates that were specific to a single site and hostfamily results showed that 42 of the isolates exhibitedthis characteristic while the remaining 58 of the generapresented a broader host-site range A small number of thegenera 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 isshown for each of the coleopteran families
Flavobacteria Chryseobacterium 1 1Fusobacteria Sebaldella 1 1Total 18 20 41 46 10 135Cer Cerambycidae Ela Elateridae Pas Passalidae Sca Scarabaeidae and Ten TenebrionidaelowastGenera that presented positive enzymatic activities in more than four pathways
(ie Rhizobium in Hitoy Cerere National Park) others wereassociated with the same beetle family but in different sites(ie Leucobacter with Passalidae) Results of the analysisof unique phylotypes per site and insect family showedthat Hitoy Cerere National Park and Elateridae respectivelypresented the highest percentages of single bacterial isolates
32 Lignocellulolytic Activity Determination Nearly 65 ofthe fungal genera and 48 of the bacterial genera presentedpositive results in at least one of the five lignocellulolyticactivities evaluated with carboxymethylcellulose degrada-tion being the most common activity observed in bothgroups (Table 5) In general fungi showedmore capability for
degrading lignocellulosic materials than bacteria with gen-era such as Trichoderma Bionectria and Trametes showingpositive results in all the assays performed Within bacteriaBacillus Enterobacter and Acinetobacter some of the mostabundant genera isolated from the larval guts tested positivefor four out of the five enzymatic activities assayed cellulase120573-glucosidase 120573-xylanase and cellobiose hydrolase activi-ties However neither these genera nor any other bacterialgroup screened were able to degrade the Remazol BrilliantBluemolecules while 30of the fungal genera tested positivefor this lignin-related degradation activity
33 Comparison of Gut Inhabitants between Families ofColeoptera We performed community analysis with Vegan
International Journal of Microbiology 7
Table 5 Results of the screening for lignocellulolytic activitiesFungal genera with positive results are shown in the upper groupand bacterial genera in the lower group
to gain insight into how the microbial gut composition of thebeetle families related to one anotherThis approach clusteredthe environments according to Bray-Curtis distances of
Figure 1 Heatmap of the abundance distribution of fungal com-munities associated with the guts of five wood-feeding familiesof Coleoptera The taxonomic relationship of the fungal genera isshown in the rows while the clustering of the coleopteran familiesdetermined by their composition similarities is shown in thecolumns Higher intensities of the color reveal higher abundancesof the isolates
the abundance distribution of the isolates considering alsotheir phylogenetic relationships The results showed that thefungal composition of the isolates associated with larvaeof Cerambycidae Scarabaeidae and Passalidae clusteredtogether Cerambycidae and Passalidae shared one orderof Basidiomycota and three orders of Ascomycota whileScarabaeidae and Passalidae had in common four ordersof Ascomycota A second cluster was formed by the fungalmicrobiotas isolated fromTenebrionidae and Elateridae theyshared two orders of Ascomycota and one of Basidiomycota(Figure 1) The analysis of the bacterial dataset showed thatthe microbial compositions associated with Scarabaeidaeand Passalidae formed part of the same cluster sharingisolates belonging to 120573- and 120574-Proteobacteria Actinobac-teria and Firmicutes The second cluster was formed byTenebrionidae Elateridae andCerambycidae that shared iso-lates assigned to Pseudomonadales Enterobacteriales andBacillales (Figure 2) In addition we performed canonicalcorrespondence analysis for exploring relationships betweenthe microbial communities of the coleopteran hosts Resultsof this analysis where consistent with results obtained withthe Bray-Curtis clustering for both the fungal and bacterialcommunities (Figure S1 in Supplementary Material availableonline at httpdxdoiorg1011552015285018)
4 Discussion
We collected larvae of five families of wood-feedingColeoptera in tropical forests of Costa Rica with the aim ofestimating the species composition of cultivable fungi andbacteria inhabiting their guts and to identify microorganisms
Figure 2 Heatmap of the abundance distribution of bacterialcommunities associated with the guts of five wood-feeding familiesof Coleoptera The taxonomic relationship of the bacterial genera isshown in the rows while the clustering of the coleopteran familiesdetermined by their composition similarities is shown in thecolumns Higher intensities of the color reveal higher abundancesof the isolates
with relevant lignocellulolytic activities The main limitationof this study is that the cultivation-dependent approachbased on artificial media covers only a small proportion ofthe total microbial diversity present in this particular nicheThe positive trade-off of this approach was the identificationof several isolates with lignocellulose-degrading capabilitieswhich can be further used for the respective enzymecharacterization for direct degradation assays on residuesfrom agriculture and forestry for the treatment of industrialeffluents and for bioprospecting novel metabolites withother biotechnological applications Despite the inherentbias of the isolation method our results suggest that gutmicrobiota of wood-feeding tropical beetles presents arelatively high diversity in terms of microbial richnessphylogenetic composition and lignocellulolytic activities
The order Hypocreales represented about 60 of thetotal number of fungal isolates Within this group the genusTrichoderma was the most abundant comprising nearly aquarter of the fungal collection This genus was a commongut inhabitant of beetle larvae regardless of the host familyor the geographic location The reason for this dominanceis not entirely clear however one possible explanation isthat several species belonging to this fungal genus containa number of glycoside hydrolases peroxidases laccasesand phenol oxidases among other enzymes related to thedegradation of lignocellulose materials This feature mightprovide some advantages for using the recalcitrant polymericmaterials passing through the gastrointestinal tract [16 38ndash40]
In addition our data indicate that guts of wood-feedinglarvae were from environments having a high representation
of Hypocreales as also observed in a similar study performedin other locations of Costa Rica [41] This is relevant forbioprospecting purposes since wood-feeding beetles mightconstitute a good source of TrichodermaMetarhiziumMeta-cordyceps Bionectria and other fungal genera known topossess a wide array of biotechnological applications [42ndash45] The remaining orders presented a lower abundanceand in most of the cases were represented by a singlegenus Nevertheless many of the genera showed the abilityto degrade lignocellulose-related hexoses and pentoses asalso shown in other studies [46ndash50] Within the phylumBasidiomycota the genus Trametes showed positive resultsin all the lignocellulolytic assays related to the degradationof structural wood components This white-rot fungus is aknownmodel for studying degradation of lignin in free-livingconditions and in this work reported in its association withthe gut microbiota of wood-feeding insects [51 52]
It is difficult to know whether these fungal isolates aretruly endosymbionts of the intestinal tracts of the coleopteranlarvae or are transitory inhabitants associated with hostfeeding habits Hence it is also possible that some ofthese microorganisms could be commensals parasites andfacultative endosymbionts They might even be using theinsect as a dispersal mechanism [15 53] It is clear howeverthat the overall taxonomic composition of the gut-inhabitingmicrobes and the proportion of lignocellulolytic-positivefungi seem to be particular to the larval microenvironmentThe structure of this endosymbiotic community is distin-guished from the fungal composition observed in otherwood-related microhabitats such as the fungal populationsin living plant tissues They are also dominated by membersof Ascomycetes but they present a different abundancedistribution of fungal families [54 55] decaying logs aredominated mainly by Basidiomycetes [56ndash58]
The analysis of the taxonomic composition of the bacte-rial isolates showed the presence of seven major phylogeneticclasses codominated by 120574-Proteobacteria and FirmicutesThis finding is consistent with results obtained in similarstudies [6 13 14 44 59] Within the 120574-Proteobacteria themost abundant genera were Enterobacter Serratia Acineto-bacter and Pseudomonas Interestingly Serratia and Pseu-domonas were isolated from all five coleopteran familiesstudied Enterobacter and Acinetobacter were present in fourout of the five insect families and they exhibited positiveresults in the lignocellulolytic assays except for lignin degra-dation Similar characteristics related to the degradation oflignocellulose and to fermentativemetabolismwere observedin Bacillus the most abundant genus within Firmicutes [1160] Together these results support the notion that somespecies of fungi and bacteria such as Trichoderma SerratiaPseudomonas and Bacillus can be common gut inhabitantsof wood-feeding larvae in tropical forests suggesting thatcertain affinities might have developed between the beetlehost and its microbiota [41 61ndash64]
When comparing the fungal and bacterial species compo-sition among the beetle families the plots of the Bray-Curtisdistances and canonical correspondence analyses producedbiologically meaningful clusters to group the environmentsthat share similar microbial compositions The first fungal
International Journal of Microbiology 9
cluster relates the microbiota associated with the guts ofCerambycidae Passalidae and Scarabaeidae This is con-sistent with the observation of a high diversity of isolatesfrom Cerambycidae that shared members of the fungalphyla Basidiomycota and Ascomycota with Passalidae andmembers of Zygomycota and Ascomycota with ScarabaeidaeThe cluster formed by Tenebrionidae-Elateridae shared ina lower proportion members of the Basidiomycota andAscomycotaThe bacterial microbiota associated with Passal-idae and Scarabaeidae also formed a cluster sharingmembersof five major bacterial clades microbiota of CerambycidaeElateridae and Tenebrionidae shared members only of 120574-Proteobacteria and Firmicutes
The clustering analyses revealed that Cerambycidae pre-sented a high diversity of fungi but not of bacteria whilePassalidae and Elateridae exhibited a high diversity of bac-teria and moderate diversity of fungi Scarabaeidae andTenebrionidae contained a similar composition of bothThese results suggest that the nature of the beetle hosthas an important effect on the phylogenetic diversity of itsassociated microbiota and that many factors can influenceits configuration These factors may include the biology ofthe host the physical and chemical characteristics of thegut compartments the feeding habits of the insects and themicrobial diversity associated with the environment in whichthe insect is living [23 26 65 66]
Our results consistently showed that both the fungaland bacterial populations associated with the guts of beetlelarvae are highly diverse in terms of the number of speciesobtained and in their phylogenetic composition Thesemicrobial inhabitants could be forming complex consortiathat would be acting synergistically to provide many of thenutritional needs of the beetle host Some of these functionsinclude the degradation and fermentation of lignocellulosicmaterials as shown by the high percentage of fungal andbacterial genera that presented positive activities or by theproduction of proteins and other metabolites necessary forthe development of the insect [25 44 67ndash69] Furthermorecertain affinities for substrates can be expected according tothe nature of the gut inhabitant For example members ofthe Basidiomycota could possibly degrade larger polymericmolecules the Ascomycota deplete diverse lignocellulosicconstituents while the bacteria degrade and ferment thesmaller monomeric and dimeric hexoses and pentoses pro-duced by the fungal counterparts The bacteria also likelyuse these sugars to produce other nutrients and metabo-lites Consequently the present work raises new lines ofinvestigation concerning the existence of microbial consortiaacting synergistically to provide the nutritional needs of thehosts the nature of the ecological and evolutionary processesthat contribute to ensure the fitness of the insect and themechanisms that rule the interactions among the fungi thebacteria and the beetle host
Conflict of Interests
The authors declared that there is no conflict of interestsregarding this paper
Acknowledgments
The authors acknowledge the contribution of the follow-ing people during the development of this project LuisGuillermoAcosta for the field sampling and early insect iden-tification Angel Solis Carlos Hernandez and Elena Ulate forthe identification of some adult specimens Jorge Blanco forthe fungal isolation Angelica Acuna and Beatriz Rivera fortheDNA extraction and enzymatic assaysManuel Ferrer andCesar Mateo for their advices on the lignocellulolytic activitydetermination Ana Lorena Guevara and Giselle Tamayofor the overall support the editor and reviewers of thisjournal for critical comments on the paperThis research wasfunded by the support of the National Council of Science andTechnology (CONICIT FV-027-2007) the CSIC and CRUSAFoundation (2007 CR0034) and Florida Ice amp FarmCoTheythank ACLAC ACOPAC ACOSA ACTo ACLAP ACCVCACAT ACAHN ACG and ACLAC National ConservationAreas and CONAGEBIO for granting the sample collectingpermits (R-CM-INBio-40-2008-OT R-CM-INBio-48-2008-OT)
References
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[2] P Beguin ldquoMolecular biology of cellulose degradationrdquo AnnualReview of Microbiology vol 44 pp 219ndash248 1990
[3] J Perez J Munoz-Dorado T de la Rubia and J MartınezldquoBiodegradation and biological treatments of cellulose hemi-cellulose and lignin an overviewrdquo International Microbiologyvol 5 no 2 pp 53ndash63 2002
[4] M R Berenbaum and T Eisner ldquoEcology Bugsrsquo bugsrdquo Sciencevol 322 no 5898 pp 52ndash53 2008
[5] Z Zhang ldquoPhylum Arthropoda von Siebold 1948 In animalbiodiversity an outline of higher-level classification and surveyof taxonomic richnessrdquo Zootaxa vol 3148 pp 99ndash103 1948
[6] J Morales-Jimenez G Zuniga L Villa-Tanaca and CHernandez-Rodrıguez ldquoBacterial community and nitrogenfixation in the red turpentine beetle Dendroctonus valensLeConte (Coleoptera Curculionidae Scolytinae)rdquo MicrobialEcology vol 58 no 4 pp 879ndash891 2009
[7] S M Geib M del Mar Jimenez-Gasco J E Carlson M TienR Jabbour and K Hoover ldquoMicrobial community profilingto investigate transmission of bacteria between life stages ofthe wood-boring beetle Anoplophora glabripennisrdquo MicrobialEcology vol 58 no 1 pp 199ndash211 2009
[8] P Engel and N A Moran ldquoThe gut microbiota of insectsmdashdiversity in structure and functionrdquo FEMS MicrobiologyReviews vol 37 no 5 pp 699ndash735 2013
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10 International Journal of Microbiology
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[14] A Vasanthakumar J O Handelsman P D Schloss L S Bauerand K F Raffa ldquoGut microbiota of an invasive subcorticalbeetle Agrilus planipennis Fairmaire across various life stagesrdquoEnvironmental Entomology vol 37 no 5 pp 1344ndash1353 2008
[15] J Morales-Jimenez G Zuniga H C Ramırez-Saad and CHernandez-Rodrıguez ldquoGut-associated bacteria throughoutthe life cycle of the bark beetle Dendroctonus rhizophagusThomas and Bright (Curculionidae Scolytinae) and their cellu-lolytic activitiesrdquoMicrobial Ecology vol 64 no 1 pp 268ndash2782012
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[19] T L Erwin ldquoTropical forest their richness in Coleoptera andother arthropod speciesrdquoThe Coleopterists Bulletin vol 36 no1 pp 74ndash75 1982
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Pleurotus ostreatus in the decolorization of Remazol brilliantblue Rrdquo Applied and Environmental Microbiology vol 61 no 11pp 3919ndash3927 1995
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[31] K M G Machado D R Matheus and V L R BononildquoLigninolytic enzymes production and Remazol Brilliant BlueR decolorization by tropical Brazilian basidiomycetes fungirdquoBrazilian Journal of Microbiology vol 36 no 3 pp 246ndash2522005
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[39] E D Scully K Hoover J E Carlson M Tien and S M GeibldquoMidgut transcriptome profiling of Anoplophora glabripennisa lignocellulose degrading cerambycid beetlerdquo BMC Genomicsvol 14 article 850 2013
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[41] G Vargas-Asensio A Pinto-Tomas B Rivera et al ldquoUncover-ing the cultivable microbial diversity of costa rican beetles andits ability to break down plant cell wall componentsrdquoPLoSONEvol 9 no 11 Article ID e113303 2014
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of Sciences of the United States of America vol 105 no 35 pp12932ndash12937 2008
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[47] L Ayed N Assas S Sayadi and M Hamdi ldquoInvolvementof lignin peroxidase in the decolourization of black olivemill wastewaters by Geotrichum candidumrdquo Letters in AppliedMicrobiology vol 40 no 1 pp 7ndash11 2005
[48] Y Baba A Shimonaka J Koga H Kubota and T KonoldquoAlternative splicing produces two endoglucanases with oneor two carbohydrate-binding modules in Mucor circinelloidesrdquoJournal of Bacteriology vol 187 no 9 pp 3045ndash3051 2005
[49] M Dashtban H Schraft andW Qin ldquoFungal bioconversion oflignocellulosic residues opportunities amp perspectivesrdquo Interna-tional Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009
[50] D A Ribeiro J Cota T M Alvarez et al ldquoThe Penicilliumechinulatum secretome on sugar cane bagasserdquo PLoS ONE vol7 no 12 Article ID e50571 2012
[51] O Borokhov and S Rothenburger ldquoRapid dye decolorizationmethod for screening potential wood preservativesrdquo Appliedand Environmental Microbiology vol 66 no 12 pp 5457ndash54592000
[52] E Abadulla T Tzanov S Costa K-H Robra A Cavaco-Paulo and G M Gubitz ldquoDecolorization and detoxification oftextile dyes with a laccase from Trametes hirsutardquo Applied andEnvironmental Microbiology vol 66 no 8 pp 3357ndash3362 2000
[53] T J Dreaden J M Davis Z W de Beer et al ldquoPhylogenyof ambrosia beetle symbionts in the genus Raffaeleardquo FungalBiology vol 118 no 12 pp 970ndash978 2014
[54] T S Suryanarayanan T S Murali and G Venkatesan ldquoOccur-rence and distribution of fungal endophytes in tropical forestsacross a rainfall gradientrdquo Canadian Journal of Botany vol 80no 8 pp 818ndash826 2002
[55] A E Arnold and F Lutzoni ldquoDiversity and host range of foliarfungal endophytes are tropical leaves biodiversity hotspotsrdquoEcology vol 88 no 3 pp 541ndash549 2007
[56] J Heilmann-Clausen and L Boddy ldquoInhibition and stimulationeffects in communities of wood decay fungi exudates fromcolonized wood influence growth by other speciesrdquo MicrobialEcology vol 49 no 3 pp 399ndash406 2005
[57] A Kubartova E Ottosson A Dahlberg and J Stenlid ldquoPat-terns of fungal communities among and within decaying logsrevealed by 454 sequencingrdquo Molecular Ecology vol 21 no 18pp 4514ndash4532 2012
[58] L Prewitt Y Kang M L Kakumanu andMWilliams ldquoFungaland bacterial community succession differs for three woodtypes during decay in a forest soilrdquo Microbial Ecology vol 68no 2 pp 212ndash221 2014
[59] N M Reid S L Addison L J Macdonald and G Lloyd-JonesldquoBiodiversity of active and inactive bacteria in the gut floraof wood-feeding Huhu beetle larvae (Prionoplus reticularis)rdquoApplied and Environmental Microbiology vol 77 no 19 pp7000ndash7006 2011
[60] CC Lee R E Kibblewhite-AccinelliM R Smith KWagschalW J Orts and DW SWong ldquoCloning of Bacillus licheniformisxylanase gene and characterization of recombinant enzymerdquoCurrent Microbiology vol 57 no 4 pp 301ndash305 2008
[61] T L Rhoads A T Mikell Jr and M H Eley ldquoInvestigation ofthe lignin-degrading activity of Serratia marcescens biochem-ical screening and ultrastructural evidencerdquo Canadian Journalof Microbiology vol 41 no 7 pp 592ndash600 1995
[62] T Nagy K Emami C M G A Fontes L M A FerreiraD R Humphry and H J Gilbert ldquoThe membrane-bound120572-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-O-methyl-D-glucuronoxylooligosaccharides but not 4-O-methyl-D-glucuronoxylanrdquo Journal of Bacteriology vol 184 no 17 pp4925ndash4929 2002
[63] J Weslien L B Djupstrom M Schroeder and O WidenfalkldquoLong-term priority effects among insects and fungi colonizingdecaying woodrdquo The Journal of Animal Ecology vol 80 no 6pp 1155ndash1162 2011
[64] R T Jones L G Sanchez and N Fierer ldquoA cross-taxon analysisof insect-associated bacterial diversityrdquo PLoS ONE vol 8 no 4Article ID e61218 2013
[65] T Lemke U Stingl M Egert M W Friedrich and A BruneldquoPhysicochemical conditions and microbial activities in thehighly alkaline gut of the humus-feeding larva of Pachnodaephippiata (Coleoptera Scarabaeidae)rdquo Applied and Environ-mental Microbiology vol 69 no 11 pp 6650ndash6658 2003
[66] S M Geib M D M Jimenez-Gasco J E Carlson M Tienand K Hoover ldquoEffect of host tree species on cellulase activityand bacterial community composition in the gut of larval Asianlonghorned beetlerdquo Environmental Entomology vol 38 no 3pp 686ndash699 2009
[67] A E Cazemier J C Verdoes F A G Reubsaet J H PHackstein C van der Drift and H J M Op den CampldquoPromicromonospora pachnodae sp nov a member of the(hemi)cellulolytic hindgut flora of larvae of the scarab beetlePachnoda marginatardquo Antonie van Leeuwenhoek vol 83 no 2pp 135ndash148 2003
[68] F A Genta R J Dillon W R Terra and C Ferreira ldquoPotentialrole for gut microbiota in cell wall digestion and glucosidedetoxification in Tenebrio molitor larvaerdquo Journal of InsectPhysiology vol 52 no 6 pp 593ndash601 2006
[69] D-C Oh J J Scott C R Currie and J Clardy ldquoMycangimycina polyene peroxide from a mutualist Streptomyces sprdquo OrganicLetters vol 11 no 3 pp 633ndash636 2009
Table 3 Taxonomic distribution of the fungal isolates identified in this study The number of isolates at the order and genera level is shownfor each of the coleopteran families
Order Genus Cer Ela Pas Sca Ten TotalBotryosphaeriales Botryosphaeria 1 1Capnodiales Ramichloridium 1 1
Total 25 12 19 29 7 92Cer Cerambycidae Ela Elateridae Pas Passalidae Sca Scarabaeidae and Ten TenebrionidaelowastGenera that presented positive enzymatic activities in more than four pathways
a common gut inhabitant of all the insect families and waspresent at almost all the sites sampled (Table 4)
The remaining bacterial classes obtained in this studywere less represented For example members of Actinobac-teria accounted for 11 of the isolates whereas 120572- and 120573-Proteobacteria Flavobacteria and Fusobacteria represented
less than 6 of the isolates When calculating the percent-age of isolates that were specific to a single site and hostfamily results showed that 42 of the isolates exhibitedthis characteristic while the remaining 58 of the generapresented a broader host-site range A small number of thegenera 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 isshown for each of the coleopteran families
Flavobacteria Chryseobacterium 1 1Fusobacteria Sebaldella 1 1Total 18 20 41 46 10 135Cer Cerambycidae Ela Elateridae Pas Passalidae Sca Scarabaeidae and Ten TenebrionidaelowastGenera that presented positive enzymatic activities in more than four pathways
(ie Rhizobium in Hitoy Cerere National Park) others wereassociated with the same beetle family but in different sites(ie Leucobacter with Passalidae) Results of the analysisof unique phylotypes per site and insect family showedthat Hitoy Cerere National Park and Elateridae respectivelypresented the highest percentages of single bacterial isolates
32 Lignocellulolytic Activity Determination Nearly 65 ofthe fungal genera and 48 of the bacterial genera presentedpositive results in at least one of the five lignocellulolyticactivities evaluated with carboxymethylcellulose degrada-tion being the most common activity observed in bothgroups (Table 5) In general fungi showedmore capability for
degrading lignocellulosic materials than bacteria with gen-era such as Trichoderma Bionectria and Trametes showingpositive results in all the assays performed Within bacteriaBacillus Enterobacter and Acinetobacter some of the mostabundant genera isolated from the larval guts tested positivefor four out of the five enzymatic activities assayed cellulase120573-glucosidase 120573-xylanase and cellobiose hydrolase activi-ties However neither these genera nor any other bacterialgroup screened were able to degrade the Remazol BrilliantBluemolecules while 30of the fungal genera tested positivefor this lignin-related degradation activity
33 Comparison of Gut Inhabitants between Families ofColeoptera We performed community analysis with Vegan
International Journal of Microbiology 7
Table 5 Results of the screening for lignocellulolytic activitiesFungal genera with positive results are shown in the upper groupand bacterial genera in the lower group
to gain insight into how the microbial gut composition of thebeetle families related to one anotherThis approach clusteredthe environments according to Bray-Curtis distances of
Figure 1 Heatmap of the abundance distribution of fungal com-munities associated with the guts of five wood-feeding familiesof Coleoptera The taxonomic relationship of the fungal genera isshown in the rows while the clustering of the coleopteran familiesdetermined by their composition similarities is shown in thecolumns Higher intensities of the color reveal higher abundancesof the isolates
the abundance distribution of the isolates considering alsotheir phylogenetic relationships The results showed that thefungal composition of the isolates associated with larvaeof Cerambycidae Scarabaeidae and Passalidae clusteredtogether Cerambycidae and Passalidae shared one orderof Basidiomycota and three orders of Ascomycota whileScarabaeidae and Passalidae had in common four ordersof Ascomycota A second cluster was formed by the fungalmicrobiotas isolated fromTenebrionidae and Elateridae theyshared two orders of Ascomycota and one of Basidiomycota(Figure 1) The analysis of the bacterial dataset showed thatthe microbial compositions associated with Scarabaeidaeand Passalidae formed part of the same cluster sharingisolates belonging to 120573- and 120574-Proteobacteria Actinobac-teria and Firmicutes The second cluster was formed byTenebrionidae Elateridae andCerambycidae that shared iso-lates assigned to Pseudomonadales Enterobacteriales andBacillales (Figure 2) In addition we performed canonicalcorrespondence analysis for exploring relationships betweenthe microbial communities of the coleopteran hosts Resultsof this analysis where consistent with results obtained withthe Bray-Curtis clustering for both the fungal and bacterialcommunities (Figure S1 in Supplementary Material availableonline at httpdxdoiorg1011552015285018)
4 Discussion
We collected larvae of five families of wood-feedingColeoptera in tropical forests of Costa Rica with the aim ofestimating the species composition of cultivable fungi andbacteria inhabiting their guts and to identify microorganisms
Figure 2 Heatmap of the abundance distribution of bacterialcommunities associated with the guts of five wood-feeding familiesof Coleoptera The taxonomic relationship of the bacterial genera isshown in the rows while the clustering of the coleopteran familiesdetermined by their composition similarities is shown in thecolumns Higher intensities of the color reveal higher abundancesof the isolates
with relevant lignocellulolytic activities The main limitationof this study is that the cultivation-dependent approachbased on artificial media covers only a small proportion ofthe total microbial diversity present in this particular nicheThe positive trade-off of this approach was the identificationof several isolates with lignocellulose-degrading capabilitieswhich can be further used for the respective enzymecharacterization for direct degradation assays on residuesfrom agriculture and forestry for the treatment of industrialeffluents and for bioprospecting novel metabolites withother biotechnological applications Despite the inherentbias of the isolation method our results suggest that gutmicrobiota of wood-feeding tropical beetles presents arelatively high diversity in terms of microbial richnessphylogenetic composition and lignocellulolytic activities
The order Hypocreales represented about 60 of thetotal number of fungal isolates Within this group the genusTrichoderma was the most abundant comprising nearly aquarter of the fungal collection This genus was a commongut inhabitant of beetle larvae regardless of the host familyor the geographic location The reason for this dominanceis not entirely clear however one possible explanation isthat several species belonging to this fungal genus containa number of glycoside hydrolases peroxidases laccasesand phenol oxidases among other enzymes related to thedegradation of lignocellulose materials This feature mightprovide some advantages for using the recalcitrant polymericmaterials passing through the gastrointestinal tract [16 38ndash40]
In addition our data indicate that guts of wood-feedinglarvae were from environments having a high representation
of Hypocreales as also observed in a similar study performedin other locations of Costa Rica [41] This is relevant forbioprospecting purposes since wood-feeding beetles mightconstitute a good source of TrichodermaMetarhiziumMeta-cordyceps Bionectria and other fungal genera known topossess a wide array of biotechnological applications [42ndash45] The remaining orders presented a lower abundanceand in most of the cases were represented by a singlegenus Nevertheless many of the genera showed the abilityto degrade lignocellulose-related hexoses and pentoses asalso shown in other studies [46ndash50] Within the phylumBasidiomycota the genus Trametes showed positive resultsin all the lignocellulolytic assays related to the degradationof structural wood components This white-rot fungus is aknownmodel for studying degradation of lignin in free-livingconditions and in this work reported in its association withthe gut microbiota of wood-feeding insects [51 52]
It is difficult to know whether these fungal isolates aretruly endosymbionts of the intestinal tracts of the coleopteranlarvae or are transitory inhabitants associated with hostfeeding habits Hence it is also possible that some ofthese microorganisms could be commensals parasites andfacultative endosymbionts They might even be using theinsect as a dispersal mechanism [15 53] It is clear howeverthat the overall taxonomic composition of the gut-inhabitingmicrobes and the proportion of lignocellulolytic-positivefungi seem to be particular to the larval microenvironmentThe structure of this endosymbiotic community is distin-guished from the fungal composition observed in otherwood-related microhabitats such as the fungal populationsin living plant tissues They are also dominated by membersof Ascomycetes but they present a different abundancedistribution of fungal families [54 55] decaying logs aredominated mainly by Basidiomycetes [56ndash58]
The analysis of the taxonomic composition of the bacte-rial isolates showed the presence of seven major phylogeneticclasses codominated by 120574-Proteobacteria and FirmicutesThis finding is consistent with results obtained in similarstudies [6 13 14 44 59] Within the 120574-Proteobacteria themost abundant genera were Enterobacter Serratia Acineto-bacter and Pseudomonas Interestingly Serratia and Pseu-domonas were isolated from all five coleopteran familiesstudied Enterobacter and Acinetobacter were present in fourout of the five insect families and they exhibited positiveresults in the lignocellulolytic assays except for lignin degra-dation Similar characteristics related to the degradation oflignocellulose and to fermentativemetabolismwere observedin Bacillus the most abundant genus within Firmicutes [1160] Together these results support the notion that somespecies of fungi and bacteria such as Trichoderma SerratiaPseudomonas and Bacillus can be common gut inhabitantsof wood-feeding larvae in tropical forests suggesting thatcertain affinities might have developed between the beetlehost and its microbiota [41 61ndash64]
When comparing the fungal and bacterial species compo-sition among the beetle families the plots of the Bray-Curtisdistances and canonical correspondence analyses producedbiologically meaningful clusters to group the environmentsthat share similar microbial compositions The first fungal
International Journal of Microbiology 9
cluster relates the microbiota associated with the guts ofCerambycidae Passalidae and Scarabaeidae This is con-sistent with the observation of a high diversity of isolatesfrom Cerambycidae that shared members of the fungalphyla Basidiomycota and Ascomycota with Passalidae andmembers of Zygomycota and Ascomycota with ScarabaeidaeThe cluster formed by Tenebrionidae-Elateridae shared ina lower proportion members of the Basidiomycota andAscomycotaThe bacterial microbiota associated with Passal-idae and Scarabaeidae also formed a cluster sharingmembersof five major bacterial clades microbiota of CerambycidaeElateridae and Tenebrionidae shared members only of 120574-Proteobacteria and Firmicutes
The clustering analyses revealed that Cerambycidae pre-sented a high diversity of fungi but not of bacteria whilePassalidae and Elateridae exhibited a high diversity of bac-teria and moderate diversity of fungi Scarabaeidae andTenebrionidae contained a similar composition of bothThese results suggest that the nature of the beetle hosthas an important effect on the phylogenetic diversity of itsassociated microbiota and that many factors can influenceits configuration These factors may include the biology ofthe host the physical and chemical characteristics of thegut compartments the feeding habits of the insects and themicrobial diversity associated with the environment in whichthe insect is living [23 26 65 66]
Our results consistently showed that both the fungaland bacterial populations associated with the guts of beetlelarvae are highly diverse in terms of the number of speciesobtained and in their phylogenetic composition Thesemicrobial inhabitants could be forming complex consortiathat would be acting synergistically to provide many of thenutritional needs of the beetle host Some of these functionsinclude the degradation and fermentation of lignocellulosicmaterials as shown by the high percentage of fungal andbacterial genera that presented positive activities or by theproduction of proteins and other metabolites necessary forthe development of the insect [25 44 67ndash69] Furthermorecertain affinities for substrates can be expected according tothe nature of the gut inhabitant For example members ofthe Basidiomycota could possibly degrade larger polymericmolecules the Ascomycota deplete diverse lignocellulosicconstituents while the bacteria degrade and ferment thesmaller monomeric and dimeric hexoses and pentoses pro-duced by the fungal counterparts The bacteria also likelyuse these sugars to produce other nutrients and metabo-lites Consequently the present work raises new lines ofinvestigation concerning the existence of microbial consortiaacting synergistically to provide the nutritional needs of thehosts the nature of the ecological and evolutionary processesthat contribute to ensure the fitness of the insect and themechanisms that rule the interactions among the fungi thebacteria and the beetle host
Conflict of Interests
The authors declared that there is no conflict of interestsregarding this paper
Acknowledgments
The authors acknowledge the contribution of the follow-ing people during the development of this project LuisGuillermoAcosta for the field sampling and early insect iden-tification Angel Solis Carlos Hernandez and Elena Ulate forthe identification of some adult specimens Jorge Blanco forthe fungal isolation Angelica Acuna and Beatriz Rivera fortheDNA extraction and enzymatic assaysManuel Ferrer andCesar Mateo for their advices on the lignocellulolytic activitydetermination Ana Lorena Guevara and Giselle Tamayofor the overall support the editor and reviewers of thisjournal for critical comments on the paperThis research wasfunded by the support of the National Council of Science andTechnology (CONICIT FV-027-2007) the CSIC and CRUSAFoundation (2007 CR0034) and Florida Ice amp FarmCoTheythank ACLAC ACOPAC ACOSA ACTo ACLAP ACCVCACAT ACAHN ACG and ACLAC National ConservationAreas and CONAGEBIO for granting the sample collectingpermits (R-CM-INBio-40-2008-OT R-CM-INBio-48-2008-OT)
References
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[2] P Beguin ldquoMolecular biology of cellulose degradationrdquo AnnualReview of Microbiology vol 44 pp 219ndash248 1990
[3] J Perez J Munoz-Dorado T de la Rubia and J MartınezldquoBiodegradation and biological treatments of cellulose hemi-cellulose and lignin an overviewrdquo International Microbiologyvol 5 no 2 pp 53ndash63 2002
[4] M R Berenbaum and T Eisner ldquoEcology Bugsrsquo bugsrdquo Sciencevol 322 no 5898 pp 52ndash53 2008
[5] Z Zhang ldquoPhylum Arthropoda von Siebold 1948 In animalbiodiversity an outline of higher-level classification and surveyof taxonomic richnessrdquo Zootaxa vol 3148 pp 99ndash103 1948
[6] J Morales-Jimenez G Zuniga L Villa-Tanaca and CHernandez-Rodrıguez ldquoBacterial community and nitrogenfixation in the red turpentine beetle Dendroctonus valensLeConte (Coleoptera Curculionidae Scolytinae)rdquo MicrobialEcology vol 58 no 4 pp 879ndash891 2009
[7] S M Geib M del Mar Jimenez-Gasco J E Carlson M TienR Jabbour and K Hoover ldquoMicrobial community profilingto investigate transmission of bacteria between life stages ofthe wood-boring beetle Anoplophora glabripennisrdquo MicrobialEcology vol 58 no 1 pp 199ndash211 2009
[8] P Engel and N A Moran ldquoThe gut microbiota of insectsmdashdiversity in structure and functionrdquo FEMS MicrobiologyReviews vol 37 no 5 pp 699ndash735 2013
[9] W Shi S Xie X Chen et al ldquoComparative genomic anal-ysis of the microbiome of herbivorous insects reveals eco-environmental adaptations biotechnology applicationsrdquo PLoSGenetics vol 9 no 1 Article ID e1003131 2013
[10] I Hanski and Y Cambefort Dung Beetle Ecology PrincetonUniversity Press Princeton NJ USA 1991
[11] M Egert B Wagner T Lemke A Brune and M W FriedrichldquoMicrobial community structure in midgut and hindgut ofthe humus-feeding larva of Pachnoda ephippiata (Coleoptera
10 International Journal of Microbiology
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[12] R N Coulson ldquoPopulation dynamics of bark beetlesrdquo AnnualReview of Entomology vol 24 no 1 pp 417ndash447 1979
[13] P D Schloss I Delalibera Jr J Handelsman and K F RaffaldquoBacteria associated with the guts of two wood-boring beetlesanoplophora glabripennis and Saperda vestita (Cerambycidae)rdquoEnvironmental Entomology vol 35 no 3 pp 625ndash629 2006
[14] A Vasanthakumar J O Handelsman P D Schloss L S Bauerand K F Raffa ldquoGut microbiota of an invasive subcorticalbeetle Agrilus planipennis Fairmaire across various life stagesrdquoEnvironmental Entomology vol 37 no 5 pp 1344ndash1353 2008
[15] J Morales-Jimenez G Zuniga H C Ramırez-Saad and CHernandez-Rodrıguez ldquoGut-associated bacteria throughoutthe life cycle of the bark beetle Dendroctonus rhizophagusThomas and Bright (Curculionidae Scolytinae) and their cellu-lolytic activitiesrdquoMicrobial Ecology vol 64 no 1 pp 268ndash2782012
[16] R Kumar S Singh and O V Singh ldquoBioconversion of lig-nocellulosic biomass biochemical and molecular perspectivesrdquoJournal of IndustrialMicrobiology and Biotechnology vol 35 no5 pp 377ndash391 2008
[17] C Sanchez ldquoLignocellulosic residues biodegradation and bio-conversion by fungirdquo Biotechnology Advances vol 27 no 2 pp185ndash194 2009
[18] E D Scully S M Geib K Hoover et al ldquoMetagenomicprofiling reveals lignocellulose degrading system in a microbialcommunity associated with a wood-feeding beetlerdquo PLoS ONEvol 8 no 9 Article ID e73827 2013
[19] T L Erwin ldquoTropical forest their richness in Coleoptera andother arthropod speciesrdquoThe Coleopterists Bulletin vol 36 no1 pp 74ndash75 1982
[20] Y Basset L Cizek P Cuenoud et al ldquoArthropod diversity in atropical forestrdquo Science vol 338 no 6113 pp 1481ndash1484 2012
[21] A Solis Escarabajos de Costa Rica Las Familias Mas ComunesHeredia Costa Rica Editorial Inbio 1999
[22] D Borror and R White A Field Guide to the Insects HoughtonMifflin Company New York NY USA 1987
[23] M Egert U Stingl L D Bruun B Pommerenke A Bruneand M W Friedrich ldquoStructure and topology of microbialcommunities in the major gut compartments of Melolonthamelolontha larvae (Coleoptera Scarabaeidae)rdquo Applied andEnvironmentalMicrobiology vol 71 no 8 pp 4556ndash4566 2005
[24] S-O Suh J V McHugh D D Pollock and M Blackwell ldquoThebeetle gut a hyperdiverse source of novel yeastsrdquo MycologicalResearch vol 109 no 3 pp 261ndash265 2005
[25] J J Scott D-C Oh M C Yuceer K D Klepzig J Clardy andC R Currie ldquoBacterial protection of beetle-fungusmutualismrdquoScience vol 322 no 5898 p 63 2008
[26] J A Ceja-Navarro N H Nguyen U Karaoz et al ldquoCom-partmentalized microbial composition oxygen gradients andnitrogen fixation in the gut of Odontotaenius disjunctusrdquo TheISME Journal vol 8 no 1 pp 6ndash18 2014
[27] J B Nardi C M Bee L A Miller N H Nguyen S-O Suhand M Blackwell ldquoCommunities of microbes that inhabit thechanging hindgut landscape of a subsocial beetlerdquo ArthropodStructure and Development vol 35 no 1 pp 57ndash68 2006
[28] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982
[29] B RMVyas andH PMolitoris ldquoInvolvement of an extracellu-lar H2O2-dependent ligninolytic activity of the white rot fungus
Pleurotus ostreatus in the decolorization of Remazol brilliantblue Rrdquo Applied and Environmental Microbiology vol 61 no 11pp 3919ndash3927 1995
[30] J-D Bok D A Yernool and D E Eveleigh ldquoPurificationcharacterization and molecular analysis of thermostable cellu-lases CelA andCelB fromThermotoga neapolitanardquoApplied andEnvironmentalMicrobiology vol 64 no 12 pp 4774ndash4781 1998
[31] K M G Machado D R Matheus and V L R BononildquoLigninolytic enzymes production and Remazol Brilliant BlueR decolorization by tropical Brazilian basidiomycetes fungirdquoBrazilian Journal of Microbiology vol 36 no 3 pp 246ndash2522005
[32] G T Howard and B A White ldquoMolecular cloning and expres-sion of cellulase genes fromRuminococcus albus 8 in Escherichiacoli bacteriophage 120582rdquo Applied and Environmental Microbiologyvol 54 no 7 pp 1752ndash1755 1988
[33] N L Glass and G C Donaldson ldquoDevelopment of primersets designed for use with the PCR to amplify conserved genesfrom filamentous ascomycetesrdquo Applied and EnvironmentalMicrobiology vol 61 no 4 pp 1323ndash1330 1995
[34] D Lane ldquo16S23S rRNA sequencingrdquo inNucleic Acid Techniquesin Bacterial Systematics E Stachebrandt and M GoodfellowEds Wiley Chichester UK 1991
[35] Q Wang G M Garrity J M Tiedje and J R Cole ldquoNaıveBayesian classifier for rapid assignment of rRNA sequencesinto the new bacterial taxonomyrdquo Applied and EnvironmentalMicrobiology vol 73 no 16 pp 5261ndash5267 2007
[36] J Oksanen G Blanchet R Kindt et al Vegan CommunityEcology Package 2014
[37] P L Buttigieg and A Ramette ldquoA guide to statistical analysisin microbial ecology a community-focused living review ofmultivariate data analysesrdquo FEMSMicrobiology Ecology vol 90no 3 pp 543ndash550 2014
[38] DMartinez RM Berka B Henrissat et al ldquoGenome sequenc-ing and analysis of the biomass-degrading fungus Trichodermareesei (syn Hypocrea jecorina)rdquo Nature Biotechnology vol 26no 5 pp 553ndash560 2008
[39] E D Scully K Hoover J E Carlson M Tien and S M GeibldquoMidgut transcriptome profiling of Anoplophora glabripennisa lignocellulose degrading cerambycid beetlerdquo BMC Genomicsvol 14 article 850 2013
[40] P K Foreman D Brown L Dankmeyer et al ldquoTranscriptionalregulation of biomass-degrading enzymes in the filamentousfungus Trichoderma reeseirdquoThe Journal of Biological Chemistryvol 278 no 34 pp 31988ndash31997 2003
[41] G Vargas-Asensio A Pinto-Tomas B Rivera et al ldquoUncover-ing the cultivable microbial diversity of costa rican beetles andits ability to break down plant cell wall componentsrdquoPLoSONEvol 9 no 11 Article ID e113303 2014
[42] M P Coughlan ldquoThe properties of fungal and bacterial cel-lulases with comment on their production and applicationrdquoBiotechnology and Genetic Engineering Reviews vol 3 no 1 pp39ndash110 1985
[43] L R Lynd P J Weimer W H Van Zyl and I S PretoriusldquoMicrobial cellulose utilization fundamentals and biotechnol-ogyrdquoMicrobiology andMolecular Biology Reviews vol 66 no 3pp 506ndash577 2002
[44] S M Geib T R Filley P G Hatcher et al ldquoLignin degradationin wood-feeding insectsrdquo Proceedings of the National Academy
International Journal of Microbiology 11
of Sciences of the United States of America vol 105 no 35 pp12932ndash12937 2008
[45] H Alper and G Stephanopoulos ldquoEngineering for biofuelsexploiting innate microbial capacity or importing biosyntheticpotentialrdquo Nature Reviews Microbiology vol 7 no 10 pp 715ndash723 2009
[46] K Yaoi and Y Mitsuishi ldquoPurification characterizationcloning and expression of a novel xyloglucan-specific glycosi-dase oligoxyloglucan reducing end-specific cellobiohydrolaserdquoThe Journal of Biological Chemistry vol 277 no 50 pp 48276ndash48281 2002
[47] L Ayed N Assas S Sayadi and M Hamdi ldquoInvolvementof lignin peroxidase in the decolourization of black olivemill wastewaters by Geotrichum candidumrdquo Letters in AppliedMicrobiology vol 40 no 1 pp 7ndash11 2005
[48] Y Baba A Shimonaka J Koga H Kubota and T KonoldquoAlternative splicing produces two endoglucanases with oneor two carbohydrate-binding modules in Mucor circinelloidesrdquoJournal of Bacteriology vol 187 no 9 pp 3045ndash3051 2005
[49] M Dashtban H Schraft andW Qin ldquoFungal bioconversion oflignocellulosic residues opportunities amp perspectivesrdquo Interna-tional Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009
[50] D A Ribeiro J Cota T M Alvarez et al ldquoThe Penicilliumechinulatum secretome on sugar cane bagasserdquo PLoS ONE vol7 no 12 Article ID e50571 2012
[51] O Borokhov and S Rothenburger ldquoRapid dye decolorizationmethod for screening potential wood preservativesrdquo Appliedand Environmental Microbiology vol 66 no 12 pp 5457ndash54592000
[52] E Abadulla T Tzanov S Costa K-H Robra A Cavaco-Paulo and G M Gubitz ldquoDecolorization and detoxification oftextile dyes with a laccase from Trametes hirsutardquo Applied andEnvironmental Microbiology vol 66 no 8 pp 3357ndash3362 2000
[53] T J Dreaden J M Davis Z W de Beer et al ldquoPhylogenyof ambrosia beetle symbionts in the genus Raffaeleardquo FungalBiology vol 118 no 12 pp 970ndash978 2014
[54] T S Suryanarayanan T S Murali and G Venkatesan ldquoOccur-rence and distribution of fungal endophytes in tropical forestsacross a rainfall gradientrdquo Canadian Journal of Botany vol 80no 8 pp 818ndash826 2002
[55] A E Arnold and F Lutzoni ldquoDiversity and host range of foliarfungal endophytes are tropical leaves biodiversity hotspotsrdquoEcology vol 88 no 3 pp 541ndash549 2007
[56] J Heilmann-Clausen and L Boddy ldquoInhibition and stimulationeffects in communities of wood decay fungi exudates fromcolonized wood influence growth by other speciesrdquo MicrobialEcology vol 49 no 3 pp 399ndash406 2005
[57] A Kubartova E Ottosson A Dahlberg and J Stenlid ldquoPat-terns of fungal communities among and within decaying logsrevealed by 454 sequencingrdquo Molecular Ecology vol 21 no 18pp 4514ndash4532 2012
[58] L Prewitt Y Kang M L Kakumanu andMWilliams ldquoFungaland bacterial community succession differs for three woodtypes during decay in a forest soilrdquo Microbial Ecology vol 68no 2 pp 212ndash221 2014
[59] N M Reid S L Addison L J Macdonald and G Lloyd-JonesldquoBiodiversity of active and inactive bacteria in the gut floraof wood-feeding Huhu beetle larvae (Prionoplus reticularis)rdquoApplied and Environmental Microbiology vol 77 no 19 pp7000ndash7006 2011
[60] CC Lee R E Kibblewhite-AccinelliM R Smith KWagschalW J Orts and DW SWong ldquoCloning of Bacillus licheniformisxylanase gene and characterization of recombinant enzymerdquoCurrent Microbiology vol 57 no 4 pp 301ndash305 2008
[61] T L Rhoads A T Mikell Jr and M H Eley ldquoInvestigation ofthe lignin-degrading activity of Serratia marcescens biochem-ical screening and ultrastructural evidencerdquo Canadian Journalof Microbiology vol 41 no 7 pp 592ndash600 1995
[62] T Nagy K Emami C M G A Fontes L M A FerreiraD R Humphry and H J Gilbert ldquoThe membrane-bound120572-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-O-methyl-D-glucuronoxylooligosaccharides but not 4-O-methyl-D-glucuronoxylanrdquo Journal of Bacteriology vol 184 no 17 pp4925ndash4929 2002
[63] J Weslien L B Djupstrom M Schroeder and O WidenfalkldquoLong-term priority effects among insects and fungi colonizingdecaying woodrdquo The Journal of Animal Ecology vol 80 no 6pp 1155ndash1162 2011
[64] R T Jones L G Sanchez and N Fierer ldquoA cross-taxon analysisof insect-associated bacterial diversityrdquo PLoS ONE vol 8 no 4Article ID e61218 2013
[65] T Lemke U Stingl M Egert M W Friedrich and A BruneldquoPhysicochemical conditions and microbial activities in thehighly alkaline gut of the humus-feeding larva of Pachnodaephippiata (Coleoptera Scarabaeidae)rdquo Applied and Environ-mental Microbiology vol 69 no 11 pp 6650ndash6658 2003
[66] S M Geib M D M Jimenez-Gasco J E Carlson M Tienand K Hoover ldquoEffect of host tree species on cellulase activityand bacterial community composition in the gut of larval Asianlonghorned beetlerdquo Environmental Entomology vol 38 no 3pp 686ndash699 2009
[67] A E Cazemier J C Verdoes F A G Reubsaet J H PHackstein C van der Drift and H J M Op den CampldquoPromicromonospora pachnodae sp nov a member of the(hemi)cellulolytic hindgut flora of larvae of the scarab beetlePachnoda marginatardquo Antonie van Leeuwenhoek vol 83 no 2pp 135ndash148 2003
[68] F A Genta R J Dillon W R Terra and C Ferreira ldquoPotentialrole for gut microbiota in cell wall digestion and glucosidedetoxification in Tenebrio molitor larvaerdquo Journal of InsectPhysiology vol 52 no 6 pp 593ndash601 2006
[69] D-C Oh J J Scott C R Currie and J Clardy ldquoMycangimycina polyene peroxide from a mutualist Streptomyces sprdquo OrganicLetters vol 11 no 3 pp 633ndash636 2009
Table 4 Taxonomic distribution of the bacterial isolates obtained in this study The number of isolates at the phylum and genera level isshown for each of the coleopteran families
Flavobacteria Chryseobacterium 1 1Fusobacteria Sebaldella 1 1Total 18 20 41 46 10 135Cer Cerambycidae Ela Elateridae Pas Passalidae Sca Scarabaeidae and Ten TenebrionidaelowastGenera that presented positive enzymatic activities in more than four pathways
(ie Rhizobium in Hitoy Cerere National Park) others wereassociated with the same beetle family but in different sites(ie Leucobacter with Passalidae) Results of the analysisof unique phylotypes per site and insect family showedthat Hitoy Cerere National Park and Elateridae respectivelypresented the highest percentages of single bacterial isolates
32 Lignocellulolytic Activity Determination Nearly 65 ofthe fungal genera and 48 of the bacterial genera presentedpositive results in at least one of the five lignocellulolyticactivities evaluated with carboxymethylcellulose degrada-tion being the most common activity observed in bothgroups (Table 5) In general fungi showedmore capability for
degrading lignocellulosic materials than bacteria with gen-era such as Trichoderma Bionectria and Trametes showingpositive results in all the assays performed Within bacteriaBacillus Enterobacter and Acinetobacter some of the mostabundant genera isolated from the larval guts tested positivefor four out of the five enzymatic activities assayed cellulase120573-glucosidase 120573-xylanase and cellobiose hydrolase activi-ties However neither these genera nor any other bacterialgroup screened were able to degrade the Remazol BrilliantBluemolecules while 30of the fungal genera tested positivefor this lignin-related degradation activity
33 Comparison of Gut Inhabitants between Families ofColeoptera We performed community analysis with Vegan
International Journal of Microbiology 7
Table 5 Results of the screening for lignocellulolytic activitiesFungal genera with positive results are shown in the upper groupand bacterial genera in the lower group
to gain insight into how the microbial gut composition of thebeetle families related to one anotherThis approach clusteredthe environments according to Bray-Curtis distances of
Figure 1 Heatmap of the abundance distribution of fungal com-munities associated with the guts of five wood-feeding familiesof Coleoptera The taxonomic relationship of the fungal genera isshown in the rows while the clustering of the coleopteran familiesdetermined by their composition similarities is shown in thecolumns Higher intensities of the color reveal higher abundancesof the isolates
the abundance distribution of the isolates considering alsotheir phylogenetic relationships The results showed that thefungal composition of the isolates associated with larvaeof Cerambycidae Scarabaeidae and Passalidae clusteredtogether Cerambycidae and Passalidae shared one orderof Basidiomycota and three orders of Ascomycota whileScarabaeidae and Passalidae had in common four ordersof Ascomycota A second cluster was formed by the fungalmicrobiotas isolated fromTenebrionidae and Elateridae theyshared two orders of Ascomycota and one of Basidiomycota(Figure 1) The analysis of the bacterial dataset showed thatthe microbial compositions associated with Scarabaeidaeand Passalidae formed part of the same cluster sharingisolates belonging to 120573- and 120574-Proteobacteria Actinobac-teria and Firmicutes The second cluster was formed byTenebrionidae Elateridae andCerambycidae that shared iso-lates assigned to Pseudomonadales Enterobacteriales andBacillales (Figure 2) In addition we performed canonicalcorrespondence analysis for exploring relationships betweenthe microbial communities of the coleopteran hosts Resultsof this analysis where consistent with results obtained withthe Bray-Curtis clustering for both the fungal and bacterialcommunities (Figure S1 in Supplementary Material availableonline at httpdxdoiorg1011552015285018)
4 Discussion
We collected larvae of five families of wood-feedingColeoptera in tropical forests of Costa Rica with the aim ofestimating the species composition of cultivable fungi andbacteria inhabiting their guts and to identify microorganisms
Figure 2 Heatmap of the abundance distribution of bacterialcommunities associated with the guts of five wood-feeding familiesof Coleoptera The taxonomic relationship of the bacterial genera isshown in the rows while the clustering of the coleopteran familiesdetermined by their composition similarities is shown in thecolumns Higher intensities of the color reveal higher abundancesof the isolates
with relevant lignocellulolytic activities The main limitationof this study is that the cultivation-dependent approachbased on artificial media covers only a small proportion ofthe total microbial diversity present in this particular nicheThe positive trade-off of this approach was the identificationof several isolates with lignocellulose-degrading capabilitieswhich can be further used for the respective enzymecharacterization for direct degradation assays on residuesfrom agriculture and forestry for the treatment of industrialeffluents and for bioprospecting novel metabolites withother biotechnological applications Despite the inherentbias of the isolation method our results suggest that gutmicrobiota of wood-feeding tropical beetles presents arelatively high diversity in terms of microbial richnessphylogenetic composition and lignocellulolytic activities
The order Hypocreales represented about 60 of thetotal number of fungal isolates Within this group the genusTrichoderma was the most abundant comprising nearly aquarter of the fungal collection This genus was a commongut inhabitant of beetle larvae regardless of the host familyor the geographic location The reason for this dominanceis not entirely clear however one possible explanation isthat several species belonging to this fungal genus containa number of glycoside hydrolases peroxidases laccasesand phenol oxidases among other enzymes related to thedegradation of lignocellulose materials This feature mightprovide some advantages for using the recalcitrant polymericmaterials passing through the gastrointestinal tract [16 38ndash40]
In addition our data indicate that guts of wood-feedinglarvae were from environments having a high representation
of Hypocreales as also observed in a similar study performedin other locations of Costa Rica [41] This is relevant forbioprospecting purposes since wood-feeding beetles mightconstitute a good source of TrichodermaMetarhiziumMeta-cordyceps Bionectria and other fungal genera known topossess a wide array of biotechnological applications [42ndash45] The remaining orders presented a lower abundanceand in most of the cases were represented by a singlegenus Nevertheless many of the genera showed the abilityto degrade lignocellulose-related hexoses and pentoses asalso shown in other studies [46ndash50] Within the phylumBasidiomycota the genus Trametes showed positive resultsin all the lignocellulolytic assays related to the degradationof structural wood components This white-rot fungus is aknownmodel for studying degradation of lignin in free-livingconditions and in this work reported in its association withthe gut microbiota of wood-feeding insects [51 52]
It is difficult to know whether these fungal isolates aretruly endosymbionts of the intestinal tracts of the coleopteranlarvae or are transitory inhabitants associated with hostfeeding habits Hence it is also possible that some ofthese microorganisms could be commensals parasites andfacultative endosymbionts They might even be using theinsect as a dispersal mechanism [15 53] It is clear howeverthat the overall taxonomic composition of the gut-inhabitingmicrobes and the proportion of lignocellulolytic-positivefungi seem to be particular to the larval microenvironmentThe structure of this endosymbiotic community is distin-guished from the fungal composition observed in otherwood-related microhabitats such as the fungal populationsin living plant tissues They are also dominated by membersof Ascomycetes but they present a different abundancedistribution of fungal families [54 55] decaying logs aredominated mainly by Basidiomycetes [56ndash58]
The analysis of the taxonomic composition of the bacte-rial isolates showed the presence of seven major phylogeneticclasses codominated by 120574-Proteobacteria and FirmicutesThis finding is consistent with results obtained in similarstudies [6 13 14 44 59] Within the 120574-Proteobacteria themost abundant genera were Enterobacter Serratia Acineto-bacter and Pseudomonas Interestingly Serratia and Pseu-domonas were isolated from all five coleopteran familiesstudied Enterobacter and Acinetobacter were present in fourout of the five insect families and they exhibited positiveresults in the lignocellulolytic assays except for lignin degra-dation Similar characteristics related to the degradation oflignocellulose and to fermentativemetabolismwere observedin Bacillus the most abundant genus within Firmicutes [1160] Together these results support the notion that somespecies of fungi and bacteria such as Trichoderma SerratiaPseudomonas and Bacillus can be common gut inhabitantsof wood-feeding larvae in tropical forests suggesting thatcertain affinities might have developed between the beetlehost and its microbiota [41 61ndash64]
When comparing the fungal and bacterial species compo-sition among the beetle families the plots of the Bray-Curtisdistances and canonical correspondence analyses producedbiologically meaningful clusters to group the environmentsthat share similar microbial compositions The first fungal
International Journal of Microbiology 9
cluster relates the microbiota associated with the guts ofCerambycidae Passalidae and Scarabaeidae This is con-sistent with the observation of a high diversity of isolatesfrom Cerambycidae that shared members of the fungalphyla Basidiomycota and Ascomycota with Passalidae andmembers of Zygomycota and Ascomycota with ScarabaeidaeThe cluster formed by Tenebrionidae-Elateridae shared ina lower proportion members of the Basidiomycota andAscomycotaThe bacterial microbiota associated with Passal-idae and Scarabaeidae also formed a cluster sharingmembersof five major bacterial clades microbiota of CerambycidaeElateridae and Tenebrionidae shared members only of 120574-Proteobacteria and Firmicutes
The clustering analyses revealed that Cerambycidae pre-sented a high diversity of fungi but not of bacteria whilePassalidae and Elateridae exhibited a high diversity of bac-teria and moderate diversity of fungi Scarabaeidae andTenebrionidae contained a similar composition of bothThese results suggest that the nature of the beetle hosthas an important effect on the phylogenetic diversity of itsassociated microbiota and that many factors can influenceits configuration These factors may include the biology ofthe host the physical and chemical characteristics of thegut compartments the feeding habits of the insects and themicrobial diversity associated with the environment in whichthe insect is living [23 26 65 66]
Our results consistently showed that both the fungaland bacterial populations associated with the guts of beetlelarvae are highly diverse in terms of the number of speciesobtained and in their phylogenetic composition Thesemicrobial inhabitants could be forming complex consortiathat would be acting synergistically to provide many of thenutritional needs of the beetle host Some of these functionsinclude the degradation and fermentation of lignocellulosicmaterials as shown by the high percentage of fungal andbacterial genera that presented positive activities or by theproduction of proteins and other metabolites necessary forthe development of the insect [25 44 67ndash69] Furthermorecertain affinities for substrates can be expected according tothe nature of the gut inhabitant For example members ofthe Basidiomycota could possibly degrade larger polymericmolecules the Ascomycota deplete diverse lignocellulosicconstituents while the bacteria degrade and ferment thesmaller monomeric and dimeric hexoses and pentoses pro-duced by the fungal counterparts The bacteria also likelyuse these sugars to produce other nutrients and metabo-lites Consequently the present work raises new lines ofinvestigation concerning the existence of microbial consortiaacting synergistically to provide the nutritional needs of thehosts the nature of the ecological and evolutionary processesthat contribute to ensure the fitness of the insect and themechanisms that rule the interactions among the fungi thebacteria and the beetle host
Conflict of Interests
The authors declared that there is no conflict of interestsregarding this paper
Acknowledgments
The authors acknowledge the contribution of the follow-ing people during the development of this project LuisGuillermoAcosta for the field sampling and early insect iden-tification Angel Solis Carlos Hernandez and Elena Ulate forthe identification of some adult specimens Jorge Blanco forthe fungal isolation Angelica Acuna and Beatriz Rivera fortheDNA extraction and enzymatic assaysManuel Ferrer andCesar Mateo for their advices on the lignocellulolytic activitydetermination Ana Lorena Guevara and Giselle Tamayofor the overall support the editor and reviewers of thisjournal for critical comments on the paperThis research wasfunded by the support of the National Council of Science andTechnology (CONICIT FV-027-2007) the CSIC and CRUSAFoundation (2007 CR0034) and Florida Ice amp FarmCoTheythank ACLAC ACOPAC ACOSA ACTo ACLAP ACCVCACAT ACAHN ACG and ACLAC National ConservationAreas and CONAGEBIO for granting the sample collectingpermits (R-CM-INBio-40-2008-OT R-CM-INBio-48-2008-OT)
References
[1] M Tien and C-P D Tu ldquoCloning and sequencing of a cDNAfor a ligninase from Phanerochaete chrysosporiumrdquo Nature vol326 no 6112 pp 520ndash523 1987
[2] P Beguin ldquoMolecular biology of cellulose degradationrdquo AnnualReview of Microbiology vol 44 pp 219ndash248 1990
[3] J Perez J Munoz-Dorado T de la Rubia and J MartınezldquoBiodegradation and biological treatments of cellulose hemi-cellulose and lignin an overviewrdquo International Microbiologyvol 5 no 2 pp 53ndash63 2002
[4] M R Berenbaum and T Eisner ldquoEcology Bugsrsquo bugsrdquo Sciencevol 322 no 5898 pp 52ndash53 2008
[5] Z Zhang ldquoPhylum Arthropoda von Siebold 1948 In animalbiodiversity an outline of higher-level classification and surveyof taxonomic richnessrdquo Zootaxa vol 3148 pp 99ndash103 1948
[6] J Morales-Jimenez G Zuniga L Villa-Tanaca and CHernandez-Rodrıguez ldquoBacterial community and nitrogenfixation in the red turpentine beetle Dendroctonus valensLeConte (Coleoptera Curculionidae Scolytinae)rdquo MicrobialEcology vol 58 no 4 pp 879ndash891 2009
[7] S M Geib M del Mar Jimenez-Gasco J E Carlson M TienR Jabbour and K Hoover ldquoMicrobial community profilingto investigate transmission of bacteria between life stages ofthe wood-boring beetle Anoplophora glabripennisrdquo MicrobialEcology vol 58 no 1 pp 199ndash211 2009
[8] P Engel and N A Moran ldquoThe gut microbiota of insectsmdashdiversity in structure and functionrdquo FEMS MicrobiologyReviews vol 37 no 5 pp 699ndash735 2013
[9] W Shi S Xie X Chen et al ldquoComparative genomic anal-ysis of the microbiome of herbivorous insects reveals eco-environmental adaptations biotechnology applicationsrdquo PLoSGenetics vol 9 no 1 Article ID e1003131 2013
[10] I Hanski and Y Cambefort Dung Beetle Ecology PrincetonUniversity Press Princeton NJ USA 1991
[11] M Egert B Wagner T Lemke A Brune and M W FriedrichldquoMicrobial community structure in midgut and hindgut ofthe humus-feeding larva of Pachnoda ephippiata (Coleoptera
10 International Journal of Microbiology
Scarabaeidae)rdquo Applied and Environmental Microbiology vol69 no 11 pp 6659ndash6668 2003
[12] R N Coulson ldquoPopulation dynamics of bark beetlesrdquo AnnualReview of Entomology vol 24 no 1 pp 417ndash447 1979
[13] P D Schloss I Delalibera Jr J Handelsman and K F RaffaldquoBacteria associated with the guts of two wood-boring beetlesanoplophora glabripennis and Saperda vestita (Cerambycidae)rdquoEnvironmental Entomology vol 35 no 3 pp 625ndash629 2006
[14] A Vasanthakumar J O Handelsman P D Schloss L S Bauerand K F Raffa ldquoGut microbiota of an invasive subcorticalbeetle Agrilus planipennis Fairmaire across various life stagesrdquoEnvironmental Entomology vol 37 no 5 pp 1344ndash1353 2008
[15] J Morales-Jimenez G Zuniga H C Ramırez-Saad and CHernandez-Rodrıguez ldquoGut-associated bacteria throughoutthe life cycle of the bark beetle Dendroctonus rhizophagusThomas and Bright (Curculionidae Scolytinae) and their cellu-lolytic activitiesrdquoMicrobial Ecology vol 64 no 1 pp 268ndash2782012
[16] R Kumar S Singh and O V Singh ldquoBioconversion of lig-nocellulosic biomass biochemical and molecular perspectivesrdquoJournal of IndustrialMicrobiology and Biotechnology vol 35 no5 pp 377ndash391 2008
[17] C Sanchez ldquoLignocellulosic residues biodegradation and bio-conversion by fungirdquo Biotechnology Advances vol 27 no 2 pp185ndash194 2009
[18] E D Scully S M Geib K Hoover et al ldquoMetagenomicprofiling reveals lignocellulose degrading system in a microbialcommunity associated with a wood-feeding beetlerdquo PLoS ONEvol 8 no 9 Article ID e73827 2013
[19] T L Erwin ldquoTropical forest their richness in Coleoptera andother arthropod speciesrdquoThe Coleopterists Bulletin vol 36 no1 pp 74ndash75 1982
[20] Y Basset L Cizek P Cuenoud et al ldquoArthropod diversity in atropical forestrdquo Science vol 338 no 6113 pp 1481ndash1484 2012
[21] A Solis Escarabajos de Costa Rica Las Familias Mas ComunesHeredia Costa Rica Editorial Inbio 1999
[22] D Borror and R White A Field Guide to the Insects HoughtonMifflin Company New York NY USA 1987
[23] M Egert U Stingl L D Bruun B Pommerenke A Bruneand M W Friedrich ldquoStructure and topology of microbialcommunities in the major gut compartments of Melolonthamelolontha larvae (Coleoptera Scarabaeidae)rdquo Applied andEnvironmentalMicrobiology vol 71 no 8 pp 4556ndash4566 2005
[24] S-O Suh J V McHugh D D Pollock and M Blackwell ldquoThebeetle gut a hyperdiverse source of novel yeastsrdquo MycologicalResearch vol 109 no 3 pp 261ndash265 2005
[25] J J Scott D-C Oh M C Yuceer K D Klepzig J Clardy andC R Currie ldquoBacterial protection of beetle-fungusmutualismrdquoScience vol 322 no 5898 p 63 2008
[26] J A Ceja-Navarro N H Nguyen U Karaoz et al ldquoCom-partmentalized microbial composition oxygen gradients andnitrogen fixation in the gut of Odontotaenius disjunctusrdquo TheISME Journal vol 8 no 1 pp 6ndash18 2014
[27] J B Nardi C M Bee L A Miller N H Nguyen S-O Suhand M Blackwell ldquoCommunities of microbes that inhabit thechanging hindgut landscape of a subsocial beetlerdquo ArthropodStructure and Development vol 35 no 1 pp 57ndash68 2006
[28] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982
[29] B RMVyas andH PMolitoris ldquoInvolvement of an extracellu-lar H2O2-dependent ligninolytic activity of the white rot fungus
Pleurotus ostreatus in the decolorization of Remazol brilliantblue Rrdquo Applied and Environmental Microbiology vol 61 no 11pp 3919ndash3927 1995
[30] J-D Bok D A Yernool and D E Eveleigh ldquoPurificationcharacterization and molecular analysis of thermostable cellu-lases CelA andCelB fromThermotoga neapolitanardquoApplied andEnvironmentalMicrobiology vol 64 no 12 pp 4774ndash4781 1998
[31] K M G Machado D R Matheus and V L R BononildquoLigninolytic enzymes production and Remazol Brilliant BlueR decolorization by tropical Brazilian basidiomycetes fungirdquoBrazilian Journal of Microbiology vol 36 no 3 pp 246ndash2522005
[32] G T Howard and B A White ldquoMolecular cloning and expres-sion of cellulase genes fromRuminococcus albus 8 in Escherichiacoli bacteriophage 120582rdquo Applied and Environmental Microbiologyvol 54 no 7 pp 1752ndash1755 1988
[33] N L Glass and G C Donaldson ldquoDevelopment of primersets designed for use with the PCR to amplify conserved genesfrom filamentous ascomycetesrdquo Applied and EnvironmentalMicrobiology vol 61 no 4 pp 1323ndash1330 1995
[34] D Lane ldquo16S23S rRNA sequencingrdquo inNucleic Acid Techniquesin Bacterial Systematics E Stachebrandt and M GoodfellowEds Wiley Chichester UK 1991
[35] Q Wang G M Garrity J M Tiedje and J R Cole ldquoNaıveBayesian classifier for rapid assignment of rRNA sequencesinto the new bacterial taxonomyrdquo Applied and EnvironmentalMicrobiology vol 73 no 16 pp 5261ndash5267 2007
[36] J Oksanen G Blanchet R Kindt et al Vegan CommunityEcology Package 2014
[37] P L Buttigieg and A Ramette ldquoA guide to statistical analysisin microbial ecology a community-focused living review ofmultivariate data analysesrdquo FEMSMicrobiology Ecology vol 90no 3 pp 543ndash550 2014
[38] DMartinez RM Berka B Henrissat et al ldquoGenome sequenc-ing and analysis of the biomass-degrading fungus Trichodermareesei (syn Hypocrea jecorina)rdquo Nature Biotechnology vol 26no 5 pp 553ndash560 2008
[39] E D Scully K Hoover J E Carlson M Tien and S M GeibldquoMidgut transcriptome profiling of Anoplophora glabripennisa lignocellulose degrading cerambycid beetlerdquo BMC Genomicsvol 14 article 850 2013
[40] P K Foreman D Brown L Dankmeyer et al ldquoTranscriptionalregulation of biomass-degrading enzymes in the filamentousfungus Trichoderma reeseirdquoThe Journal of Biological Chemistryvol 278 no 34 pp 31988ndash31997 2003
[41] G Vargas-Asensio A Pinto-Tomas B Rivera et al ldquoUncover-ing the cultivable microbial diversity of costa rican beetles andits ability to break down plant cell wall componentsrdquoPLoSONEvol 9 no 11 Article ID e113303 2014
[42] M P Coughlan ldquoThe properties of fungal and bacterial cel-lulases with comment on their production and applicationrdquoBiotechnology and Genetic Engineering Reviews vol 3 no 1 pp39ndash110 1985
[43] L R Lynd P J Weimer W H Van Zyl and I S PretoriusldquoMicrobial cellulose utilization fundamentals and biotechnol-ogyrdquoMicrobiology andMolecular Biology Reviews vol 66 no 3pp 506ndash577 2002
[44] S M Geib T R Filley P G Hatcher et al ldquoLignin degradationin wood-feeding insectsrdquo Proceedings of the National Academy
International Journal of Microbiology 11
of Sciences of the United States of America vol 105 no 35 pp12932ndash12937 2008
[45] H Alper and G Stephanopoulos ldquoEngineering for biofuelsexploiting innate microbial capacity or importing biosyntheticpotentialrdquo Nature Reviews Microbiology vol 7 no 10 pp 715ndash723 2009
[46] K Yaoi and Y Mitsuishi ldquoPurification characterizationcloning and expression of a novel xyloglucan-specific glycosi-dase oligoxyloglucan reducing end-specific cellobiohydrolaserdquoThe Journal of Biological Chemistry vol 277 no 50 pp 48276ndash48281 2002
[47] L Ayed N Assas S Sayadi and M Hamdi ldquoInvolvementof lignin peroxidase in the decolourization of black olivemill wastewaters by Geotrichum candidumrdquo Letters in AppliedMicrobiology vol 40 no 1 pp 7ndash11 2005
[48] Y Baba A Shimonaka J Koga H Kubota and T KonoldquoAlternative splicing produces two endoglucanases with oneor two carbohydrate-binding modules in Mucor circinelloidesrdquoJournal of Bacteriology vol 187 no 9 pp 3045ndash3051 2005
[49] M Dashtban H Schraft andW Qin ldquoFungal bioconversion oflignocellulosic residues opportunities amp perspectivesrdquo Interna-tional Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009
[50] D A Ribeiro J Cota T M Alvarez et al ldquoThe Penicilliumechinulatum secretome on sugar cane bagasserdquo PLoS ONE vol7 no 12 Article ID e50571 2012
[51] O Borokhov and S Rothenburger ldquoRapid dye decolorizationmethod for screening potential wood preservativesrdquo Appliedand Environmental Microbiology vol 66 no 12 pp 5457ndash54592000
[52] E Abadulla T Tzanov S Costa K-H Robra A Cavaco-Paulo and G M Gubitz ldquoDecolorization and detoxification oftextile dyes with a laccase from Trametes hirsutardquo Applied andEnvironmental Microbiology vol 66 no 8 pp 3357ndash3362 2000
[53] T J Dreaden J M Davis Z W de Beer et al ldquoPhylogenyof ambrosia beetle symbionts in the genus Raffaeleardquo FungalBiology vol 118 no 12 pp 970ndash978 2014
[54] T S Suryanarayanan T S Murali and G Venkatesan ldquoOccur-rence and distribution of fungal endophytes in tropical forestsacross a rainfall gradientrdquo Canadian Journal of Botany vol 80no 8 pp 818ndash826 2002
[55] A E Arnold and F Lutzoni ldquoDiversity and host range of foliarfungal endophytes are tropical leaves biodiversity hotspotsrdquoEcology vol 88 no 3 pp 541ndash549 2007
[56] J Heilmann-Clausen and L Boddy ldquoInhibition and stimulationeffects in communities of wood decay fungi exudates fromcolonized wood influence growth by other speciesrdquo MicrobialEcology vol 49 no 3 pp 399ndash406 2005
[57] A Kubartova E Ottosson A Dahlberg and J Stenlid ldquoPat-terns of fungal communities among and within decaying logsrevealed by 454 sequencingrdquo Molecular Ecology vol 21 no 18pp 4514ndash4532 2012
[58] L Prewitt Y Kang M L Kakumanu andMWilliams ldquoFungaland bacterial community succession differs for three woodtypes during decay in a forest soilrdquo Microbial Ecology vol 68no 2 pp 212ndash221 2014
[59] N M Reid S L Addison L J Macdonald and G Lloyd-JonesldquoBiodiversity of active and inactive bacteria in the gut floraof wood-feeding Huhu beetle larvae (Prionoplus reticularis)rdquoApplied and Environmental Microbiology vol 77 no 19 pp7000ndash7006 2011
[60] CC Lee R E Kibblewhite-AccinelliM R Smith KWagschalW J Orts and DW SWong ldquoCloning of Bacillus licheniformisxylanase gene and characterization of recombinant enzymerdquoCurrent Microbiology vol 57 no 4 pp 301ndash305 2008
[61] T L Rhoads A T Mikell Jr and M H Eley ldquoInvestigation ofthe lignin-degrading activity of Serratia marcescens biochem-ical screening and ultrastructural evidencerdquo Canadian Journalof Microbiology vol 41 no 7 pp 592ndash600 1995
[62] T Nagy K Emami C M G A Fontes L M A FerreiraD R Humphry and H J Gilbert ldquoThe membrane-bound120572-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-O-methyl-D-glucuronoxylooligosaccharides but not 4-O-methyl-D-glucuronoxylanrdquo Journal of Bacteriology vol 184 no 17 pp4925ndash4929 2002
[63] J Weslien L B Djupstrom M Schroeder and O WidenfalkldquoLong-term priority effects among insects and fungi colonizingdecaying woodrdquo The Journal of Animal Ecology vol 80 no 6pp 1155ndash1162 2011
[64] R T Jones L G Sanchez and N Fierer ldquoA cross-taxon analysisof insect-associated bacterial diversityrdquo PLoS ONE vol 8 no 4Article ID e61218 2013
[65] T Lemke U Stingl M Egert M W Friedrich and A BruneldquoPhysicochemical conditions and microbial activities in thehighly alkaline gut of the humus-feeding larva of Pachnodaephippiata (Coleoptera Scarabaeidae)rdquo Applied and Environ-mental Microbiology vol 69 no 11 pp 6650ndash6658 2003
[66] S M Geib M D M Jimenez-Gasco J E Carlson M Tienand K Hoover ldquoEffect of host tree species on cellulase activityand bacterial community composition in the gut of larval Asianlonghorned beetlerdquo Environmental Entomology vol 38 no 3pp 686ndash699 2009
[67] A E Cazemier J C Verdoes F A G Reubsaet J H PHackstein C van der Drift and H J M Op den CampldquoPromicromonospora pachnodae sp nov a member of the(hemi)cellulolytic hindgut flora of larvae of the scarab beetlePachnoda marginatardquo Antonie van Leeuwenhoek vol 83 no 2pp 135ndash148 2003
[68] F A Genta R J Dillon W R Terra and C Ferreira ldquoPotentialrole for gut microbiota in cell wall digestion and glucosidedetoxification in Tenebrio molitor larvaerdquo Journal of InsectPhysiology vol 52 no 6 pp 593ndash601 2006
[69] D-C Oh J J Scott C R Currie and J Clardy ldquoMycangimycina polyene peroxide from a mutualist Streptomyces sprdquo OrganicLetters vol 11 no 3 pp 633ndash636 2009
Table 5 Results of the screening for lignocellulolytic activitiesFungal genera with positive results are shown in the upper groupand bacterial genera in the lower group
to gain insight into how the microbial gut composition of thebeetle families related to one anotherThis approach clusteredthe environments according to Bray-Curtis distances of
Figure 1 Heatmap of the abundance distribution of fungal com-munities associated with the guts of five wood-feeding familiesof Coleoptera The taxonomic relationship of the fungal genera isshown in the rows while the clustering of the coleopteran familiesdetermined by their composition similarities is shown in thecolumns Higher intensities of the color reveal higher abundancesof the isolates
the abundance distribution of the isolates considering alsotheir phylogenetic relationships The results showed that thefungal composition of the isolates associated with larvaeof Cerambycidae Scarabaeidae and Passalidae clusteredtogether Cerambycidae and Passalidae shared one orderof Basidiomycota and three orders of Ascomycota whileScarabaeidae and Passalidae had in common four ordersof Ascomycota A second cluster was formed by the fungalmicrobiotas isolated fromTenebrionidae and Elateridae theyshared two orders of Ascomycota and one of Basidiomycota(Figure 1) The analysis of the bacterial dataset showed thatthe microbial compositions associated with Scarabaeidaeand Passalidae formed part of the same cluster sharingisolates belonging to 120573- and 120574-Proteobacteria Actinobac-teria and Firmicutes The second cluster was formed byTenebrionidae Elateridae andCerambycidae that shared iso-lates assigned to Pseudomonadales Enterobacteriales andBacillales (Figure 2) In addition we performed canonicalcorrespondence analysis for exploring relationships betweenthe microbial communities of the coleopteran hosts Resultsof this analysis where consistent with results obtained withthe Bray-Curtis clustering for both the fungal and bacterialcommunities (Figure S1 in Supplementary Material availableonline at httpdxdoiorg1011552015285018)
4 Discussion
We collected larvae of five families of wood-feedingColeoptera in tropical forests of Costa Rica with the aim ofestimating the species composition of cultivable fungi andbacteria inhabiting their guts and to identify microorganisms
Figure 2 Heatmap of the abundance distribution of bacterialcommunities associated with the guts of five wood-feeding familiesof Coleoptera The taxonomic relationship of the bacterial genera isshown in the rows while the clustering of the coleopteran familiesdetermined by their composition similarities is shown in thecolumns Higher intensities of the color reveal higher abundancesof the isolates
with relevant lignocellulolytic activities The main limitationof this study is that the cultivation-dependent approachbased on artificial media covers only a small proportion ofthe total microbial diversity present in this particular nicheThe positive trade-off of this approach was the identificationof several isolates with lignocellulose-degrading capabilitieswhich can be further used for the respective enzymecharacterization for direct degradation assays on residuesfrom agriculture and forestry for the treatment of industrialeffluents and for bioprospecting novel metabolites withother biotechnological applications Despite the inherentbias of the isolation method our results suggest that gutmicrobiota of wood-feeding tropical beetles presents arelatively high diversity in terms of microbial richnessphylogenetic composition and lignocellulolytic activities
The order Hypocreales represented about 60 of thetotal number of fungal isolates Within this group the genusTrichoderma was the most abundant comprising nearly aquarter of the fungal collection This genus was a commongut inhabitant of beetle larvae regardless of the host familyor the geographic location The reason for this dominanceis not entirely clear however one possible explanation isthat several species belonging to this fungal genus containa number of glycoside hydrolases peroxidases laccasesand phenol oxidases among other enzymes related to thedegradation of lignocellulose materials This feature mightprovide some advantages for using the recalcitrant polymericmaterials passing through the gastrointestinal tract [16 38ndash40]
In addition our data indicate that guts of wood-feedinglarvae were from environments having a high representation
of Hypocreales as also observed in a similar study performedin other locations of Costa Rica [41] This is relevant forbioprospecting purposes since wood-feeding beetles mightconstitute a good source of TrichodermaMetarhiziumMeta-cordyceps Bionectria and other fungal genera known topossess a wide array of biotechnological applications [42ndash45] The remaining orders presented a lower abundanceand in most of the cases were represented by a singlegenus Nevertheless many of the genera showed the abilityto degrade lignocellulose-related hexoses and pentoses asalso shown in other studies [46ndash50] Within the phylumBasidiomycota the genus Trametes showed positive resultsin all the lignocellulolytic assays related to the degradationof structural wood components This white-rot fungus is aknownmodel for studying degradation of lignin in free-livingconditions and in this work reported in its association withthe gut microbiota of wood-feeding insects [51 52]
It is difficult to know whether these fungal isolates aretruly endosymbionts of the intestinal tracts of the coleopteranlarvae or are transitory inhabitants associated with hostfeeding habits Hence it is also possible that some ofthese microorganisms could be commensals parasites andfacultative endosymbionts They might even be using theinsect as a dispersal mechanism [15 53] It is clear howeverthat the overall taxonomic composition of the gut-inhabitingmicrobes and the proportion of lignocellulolytic-positivefungi seem to be particular to the larval microenvironmentThe structure of this endosymbiotic community is distin-guished from the fungal composition observed in otherwood-related microhabitats such as the fungal populationsin living plant tissues They are also dominated by membersof Ascomycetes but they present a different abundancedistribution of fungal families [54 55] decaying logs aredominated mainly by Basidiomycetes [56ndash58]
The analysis of the taxonomic composition of the bacte-rial isolates showed the presence of seven major phylogeneticclasses codominated by 120574-Proteobacteria and FirmicutesThis finding is consistent with results obtained in similarstudies [6 13 14 44 59] Within the 120574-Proteobacteria themost abundant genera were Enterobacter Serratia Acineto-bacter and Pseudomonas Interestingly Serratia and Pseu-domonas were isolated from all five coleopteran familiesstudied Enterobacter and Acinetobacter were present in fourout of the five insect families and they exhibited positiveresults in the lignocellulolytic assays except for lignin degra-dation Similar characteristics related to the degradation oflignocellulose and to fermentativemetabolismwere observedin Bacillus the most abundant genus within Firmicutes [1160] Together these results support the notion that somespecies of fungi and bacteria such as Trichoderma SerratiaPseudomonas and Bacillus can be common gut inhabitantsof wood-feeding larvae in tropical forests suggesting thatcertain affinities might have developed between the beetlehost and its microbiota [41 61ndash64]
When comparing the fungal and bacterial species compo-sition among the beetle families the plots of the Bray-Curtisdistances and canonical correspondence analyses producedbiologically meaningful clusters to group the environmentsthat share similar microbial compositions The first fungal
International Journal of Microbiology 9
cluster relates the microbiota associated with the guts ofCerambycidae Passalidae and Scarabaeidae This is con-sistent with the observation of a high diversity of isolatesfrom Cerambycidae that shared members of the fungalphyla Basidiomycota and Ascomycota with Passalidae andmembers of Zygomycota and Ascomycota with ScarabaeidaeThe cluster formed by Tenebrionidae-Elateridae shared ina lower proportion members of the Basidiomycota andAscomycotaThe bacterial microbiota associated with Passal-idae and Scarabaeidae also formed a cluster sharingmembersof five major bacterial clades microbiota of CerambycidaeElateridae and Tenebrionidae shared members only of 120574-Proteobacteria and Firmicutes
The clustering analyses revealed that Cerambycidae pre-sented a high diversity of fungi but not of bacteria whilePassalidae and Elateridae exhibited a high diversity of bac-teria and moderate diversity of fungi Scarabaeidae andTenebrionidae contained a similar composition of bothThese results suggest that the nature of the beetle hosthas an important effect on the phylogenetic diversity of itsassociated microbiota and that many factors can influenceits configuration These factors may include the biology ofthe host the physical and chemical characteristics of thegut compartments the feeding habits of the insects and themicrobial diversity associated with the environment in whichthe insect is living [23 26 65 66]
Our results consistently showed that both the fungaland bacterial populations associated with the guts of beetlelarvae are highly diverse in terms of the number of speciesobtained and in their phylogenetic composition Thesemicrobial inhabitants could be forming complex consortiathat would be acting synergistically to provide many of thenutritional needs of the beetle host Some of these functionsinclude the degradation and fermentation of lignocellulosicmaterials as shown by the high percentage of fungal andbacterial genera that presented positive activities or by theproduction of proteins and other metabolites necessary forthe development of the insect [25 44 67ndash69] Furthermorecertain affinities for substrates can be expected according tothe nature of the gut inhabitant For example members ofthe Basidiomycota could possibly degrade larger polymericmolecules the Ascomycota deplete diverse lignocellulosicconstituents while the bacteria degrade and ferment thesmaller monomeric and dimeric hexoses and pentoses pro-duced by the fungal counterparts The bacteria also likelyuse these sugars to produce other nutrients and metabo-lites Consequently the present work raises new lines ofinvestigation concerning the existence of microbial consortiaacting synergistically to provide the nutritional needs of thehosts the nature of the ecological and evolutionary processesthat contribute to ensure the fitness of the insect and themechanisms that rule the interactions among the fungi thebacteria and the beetle host
Conflict of Interests
The authors declared that there is no conflict of interestsregarding this paper
Acknowledgments
The authors acknowledge the contribution of the follow-ing people during the development of this project LuisGuillermoAcosta for the field sampling and early insect iden-tification Angel Solis Carlos Hernandez and Elena Ulate forthe identification of some adult specimens Jorge Blanco forthe fungal isolation Angelica Acuna and Beatriz Rivera fortheDNA extraction and enzymatic assaysManuel Ferrer andCesar Mateo for their advices on the lignocellulolytic activitydetermination Ana Lorena Guevara and Giselle Tamayofor the overall support the editor and reviewers of thisjournal for critical comments on the paperThis research wasfunded by the support of the National Council of Science andTechnology (CONICIT FV-027-2007) the CSIC and CRUSAFoundation (2007 CR0034) and Florida Ice amp FarmCoTheythank ACLAC ACOPAC ACOSA ACTo ACLAP ACCVCACAT ACAHN ACG and ACLAC National ConservationAreas and CONAGEBIO for granting the sample collectingpermits (R-CM-INBio-40-2008-OT R-CM-INBio-48-2008-OT)
References
[1] M Tien and C-P D Tu ldquoCloning and sequencing of a cDNAfor a ligninase from Phanerochaete chrysosporiumrdquo Nature vol326 no 6112 pp 520ndash523 1987
[2] P Beguin ldquoMolecular biology of cellulose degradationrdquo AnnualReview of Microbiology vol 44 pp 219ndash248 1990
[3] J Perez J Munoz-Dorado T de la Rubia and J MartınezldquoBiodegradation and biological treatments of cellulose hemi-cellulose and lignin an overviewrdquo International Microbiologyvol 5 no 2 pp 53ndash63 2002
[4] M R Berenbaum and T Eisner ldquoEcology Bugsrsquo bugsrdquo Sciencevol 322 no 5898 pp 52ndash53 2008
[5] Z Zhang ldquoPhylum Arthropoda von Siebold 1948 In animalbiodiversity an outline of higher-level classification and surveyof taxonomic richnessrdquo Zootaxa vol 3148 pp 99ndash103 1948
[6] J Morales-Jimenez G Zuniga L Villa-Tanaca and CHernandez-Rodrıguez ldquoBacterial community and nitrogenfixation in the red turpentine beetle Dendroctonus valensLeConte (Coleoptera Curculionidae Scolytinae)rdquo MicrobialEcology vol 58 no 4 pp 879ndash891 2009
[7] S M Geib M del Mar Jimenez-Gasco J E Carlson M TienR Jabbour and K Hoover ldquoMicrobial community profilingto investigate transmission of bacteria between life stages ofthe wood-boring beetle Anoplophora glabripennisrdquo MicrobialEcology vol 58 no 1 pp 199ndash211 2009
[8] P Engel and N A Moran ldquoThe gut microbiota of insectsmdashdiversity in structure and functionrdquo FEMS MicrobiologyReviews vol 37 no 5 pp 699ndash735 2013
[9] W Shi S Xie X Chen et al ldquoComparative genomic anal-ysis of the microbiome of herbivorous insects reveals eco-environmental adaptations biotechnology applicationsrdquo PLoSGenetics vol 9 no 1 Article ID e1003131 2013
[10] I Hanski and Y Cambefort Dung Beetle Ecology PrincetonUniversity Press Princeton NJ USA 1991
[11] M Egert B Wagner T Lemke A Brune and M W FriedrichldquoMicrobial community structure in midgut and hindgut ofthe humus-feeding larva of Pachnoda ephippiata (Coleoptera
10 International Journal of Microbiology
Scarabaeidae)rdquo Applied and Environmental Microbiology vol69 no 11 pp 6659ndash6668 2003
[12] R N Coulson ldquoPopulation dynamics of bark beetlesrdquo AnnualReview of Entomology vol 24 no 1 pp 417ndash447 1979
[13] P D Schloss I Delalibera Jr J Handelsman and K F RaffaldquoBacteria associated with the guts of two wood-boring beetlesanoplophora glabripennis and Saperda vestita (Cerambycidae)rdquoEnvironmental Entomology vol 35 no 3 pp 625ndash629 2006
[14] A Vasanthakumar J O Handelsman P D Schloss L S Bauerand K F Raffa ldquoGut microbiota of an invasive subcorticalbeetle Agrilus planipennis Fairmaire across various life stagesrdquoEnvironmental Entomology vol 37 no 5 pp 1344ndash1353 2008
[15] J Morales-Jimenez G Zuniga H C Ramırez-Saad and CHernandez-Rodrıguez ldquoGut-associated bacteria throughoutthe life cycle of the bark beetle Dendroctonus rhizophagusThomas and Bright (Curculionidae Scolytinae) and their cellu-lolytic activitiesrdquoMicrobial Ecology vol 64 no 1 pp 268ndash2782012
[16] R Kumar S Singh and O V Singh ldquoBioconversion of lig-nocellulosic biomass biochemical and molecular perspectivesrdquoJournal of IndustrialMicrobiology and Biotechnology vol 35 no5 pp 377ndash391 2008
[17] C Sanchez ldquoLignocellulosic residues biodegradation and bio-conversion by fungirdquo Biotechnology Advances vol 27 no 2 pp185ndash194 2009
[18] E D Scully S M Geib K Hoover et al ldquoMetagenomicprofiling reveals lignocellulose degrading system in a microbialcommunity associated with a wood-feeding beetlerdquo PLoS ONEvol 8 no 9 Article ID e73827 2013
[19] T L Erwin ldquoTropical forest their richness in Coleoptera andother arthropod speciesrdquoThe Coleopterists Bulletin vol 36 no1 pp 74ndash75 1982
[20] Y Basset L Cizek P Cuenoud et al ldquoArthropod diversity in atropical forestrdquo Science vol 338 no 6113 pp 1481ndash1484 2012
[21] A Solis Escarabajos de Costa Rica Las Familias Mas ComunesHeredia Costa Rica Editorial Inbio 1999
[22] D Borror and R White A Field Guide to the Insects HoughtonMifflin Company New York NY USA 1987
[23] M Egert U Stingl L D Bruun B Pommerenke A Bruneand M W Friedrich ldquoStructure and topology of microbialcommunities in the major gut compartments of Melolonthamelolontha larvae (Coleoptera Scarabaeidae)rdquo Applied andEnvironmentalMicrobiology vol 71 no 8 pp 4556ndash4566 2005
[24] S-O Suh J V McHugh D D Pollock and M Blackwell ldquoThebeetle gut a hyperdiverse source of novel yeastsrdquo MycologicalResearch vol 109 no 3 pp 261ndash265 2005
[25] J J Scott D-C Oh M C Yuceer K D Klepzig J Clardy andC R Currie ldquoBacterial protection of beetle-fungusmutualismrdquoScience vol 322 no 5898 p 63 2008
[26] J A Ceja-Navarro N H Nguyen U Karaoz et al ldquoCom-partmentalized microbial composition oxygen gradients andnitrogen fixation in the gut of Odontotaenius disjunctusrdquo TheISME Journal vol 8 no 1 pp 6ndash18 2014
[27] J B Nardi C M Bee L A Miller N H Nguyen S-O Suhand M Blackwell ldquoCommunities of microbes that inhabit thechanging hindgut landscape of a subsocial beetlerdquo ArthropodStructure and Development vol 35 no 1 pp 57ndash68 2006
[28] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982
[29] B RMVyas andH PMolitoris ldquoInvolvement of an extracellu-lar H2O2-dependent ligninolytic activity of the white rot fungus
Pleurotus ostreatus in the decolorization of Remazol brilliantblue Rrdquo Applied and Environmental Microbiology vol 61 no 11pp 3919ndash3927 1995
[30] J-D Bok D A Yernool and D E Eveleigh ldquoPurificationcharacterization and molecular analysis of thermostable cellu-lases CelA andCelB fromThermotoga neapolitanardquoApplied andEnvironmentalMicrobiology vol 64 no 12 pp 4774ndash4781 1998
[31] K M G Machado D R Matheus and V L R BononildquoLigninolytic enzymes production and Remazol Brilliant BlueR decolorization by tropical Brazilian basidiomycetes fungirdquoBrazilian Journal of Microbiology vol 36 no 3 pp 246ndash2522005
[32] G T Howard and B A White ldquoMolecular cloning and expres-sion of cellulase genes fromRuminococcus albus 8 in Escherichiacoli bacteriophage 120582rdquo Applied and Environmental Microbiologyvol 54 no 7 pp 1752ndash1755 1988
[33] N L Glass and G C Donaldson ldquoDevelopment of primersets designed for use with the PCR to amplify conserved genesfrom filamentous ascomycetesrdquo Applied and EnvironmentalMicrobiology vol 61 no 4 pp 1323ndash1330 1995
[34] D Lane ldquo16S23S rRNA sequencingrdquo inNucleic Acid Techniquesin Bacterial Systematics E Stachebrandt and M GoodfellowEds Wiley Chichester UK 1991
[35] Q Wang G M Garrity J M Tiedje and J R Cole ldquoNaıveBayesian classifier for rapid assignment of rRNA sequencesinto the new bacterial taxonomyrdquo Applied and EnvironmentalMicrobiology vol 73 no 16 pp 5261ndash5267 2007
[36] J Oksanen G Blanchet R Kindt et al Vegan CommunityEcology Package 2014
[37] P L Buttigieg and A Ramette ldquoA guide to statistical analysisin microbial ecology a community-focused living review ofmultivariate data analysesrdquo FEMSMicrobiology Ecology vol 90no 3 pp 543ndash550 2014
[38] DMartinez RM Berka B Henrissat et al ldquoGenome sequenc-ing and analysis of the biomass-degrading fungus Trichodermareesei (syn Hypocrea jecorina)rdquo Nature Biotechnology vol 26no 5 pp 553ndash560 2008
[39] E D Scully K Hoover J E Carlson M Tien and S M GeibldquoMidgut transcriptome profiling of Anoplophora glabripennisa lignocellulose degrading cerambycid beetlerdquo BMC Genomicsvol 14 article 850 2013
[40] P K Foreman D Brown L Dankmeyer et al ldquoTranscriptionalregulation of biomass-degrading enzymes in the filamentousfungus Trichoderma reeseirdquoThe Journal of Biological Chemistryvol 278 no 34 pp 31988ndash31997 2003
[41] G Vargas-Asensio A Pinto-Tomas B Rivera et al ldquoUncover-ing the cultivable microbial diversity of costa rican beetles andits ability to break down plant cell wall componentsrdquoPLoSONEvol 9 no 11 Article ID e113303 2014
[42] M P Coughlan ldquoThe properties of fungal and bacterial cel-lulases with comment on their production and applicationrdquoBiotechnology and Genetic Engineering Reviews vol 3 no 1 pp39ndash110 1985
[43] L R Lynd P J Weimer W H Van Zyl and I S PretoriusldquoMicrobial cellulose utilization fundamentals and biotechnol-ogyrdquoMicrobiology andMolecular Biology Reviews vol 66 no 3pp 506ndash577 2002
[44] S M Geib T R Filley P G Hatcher et al ldquoLignin degradationin wood-feeding insectsrdquo Proceedings of the National Academy
International Journal of Microbiology 11
of Sciences of the United States of America vol 105 no 35 pp12932ndash12937 2008
[45] H Alper and G Stephanopoulos ldquoEngineering for biofuelsexploiting innate microbial capacity or importing biosyntheticpotentialrdquo Nature Reviews Microbiology vol 7 no 10 pp 715ndash723 2009
[46] K Yaoi and Y Mitsuishi ldquoPurification characterizationcloning and expression of a novel xyloglucan-specific glycosi-dase oligoxyloglucan reducing end-specific cellobiohydrolaserdquoThe Journal of Biological Chemistry vol 277 no 50 pp 48276ndash48281 2002
[47] L Ayed N Assas S Sayadi and M Hamdi ldquoInvolvementof lignin peroxidase in the decolourization of black olivemill wastewaters by Geotrichum candidumrdquo Letters in AppliedMicrobiology vol 40 no 1 pp 7ndash11 2005
[48] Y Baba A Shimonaka J Koga H Kubota and T KonoldquoAlternative splicing produces two endoglucanases with oneor two carbohydrate-binding modules in Mucor circinelloidesrdquoJournal of Bacteriology vol 187 no 9 pp 3045ndash3051 2005
[49] M Dashtban H Schraft andW Qin ldquoFungal bioconversion oflignocellulosic residues opportunities amp perspectivesrdquo Interna-tional Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009
[50] D A Ribeiro J Cota T M Alvarez et al ldquoThe Penicilliumechinulatum secretome on sugar cane bagasserdquo PLoS ONE vol7 no 12 Article ID e50571 2012
[51] O Borokhov and S Rothenburger ldquoRapid dye decolorizationmethod for screening potential wood preservativesrdquo Appliedand Environmental Microbiology vol 66 no 12 pp 5457ndash54592000
[52] E Abadulla T Tzanov S Costa K-H Robra A Cavaco-Paulo and G M Gubitz ldquoDecolorization and detoxification oftextile dyes with a laccase from Trametes hirsutardquo Applied andEnvironmental Microbiology vol 66 no 8 pp 3357ndash3362 2000
[53] T J Dreaden J M Davis Z W de Beer et al ldquoPhylogenyof ambrosia beetle symbionts in the genus Raffaeleardquo FungalBiology vol 118 no 12 pp 970ndash978 2014
[54] T S Suryanarayanan T S Murali and G Venkatesan ldquoOccur-rence and distribution of fungal endophytes in tropical forestsacross a rainfall gradientrdquo Canadian Journal of Botany vol 80no 8 pp 818ndash826 2002
[55] A E Arnold and F Lutzoni ldquoDiversity and host range of foliarfungal endophytes are tropical leaves biodiversity hotspotsrdquoEcology vol 88 no 3 pp 541ndash549 2007
[56] J Heilmann-Clausen and L Boddy ldquoInhibition and stimulationeffects in communities of wood decay fungi exudates fromcolonized wood influence growth by other speciesrdquo MicrobialEcology vol 49 no 3 pp 399ndash406 2005
[57] A Kubartova E Ottosson A Dahlberg and J Stenlid ldquoPat-terns of fungal communities among and within decaying logsrevealed by 454 sequencingrdquo Molecular Ecology vol 21 no 18pp 4514ndash4532 2012
[58] L Prewitt Y Kang M L Kakumanu andMWilliams ldquoFungaland bacterial community succession differs for three woodtypes during decay in a forest soilrdquo Microbial Ecology vol 68no 2 pp 212ndash221 2014
[59] N M Reid S L Addison L J Macdonald and G Lloyd-JonesldquoBiodiversity of active and inactive bacteria in the gut floraof wood-feeding Huhu beetle larvae (Prionoplus reticularis)rdquoApplied and Environmental Microbiology vol 77 no 19 pp7000ndash7006 2011
[60] CC Lee R E Kibblewhite-AccinelliM R Smith KWagschalW J Orts and DW SWong ldquoCloning of Bacillus licheniformisxylanase gene and characterization of recombinant enzymerdquoCurrent Microbiology vol 57 no 4 pp 301ndash305 2008
[61] T L Rhoads A T Mikell Jr and M H Eley ldquoInvestigation ofthe lignin-degrading activity of Serratia marcescens biochem-ical screening and ultrastructural evidencerdquo Canadian Journalof Microbiology vol 41 no 7 pp 592ndash600 1995
[62] T Nagy K Emami C M G A Fontes L M A FerreiraD R Humphry and H J Gilbert ldquoThe membrane-bound120572-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-O-methyl-D-glucuronoxylooligosaccharides but not 4-O-methyl-D-glucuronoxylanrdquo Journal of Bacteriology vol 184 no 17 pp4925ndash4929 2002
[63] J Weslien L B Djupstrom M Schroeder and O WidenfalkldquoLong-term priority effects among insects and fungi colonizingdecaying woodrdquo The Journal of Animal Ecology vol 80 no 6pp 1155ndash1162 2011
[64] R T Jones L G Sanchez and N Fierer ldquoA cross-taxon analysisof insect-associated bacterial diversityrdquo PLoS ONE vol 8 no 4Article ID e61218 2013
[65] T Lemke U Stingl M Egert M W Friedrich and A BruneldquoPhysicochemical conditions and microbial activities in thehighly alkaline gut of the humus-feeding larva of Pachnodaephippiata (Coleoptera Scarabaeidae)rdquo Applied and Environ-mental Microbiology vol 69 no 11 pp 6650ndash6658 2003
[66] S M Geib M D M Jimenez-Gasco J E Carlson M Tienand K Hoover ldquoEffect of host tree species on cellulase activityand bacterial community composition in the gut of larval Asianlonghorned beetlerdquo Environmental Entomology vol 38 no 3pp 686ndash699 2009
[67] A E Cazemier J C Verdoes F A G Reubsaet J H PHackstein C van der Drift and H J M Op den CampldquoPromicromonospora pachnodae sp nov a member of the(hemi)cellulolytic hindgut flora of larvae of the scarab beetlePachnoda marginatardquo Antonie van Leeuwenhoek vol 83 no 2pp 135ndash148 2003
[68] F A Genta R J Dillon W R Terra and C Ferreira ldquoPotentialrole for gut microbiota in cell wall digestion and glucosidedetoxification in Tenebrio molitor larvaerdquo Journal of InsectPhysiology vol 52 no 6 pp 593ndash601 2006
[69] D-C Oh J J Scott C R Currie and J Clardy ldquoMycangimycina polyene peroxide from a mutualist Streptomyces sprdquo OrganicLetters vol 11 no 3 pp 633ndash636 2009
Figure 2 Heatmap of the abundance distribution of bacterialcommunities associated with the guts of five wood-feeding familiesof Coleoptera The taxonomic relationship of the bacterial genera isshown in the rows while the clustering of the coleopteran familiesdetermined by their composition similarities is shown in thecolumns Higher intensities of the color reveal higher abundancesof the isolates
with relevant lignocellulolytic activities The main limitationof this study is that the cultivation-dependent approachbased on artificial media covers only a small proportion ofthe total microbial diversity present in this particular nicheThe positive trade-off of this approach was the identificationof several isolates with lignocellulose-degrading capabilitieswhich can be further used for the respective enzymecharacterization for direct degradation assays on residuesfrom agriculture and forestry for the treatment of industrialeffluents and for bioprospecting novel metabolites withother biotechnological applications Despite the inherentbias of the isolation method our results suggest that gutmicrobiota of wood-feeding tropical beetles presents arelatively high diversity in terms of microbial richnessphylogenetic composition and lignocellulolytic activities
The order Hypocreales represented about 60 of thetotal number of fungal isolates Within this group the genusTrichoderma was the most abundant comprising nearly aquarter of the fungal collection This genus was a commongut inhabitant of beetle larvae regardless of the host familyor the geographic location The reason for this dominanceis not entirely clear however one possible explanation isthat several species belonging to this fungal genus containa number of glycoside hydrolases peroxidases laccasesand phenol oxidases among other enzymes related to thedegradation of lignocellulose materials This feature mightprovide some advantages for using the recalcitrant polymericmaterials passing through the gastrointestinal tract [16 38ndash40]
In addition our data indicate that guts of wood-feedinglarvae were from environments having a high representation
of Hypocreales as also observed in a similar study performedin other locations of Costa Rica [41] This is relevant forbioprospecting purposes since wood-feeding beetles mightconstitute a good source of TrichodermaMetarhiziumMeta-cordyceps Bionectria and other fungal genera known topossess a wide array of biotechnological applications [42ndash45] The remaining orders presented a lower abundanceand in most of the cases were represented by a singlegenus Nevertheless many of the genera showed the abilityto degrade lignocellulose-related hexoses and pentoses asalso shown in other studies [46ndash50] Within the phylumBasidiomycota the genus Trametes showed positive resultsin all the lignocellulolytic assays related to the degradationof structural wood components This white-rot fungus is aknownmodel for studying degradation of lignin in free-livingconditions and in this work reported in its association withthe gut microbiota of wood-feeding insects [51 52]
It is difficult to know whether these fungal isolates aretruly endosymbionts of the intestinal tracts of the coleopteranlarvae or are transitory inhabitants associated with hostfeeding habits Hence it is also possible that some ofthese microorganisms could be commensals parasites andfacultative endosymbionts They might even be using theinsect as a dispersal mechanism [15 53] It is clear howeverthat the overall taxonomic composition of the gut-inhabitingmicrobes and the proportion of lignocellulolytic-positivefungi seem to be particular to the larval microenvironmentThe structure of this endosymbiotic community is distin-guished from the fungal composition observed in otherwood-related microhabitats such as the fungal populationsin living plant tissues They are also dominated by membersof Ascomycetes but they present a different abundancedistribution of fungal families [54 55] decaying logs aredominated mainly by Basidiomycetes [56ndash58]
The analysis of the taxonomic composition of the bacte-rial isolates showed the presence of seven major phylogeneticclasses codominated by 120574-Proteobacteria and FirmicutesThis finding is consistent with results obtained in similarstudies [6 13 14 44 59] Within the 120574-Proteobacteria themost abundant genera were Enterobacter Serratia Acineto-bacter and Pseudomonas Interestingly Serratia and Pseu-domonas were isolated from all five coleopteran familiesstudied Enterobacter and Acinetobacter were present in fourout of the five insect families and they exhibited positiveresults in the lignocellulolytic assays except for lignin degra-dation Similar characteristics related to the degradation oflignocellulose and to fermentativemetabolismwere observedin Bacillus the most abundant genus within Firmicutes [1160] Together these results support the notion that somespecies of fungi and bacteria such as Trichoderma SerratiaPseudomonas and Bacillus can be common gut inhabitantsof wood-feeding larvae in tropical forests suggesting thatcertain affinities might have developed between the beetlehost and its microbiota [41 61ndash64]
When comparing the fungal and bacterial species compo-sition among the beetle families the plots of the Bray-Curtisdistances and canonical correspondence analyses producedbiologically meaningful clusters to group the environmentsthat share similar microbial compositions The first fungal
International Journal of Microbiology 9
cluster relates the microbiota associated with the guts ofCerambycidae Passalidae and Scarabaeidae This is con-sistent with the observation of a high diversity of isolatesfrom Cerambycidae that shared members of the fungalphyla Basidiomycota and Ascomycota with Passalidae andmembers of Zygomycota and Ascomycota with ScarabaeidaeThe cluster formed by Tenebrionidae-Elateridae shared ina lower proportion members of the Basidiomycota andAscomycotaThe bacterial microbiota associated with Passal-idae and Scarabaeidae also formed a cluster sharingmembersof five major bacterial clades microbiota of CerambycidaeElateridae and Tenebrionidae shared members only of 120574-Proteobacteria and Firmicutes
The clustering analyses revealed that Cerambycidae pre-sented a high diversity of fungi but not of bacteria whilePassalidae and Elateridae exhibited a high diversity of bac-teria and moderate diversity of fungi Scarabaeidae andTenebrionidae contained a similar composition of bothThese results suggest that the nature of the beetle hosthas an important effect on the phylogenetic diversity of itsassociated microbiota and that many factors can influenceits configuration These factors may include the biology ofthe host the physical and chemical characteristics of thegut compartments the feeding habits of the insects and themicrobial diversity associated with the environment in whichthe insect is living [23 26 65 66]
Our results consistently showed that both the fungaland bacterial populations associated with the guts of beetlelarvae are highly diverse in terms of the number of speciesobtained and in their phylogenetic composition Thesemicrobial inhabitants could be forming complex consortiathat would be acting synergistically to provide many of thenutritional needs of the beetle host Some of these functionsinclude the degradation and fermentation of lignocellulosicmaterials as shown by the high percentage of fungal andbacterial genera that presented positive activities or by theproduction of proteins and other metabolites necessary forthe development of the insect [25 44 67ndash69] Furthermorecertain affinities for substrates can be expected according tothe nature of the gut inhabitant For example members ofthe Basidiomycota could possibly degrade larger polymericmolecules the Ascomycota deplete diverse lignocellulosicconstituents while the bacteria degrade and ferment thesmaller monomeric and dimeric hexoses and pentoses pro-duced by the fungal counterparts The bacteria also likelyuse these sugars to produce other nutrients and metabo-lites Consequently the present work raises new lines ofinvestigation concerning the existence of microbial consortiaacting synergistically to provide the nutritional needs of thehosts the nature of the ecological and evolutionary processesthat contribute to ensure the fitness of the insect and themechanisms that rule the interactions among the fungi thebacteria and the beetle host
Conflict of Interests
The authors declared that there is no conflict of interestsregarding this paper
Acknowledgments
The authors acknowledge the contribution of the follow-ing people during the development of this project LuisGuillermoAcosta for the field sampling and early insect iden-tification Angel Solis Carlos Hernandez and Elena Ulate forthe identification of some adult specimens Jorge Blanco forthe fungal isolation Angelica Acuna and Beatriz Rivera fortheDNA extraction and enzymatic assaysManuel Ferrer andCesar Mateo for their advices on the lignocellulolytic activitydetermination Ana Lorena Guevara and Giselle Tamayofor the overall support the editor and reviewers of thisjournal for critical comments on the paperThis research wasfunded by the support of the National Council of Science andTechnology (CONICIT FV-027-2007) the CSIC and CRUSAFoundation (2007 CR0034) and Florida Ice amp FarmCoTheythank ACLAC ACOPAC ACOSA ACTo ACLAP ACCVCACAT ACAHN ACG and ACLAC National ConservationAreas and CONAGEBIO for granting the sample collectingpermits (R-CM-INBio-40-2008-OT R-CM-INBio-48-2008-OT)
References
[1] M Tien and C-P D Tu ldquoCloning and sequencing of a cDNAfor a ligninase from Phanerochaete chrysosporiumrdquo Nature vol326 no 6112 pp 520ndash523 1987
[2] P Beguin ldquoMolecular biology of cellulose degradationrdquo AnnualReview of Microbiology vol 44 pp 219ndash248 1990
[3] J Perez J Munoz-Dorado T de la Rubia and J MartınezldquoBiodegradation and biological treatments of cellulose hemi-cellulose and lignin an overviewrdquo International Microbiologyvol 5 no 2 pp 53ndash63 2002
[4] M R Berenbaum and T Eisner ldquoEcology Bugsrsquo bugsrdquo Sciencevol 322 no 5898 pp 52ndash53 2008
[5] Z Zhang ldquoPhylum Arthropoda von Siebold 1948 In animalbiodiversity an outline of higher-level classification and surveyof taxonomic richnessrdquo Zootaxa vol 3148 pp 99ndash103 1948
[6] J Morales-Jimenez G Zuniga L Villa-Tanaca and CHernandez-Rodrıguez ldquoBacterial community and nitrogenfixation in the red turpentine beetle Dendroctonus valensLeConte (Coleoptera Curculionidae Scolytinae)rdquo MicrobialEcology vol 58 no 4 pp 879ndash891 2009
[7] S M Geib M del Mar Jimenez-Gasco J E Carlson M TienR Jabbour and K Hoover ldquoMicrobial community profilingto investigate transmission of bacteria between life stages ofthe wood-boring beetle Anoplophora glabripennisrdquo MicrobialEcology vol 58 no 1 pp 199ndash211 2009
[8] P Engel and N A Moran ldquoThe gut microbiota of insectsmdashdiversity in structure and functionrdquo FEMS MicrobiologyReviews vol 37 no 5 pp 699ndash735 2013
[9] W Shi S Xie X Chen et al ldquoComparative genomic anal-ysis of the microbiome of herbivorous insects reveals eco-environmental adaptations biotechnology applicationsrdquo PLoSGenetics vol 9 no 1 Article ID e1003131 2013
[10] I Hanski and Y Cambefort Dung Beetle Ecology PrincetonUniversity Press Princeton NJ USA 1991
[11] M Egert B Wagner T Lemke A Brune and M W FriedrichldquoMicrobial community structure in midgut and hindgut ofthe humus-feeding larva of Pachnoda ephippiata (Coleoptera
10 International Journal of Microbiology
Scarabaeidae)rdquo Applied and Environmental Microbiology vol69 no 11 pp 6659ndash6668 2003
[12] R N Coulson ldquoPopulation dynamics of bark beetlesrdquo AnnualReview of Entomology vol 24 no 1 pp 417ndash447 1979
[13] P D Schloss I Delalibera Jr J Handelsman and K F RaffaldquoBacteria associated with the guts of two wood-boring beetlesanoplophora glabripennis and Saperda vestita (Cerambycidae)rdquoEnvironmental Entomology vol 35 no 3 pp 625ndash629 2006
[14] A Vasanthakumar J O Handelsman P D Schloss L S Bauerand K F Raffa ldquoGut microbiota of an invasive subcorticalbeetle Agrilus planipennis Fairmaire across various life stagesrdquoEnvironmental Entomology vol 37 no 5 pp 1344ndash1353 2008
[15] J Morales-Jimenez G Zuniga H C Ramırez-Saad and CHernandez-Rodrıguez ldquoGut-associated bacteria throughoutthe life cycle of the bark beetle Dendroctonus rhizophagusThomas and Bright (Curculionidae Scolytinae) and their cellu-lolytic activitiesrdquoMicrobial Ecology vol 64 no 1 pp 268ndash2782012
[16] R Kumar S Singh and O V Singh ldquoBioconversion of lig-nocellulosic biomass biochemical and molecular perspectivesrdquoJournal of IndustrialMicrobiology and Biotechnology vol 35 no5 pp 377ndash391 2008
[17] C Sanchez ldquoLignocellulosic residues biodegradation and bio-conversion by fungirdquo Biotechnology Advances vol 27 no 2 pp185ndash194 2009
[18] E D Scully S M Geib K Hoover et al ldquoMetagenomicprofiling reveals lignocellulose degrading system in a microbialcommunity associated with a wood-feeding beetlerdquo PLoS ONEvol 8 no 9 Article ID e73827 2013
[19] T L Erwin ldquoTropical forest their richness in Coleoptera andother arthropod speciesrdquoThe Coleopterists Bulletin vol 36 no1 pp 74ndash75 1982
[20] Y Basset L Cizek P Cuenoud et al ldquoArthropod diversity in atropical forestrdquo Science vol 338 no 6113 pp 1481ndash1484 2012
[21] A Solis Escarabajos de Costa Rica Las Familias Mas ComunesHeredia Costa Rica Editorial Inbio 1999
[22] D Borror and R White A Field Guide to the Insects HoughtonMifflin Company New York NY USA 1987
[23] M Egert U Stingl L D Bruun B Pommerenke A Bruneand M W Friedrich ldquoStructure and topology of microbialcommunities in the major gut compartments of Melolonthamelolontha larvae (Coleoptera Scarabaeidae)rdquo Applied andEnvironmentalMicrobiology vol 71 no 8 pp 4556ndash4566 2005
[24] S-O Suh J V McHugh D D Pollock and M Blackwell ldquoThebeetle gut a hyperdiverse source of novel yeastsrdquo MycologicalResearch vol 109 no 3 pp 261ndash265 2005
[25] J J Scott D-C Oh M C Yuceer K D Klepzig J Clardy andC R Currie ldquoBacterial protection of beetle-fungusmutualismrdquoScience vol 322 no 5898 p 63 2008
[26] J A Ceja-Navarro N H Nguyen U Karaoz et al ldquoCom-partmentalized microbial composition oxygen gradients andnitrogen fixation in the gut of Odontotaenius disjunctusrdquo TheISME Journal vol 8 no 1 pp 6ndash18 2014
[27] J B Nardi C M Bee L A Miller N H Nguyen S-O Suhand M Blackwell ldquoCommunities of microbes that inhabit thechanging hindgut landscape of a subsocial beetlerdquo ArthropodStructure and Development vol 35 no 1 pp 57ndash68 2006
[28] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982
[29] B RMVyas andH PMolitoris ldquoInvolvement of an extracellu-lar H2O2-dependent ligninolytic activity of the white rot fungus
Pleurotus ostreatus in the decolorization of Remazol brilliantblue Rrdquo Applied and Environmental Microbiology vol 61 no 11pp 3919ndash3927 1995
[30] J-D Bok D A Yernool and D E Eveleigh ldquoPurificationcharacterization and molecular analysis of thermostable cellu-lases CelA andCelB fromThermotoga neapolitanardquoApplied andEnvironmentalMicrobiology vol 64 no 12 pp 4774ndash4781 1998
[31] K M G Machado D R Matheus and V L R BononildquoLigninolytic enzymes production and Remazol Brilliant BlueR decolorization by tropical Brazilian basidiomycetes fungirdquoBrazilian Journal of Microbiology vol 36 no 3 pp 246ndash2522005
[32] G T Howard and B A White ldquoMolecular cloning and expres-sion of cellulase genes fromRuminococcus albus 8 in Escherichiacoli bacteriophage 120582rdquo Applied and Environmental Microbiologyvol 54 no 7 pp 1752ndash1755 1988
[33] N L Glass and G C Donaldson ldquoDevelopment of primersets designed for use with the PCR to amplify conserved genesfrom filamentous ascomycetesrdquo Applied and EnvironmentalMicrobiology vol 61 no 4 pp 1323ndash1330 1995
[34] D Lane ldquo16S23S rRNA sequencingrdquo inNucleic Acid Techniquesin Bacterial Systematics E Stachebrandt and M GoodfellowEds Wiley Chichester UK 1991
[35] Q Wang G M Garrity J M Tiedje and J R Cole ldquoNaıveBayesian classifier for rapid assignment of rRNA sequencesinto the new bacterial taxonomyrdquo Applied and EnvironmentalMicrobiology vol 73 no 16 pp 5261ndash5267 2007
[36] J Oksanen G Blanchet R Kindt et al Vegan CommunityEcology Package 2014
[37] P L Buttigieg and A Ramette ldquoA guide to statistical analysisin microbial ecology a community-focused living review ofmultivariate data analysesrdquo FEMSMicrobiology Ecology vol 90no 3 pp 543ndash550 2014
[38] DMartinez RM Berka B Henrissat et al ldquoGenome sequenc-ing and analysis of the biomass-degrading fungus Trichodermareesei (syn Hypocrea jecorina)rdquo Nature Biotechnology vol 26no 5 pp 553ndash560 2008
[39] E D Scully K Hoover J E Carlson M Tien and S M GeibldquoMidgut transcriptome profiling of Anoplophora glabripennisa lignocellulose degrading cerambycid beetlerdquo BMC Genomicsvol 14 article 850 2013
[40] P K Foreman D Brown L Dankmeyer et al ldquoTranscriptionalregulation of biomass-degrading enzymes in the filamentousfungus Trichoderma reeseirdquoThe Journal of Biological Chemistryvol 278 no 34 pp 31988ndash31997 2003
[41] G Vargas-Asensio A Pinto-Tomas B Rivera et al ldquoUncover-ing the cultivable microbial diversity of costa rican beetles andits ability to break down plant cell wall componentsrdquoPLoSONEvol 9 no 11 Article ID e113303 2014
[42] M P Coughlan ldquoThe properties of fungal and bacterial cel-lulases with comment on their production and applicationrdquoBiotechnology and Genetic Engineering Reviews vol 3 no 1 pp39ndash110 1985
[43] L R Lynd P J Weimer W H Van Zyl and I S PretoriusldquoMicrobial cellulose utilization fundamentals and biotechnol-ogyrdquoMicrobiology andMolecular Biology Reviews vol 66 no 3pp 506ndash577 2002
[44] S M Geib T R Filley P G Hatcher et al ldquoLignin degradationin wood-feeding insectsrdquo Proceedings of the National Academy
International Journal of Microbiology 11
of Sciences of the United States of America vol 105 no 35 pp12932ndash12937 2008
[45] H Alper and G Stephanopoulos ldquoEngineering for biofuelsexploiting innate microbial capacity or importing biosyntheticpotentialrdquo Nature Reviews Microbiology vol 7 no 10 pp 715ndash723 2009
[46] K Yaoi and Y Mitsuishi ldquoPurification characterizationcloning and expression of a novel xyloglucan-specific glycosi-dase oligoxyloglucan reducing end-specific cellobiohydrolaserdquoThe Journal of Biological Chemistry vol 277 no 50 pp 48276ndash48281 2002
[47] L Ayed N Assas S Sayadi and M Hamdi ldquoInvolvementof lignin peroxidase in the decolourization of black olivemill wastewaters by Geotrichum candidumrdquo Letters in AppliedMicrobiology vol 40 no 1 pp 7ndash11 2005
[48] Y Baba A Shimonaka J Koga H Kubota and T KonoldquoAlternative splicing produces two endoglucanases with oneor two carbohydrate-binding modules in Mucor circinelloidesrdquoJournal of Bacteriology vol 187 no 9 pp 3045ndash3051 2005
[49] M Dashtban H Schraft andW Qin ldquoFungal bioconversion oflignocellulosic residues opportunities amp perspectivesrdquo Interna-tional Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009
[50] D A Ribeiro J Cota T M Alvarez et al ldquoThe Penicilliumechinulatum secretome on sugar cane bagasserdquo PLoS ONE vol7 no 12 Article ID e50571 2012
[51] O Borokhov and S Rothenburger ldquoRapid dye decolorizationmethod for screening potential wood preservativesrdquo Appliedand Environmental Microbiology vol 66 no 12 pp 5457ndash54592000
[52] E Abadulla T Tzanov S Costa K-H Robra A Cavaco-Paulo and G M Gubitz ldquoDecolorization and detoxification oftextile dyes with a laccase from Trametes hirsutardquo Applied andEnvironmental Microbiology vol 66 no 8 pp 3357ndash3362 2000
[53] T J Dreaden J M Davis Z W de Beer et al ldquoPhylogenyof ambrosia beetle symbionts in the genus Raffaeleardquo FungalBiology vol 118 no 12 pp 970ndash978 2014
[54] T S Suryanarayanan T S Murali and G Venkatesan ldquoOccur-rence and distribution of fungal endophytes in tropical forestsacross a rainfall gradientrdquo Canadian Journal of Botany vol 80no 8 pp 818ndash826 2002
[55] A E Arnold and F Lutzoni ldquoDiversity and host range of foliarfungal endophytes are tropical leaves biodiversity hotspotsrdquoEcology vol 88 no 3 pp 541ndash549 2007
[56] J Heilmann-Clausen and L Boddy ldquoInhibition and stimulationeffects in communities of wood decay fungi exudates fromcolonized wood influence growth by other speciesrdquo MicrobialEcology vol 49 no 3 pp 399ndash406 2005
[57] A Kubartova E Ottosson A Dahlberg and J Stenlid ldquoPat-terns of fungal communities among and within decaying logsrevealed by 454 sequencingrdquo Molecular Ecology vol 21 no 18pp 4514ndash4532 2012
[58] L Prewitt Y Kang M L Kakumanu andMWilliams ldquoFungaland bacterial community succession differs for three woodtypes during decay in a forest soilrdquo Microbial Ecology vol 68no 2 pp 212ndash221 2014
[59] N M Reid S L Addison L J Macdonald and G Lloyd-JonesldquoBiodiversity of active and inactive bacteria in the gut floraof wood-feeding Huhu beetle larvae (Prionoplus reticularis)rdquoApplied and Environmental Microbiology vol 77 no 19 pp7000ndash7006 2011
[60] CC Lee R E Kibblewhite-AccinelliM R Smith KWagschalW J Orts and DW SWong ldquoCloning of Bacillus licheniformisxylanase gene and characterization of recombinant enzymerdquoCurrent Microbiology vol 57 no 4 pp 301ndash305 2008
[61] T L Rhoads A T Mikell Jr and M H Eley ldquoInvestigation ofthe lignin-degrading activity of Serratia marcescens biochem-ical screening and ultrastructural evidencerdquo Canadian Journalof Microbiology vol 41 no 7 pp 592ndash600 1995
[62] T Nagy K Emami C M G A Fontes L M A FerreiraD R Humphry and H J Gilbert ldquoThe membrane-bound120572-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-O-methyl-D-glucuronoxylooligosaccharides but not 4-O-methyl-D-glucuronoxylanrdquo Journal of Bacteriology vol 184 no 17 pp4925ndash4929 2002
[63] J Weslien L B Djupstrom M Schroeder and O WidenfalkldquoLong-term priority effects among insects and fungi colonizingdecaying woodrdquo The Journal of Animal Ecology vol 80 no 6pp 1155ndash1162 2011
[64] R T Jones L G Sanchez and N Fierer ldquoA cross-taxon analysisof insect-associated bacterial diversityrdquo PLoS ONE vol 8 no 4Article ID e61218 2013
[65] T Lemke U Stingl M Egert M W Friedrich and A BruneldquoPhysicochemical conditions and microbial activities in thehighly alkaline gut of the humus-feeding larva of Pachnodaephippiata (Coleoptera Scarabaeidae)rdquo Applied and Environ-mental Microbiology vol 69 no 11 pp 6650ndash6658 2003
[66] S M Geib M D M Jimenez-Gasco J E Carlson M Tienand K Hoover ldquoEffect of host tree species on cellulase activityand bacterial community composition in the gut of larval Asianlonghorned beetlerdquo Environmental Entomology vol 38 no 3pp 686ndash699 2009
[67] A E Cazemier J C Verdoes F A G Reubsaet J H PHackstein C van der Drift and H J M Op den CampldquoPromicromonospora pachnodae sp nov a member of the(hemi)cellulolytic hindgut flora of larvae of the scarab beetlePachnoda marginatardquo Antonie van Leeuwenhoek vol 83 no 2pp 135ndash148 2003
[68] F A Genta R J Dillon W R Terra and C Ferreira ldquoPotentialrole for gut microbiota in cell wall digestion and glucosidedetoxification in Tenebrio molitor larvaerdquo Journal of InsectPhysiology vol 52 no 6 pp 593ndash601 2006
[69] D-C Oh J J Scott C R Currie and J Clardy ldquoMycangimycina polyene peroxide from a mutualist Streptomyces sprdquo OrganicLetters vol 11 no 3 pp 633ndash636 2009
cluster relates the microbiota associated with the guts ofCerambycidae Passalidae and Scarabaeidae This is con-sistent with the observation of a high diversity of isolatesfrom Cerambycidae that shared members of the fungalphyla Basidiomycota and Ascomycota with Passalidae andmembers of Zygomycota and Ascomycota with ScarabaeidaeThe cluster formed by Tenebrionidae-Elateridae shared ina lower proportion members of the Basidiomycota andAscomycotaThe bacterial microbiota associated with Passal-idae and Scarabaeidae also formed a cluster sharingmembersof five major bacterial clades microbiota of CerambycidaeElateridae and Tenebrionidae shared members only of 120574-Proteobacteria and Firmicutes
The clustering analyses revealed that Cerambycidae pre-sented a high diversity of fungi but not of bacteria whilePassalidae and Elateridae exhibited a high diversity of bac-teria and moderate diversity of fungi Scarabaeidae andTenebrionidae contained a similar composition of bothThese results suggest that the nature of the beetle hosthas an important effect on the phylogenetic diversity of itsassociated microbiota and that many factors can influenceits configuration These factors may include the biology ofthe host the physical and chemical characteristics of thegut compartments the feeding habits of the insects and themicrobial diversity associated with the environment in whichthe insect is living [23 26 65 66]
Our results consistently showed that both the fungaland bacterial populations associated with the guts of beetlelarvae are highly diverse in terms of the number of speciesobtained and in their phylogenetic composition Thesemicrobial inhabitants could be forming complex consortiathat would be acting synergistically to provide many of thenutritional needs of the beetle host Some of these functionsinclude the degradation and fermentation of lignocellulosicmaterials as shown by the high percentage of fungal andbacterial genera that presented positive activities or by theproduction of proteins and other metabolites necessary forthe development of the insect [25 44 67ndash69] Furthermorecertain affinities for substrates can be expected according tothe nature of the gut inhabitant For example members ofthe Basidiomycota could possibly degrade larger polymericmolecules the Ascomycota deplete diverse lignocellulosicconstituents while the bacteria degrade and ferment thesmaller monomeric and dimeric hexoses and pentoses pro-duced by the fungal counterparts The bacteria also likelyuse these sugars to produce other nutrients and metabo-lites Consequently the present work raises new lines ofinvestigation concerning the existence of microbial consortiaacting synergistically to provide the nutritional needs of thehosts the nature of the ecological and evolutionary processesthat contribute to ensure the fitness of the insect and themechanisms that rule the interactions among the fungi thebacteria and the beetle host
Conflict of Interests
The authors declared that there is no conflict of interestsregarding this paper
Acknowledgments
The authors acknowledge the contribution of the follow-ing people during the development of this project LuisGuillermoAcosta for the field sampling and early insect iden-tification Angel Solis Carlos Hernandez and Elena Ulate forthe identification of some adult specimens Jorge Blanco forthe fungal isolation Angelica Acuna and Beatriz Rivera fortheDNA extraction and enzymatic assaysManuel Ferrer andCesar Mateo for their advices on the lignocellulolytic activitydetermination Ana Lorena Guevara and Giselle Tamayofor the overall support the editor and reviewers of thisjournal for critical comments on the paperThis research wasfunded by the support of the National Council of Science andTechnology (CONICIT FV-027-2007) the CSIC and CRUSAFoundation (2007 CR0034) and Florida Ice amp FarmCoTheythank ACLAC ACOPAC ACOSA ACTo ACLAP ACCVCACAT ACAHN ACG and ACLAC National ConservationAreas and CONAGEBIO for granting the sample collectingpermits (R-CM-INBio-40-2008-OT R-CM-INBio-48-2008-OT)
References
[1] M Tien and C-P D Tu ldquoCloning and sequencing of a cDNAfor a ligninase from Phanerochaete chrysosporiumrdquo Nature vol326 no 6112 pp 520ndash523 1987
[2] P Beguin ldquoMolecular biology of cellulose degradationrdquo AnnualReview of Microbiology vol 44 pp 219ndash248 1990
[3] J Perez J Munoz-Dorado T de la Rubia and J MartınezldquoBiodegradation and biological treatments of cellulose hemi-cellulose and lignin an overviewrdquo International Microbiologyvol 5 no 2 pp 53ndash63 2002
[4] M R Berenbaum and T Eisner ldquoEcology Bugsrsquo bugsrdquo Sciencevol 322 no 5898 pp 52ndash53 2008
[5] Z Zhang ldquoPhylum Arthropoda von Siebold 1948 In animalbiodiversity an outline of higher-level classification and surveyof taxonomic richnessrdquo Zootaxa vol 3148 pp 99ndash103 1948
[6] J Morales-Jimenez G Zuniga L Villa-Tanaca and CHernandez-Rodrıguez ldquoBacterial community and nitrogenfixation in the red turpentine beetle Dendroctonus valensLeConte (Coleoptera Curculionidae Scolytinae)rdquo MicrobialEcology vol 58 no 4 pp 879ndash891 2009
[7] S M Geib M del Mar Jimenez-Gasco J E Carlson M TienR Jabbour and K Hoover ldquoMicrobial community profilingto investigate transmission of bacteria between life stages ofthe wood-boring beetle Anoplophora glabripennisrdquo MicrobialEcology vol 58 no 1 pp 199ndash211 2009
[8] P Engel and N A Moran ldquoThe gut microbiota of insectsmdashdiversity in structure and functionrdquo FEMS MicrobiologyReviews vol 37 no 5 pp 699ndash735 2013
[9] W Shi S Xie X Chen et al ldquoComparative genomic anal-ysis of the microbiome of herbivorous insects reveals eco-environmental adaptations biotechnology applicationsrdquo PLoSGenetics vol 9 no 1 Article ID e1003131 2013
[10] I Hanski and Y Cambefort Dung Beetle Ecology PrincetonUniversity Press Princeton NJ USA 1991
[11] M Egert B Wagner T Lemke A Brune and M W FriedrichldquoMicrobial community structure in midgut and hindgut ofthe humus-feeding larva of Pachnoda ephippiata (Coleoptera
10 International Journal of Microbiology
Scarabaeidae)rdquo Applied and Environmental Microbiology vol69 no 11 pp 6659ndash6668 2003
[12] R N Coulson ldquoPopulation dynamics of bark beetlesrdquo AnnualReview of Entomology vol 24 no 1 pp 417ndash447 1979
[13] P D Schloss I Delalibera Jr J Handelsman and K F RaffaldquoBacteria associated with the guts of two wood-boring beetlesanoplophora glabripennis and Saperda vestita (Cerambycidae)rdquoEnvironmental Entomology vol 35 no 3 pp 625ndash629 2006
[14] A Vasanthakumar J O Handelsman P D Schloss L S Bauerand K F Raffa ldquoGut microbiota of an invasive subcorticalbeetle Agrilus planipennis Fairmaire across various life stagesrdquoEnvironmental Entomology vol 37 no 5 pp 1344ndash1353 2008
[15] J Morales-Jimenez G Zuniga H C Ramırez-Saad and CHernandez-Rodrıguez ldquoGut-associated bacteria throughoutthe life cycle of the bark beetle Dendroctonus rhizophagusThomas and Bright (Curculionidae Scolytinae) and their cellu-lolytic activitiesrdquoMicrobial Ecology vol 64 no 1 pp 268ndash2782012
[16] R Kumar S Singh and O V Singh ldquoBioconversion of lig-nocellulosic biomass biochemical and molecular perspectivesrdquoJournal of IndustrialMicrobiology and Biotechnology vol 35 no5 pp 377ndash391 2008
[17] C Sanchez ldquoLignocellulosic residues biodegradation and bio-conversion by fungirdquo Biotechnology Advances vol 27 no 2 pp185ndash194 2009
[18] E D Scully S M Geib K Hoover et al ldquoMetagenomicprofiling reveals lignocellulose degrading system in a microbialcommunity associated with a wood-feeding beetlerdquo PLoS ONEvol 8 no 9 Article ID e73827 2013
[19] T L Erwin ldquoTropical forest their richness in Coleoptera andother arthropod speciesrdquoThe Coleopterists Bulletin vol 36 no1 pp 74ndash75 1982
[20] Y Basset L Cizek P Cuenoud et al ldquoArthropod diversity in atropical forestrdquo Science vol 338 no 6113 pp 1481ndash1484 2012
[21] A Solis Escarabajos de Costa Rica Las Familias Mas ComunesHeredia Costa Rica Editorial Inbio 1999
[22] D Borror and R White A Field Guide to the Insects HoughtonMifflin Company New York NY USA 1987
[23] M Egert U Stingl L D Bruun B Pommerenke A Bruneand M W Friedrich ldquoStructure and topology of microbialcommunities in the major gut compartments of Melolonthamelolontha larvae (Coleoptera Scarabaeidae)rdquo Applied andEnvironmentalMicrobiology vol 71 no 8 pp 4556ndash4566 2005
[24] S-O Suh J V McHugh D D Pollock and M Blackwell ldquoThebeetle gut a hyperdiverse source of novel yeastsrdquo MycologicalResearch vol 109 no 3 pp 261ndash265 2005
[25] J J Scott D-C Oh M C Yuceer K D Klepzig J Clardy andC R Currie ldquoBacterial protection of beetle-fungusmutualismrdquoScience vol 322 no 5898 p 63 2008
[26] J A Ceja-Navarro N H Nguyen U Karaoz et al ldquoCom-partmentalized microbial composition oxygen gradients andnitrogen fixation in the gut of Odontotaenius disjunctusrdquo TheISME Journal vol 8 no 1 pp 6ndash18 2014
[27] J B Nardi C M Bee L A Miller N H Nguyen S-O Suhand M Blackwell ldquoCommunities of microbes that inhabit thechanging hindgut landscape of a subsocial beetlerdquo ArthropodStructure and Development vol 35 no 1 pp 57ndash68 2006
[28] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982
[29] B RMVyas andH PMolitoris ldquoInvolvement of an extracellu-lar H2O2-dependent ligninolytic activity of the white rot fungus
Pleurotus ostreatus in the decolorization of Remazol brilliantblue Rrdquo Applied and Environmental Microbiology vol 61 no 11pp 3919ndash3927 1995
[30] J-D Bok D A Yernool and D E Eveleigh ldquoPurificationcharacterization and molecular analysis of thermostable cellu-lases CelA andCelB fromThermotoga neapolitanardquoApplied andEnvironmentalMicrobiology vol 64 no 12 pp 4774ndash4781 1998
[31] K M G Machado D R Matheus and V L R BononildquoLigninolytic enzymes production and Remazol Brilliant BlueR decolorization by tropical Brazilian basidiomycetes fungirdquoBrazilian Journal of Microbiology vol 36 no 3 pp 246ndash2522005
[32] G T Howard and B A White ldquoMolecular cloning and expres-sion of cellulase genes fromRuminococcus albus 8 in Escherichiacoli bacteriophage 120582rdquo Applied and Environmental Microbiologyvol 54 no 7 pp 1752ndash1755 1988
[33] N L Glass and G C Donaldson ldquoDevelopment of primersets designed for use with the PCR to amplify conserved genesfrom filamentous ascomycetesrdquo Applied and EnvironmentalMicrobiology vol 61 no 4 pp 1323ndash1330 1995
[34] D Lane ldquo16S23S rRNA sequencingrdquo inNucleic Acid Techniquesin Bacterial Systematics E Stachebrandt and M GoodfellowEds Wiley Chichester UK 1991
[35] Q Wang G M Garrity J M Tiedje and J R Cole ldquoNaıveBayesian classifier for rapid assignment of rRNA sequencesinto the new bacterial taxonomyrdquo Applied and EnvironmentalMicrobiology vol 73 no 16 pp 5261ndash5267 2007
[36] J Oksanen G Blanchet R Kindt et al Vegan CommunityEcology Package 2014
[37] P L Buttigieg and A Ramette ldquoA guide to statistical analysisin microbial ecology a community-focused living review ofmultivariate data analysesrdquo FEMSMicrobiology Ecology vol 90no 3 pp 543ndash550 2014
[38] DMartinez RM Berka B Henrissat et al ldquoGenome sequenc-ing and analysis of the biomass-degrading fungus Trichodermareesei (syn Hypocrea jecorina)rdquo Nature Biotechnology vol 26no 5 pp 553ndash560 2008
[39] E D Scully K Hoover J E Carlson M Tien and S M GeibldquoMidgut transcriptome profiling of Anoplophora glabripennisa lignocellulose degrading cerambycid beetlerdquo BMC Genomicsvol 14 article 850 2013
[40] P K Foreman D Brown L Dankmeyer et al ldquoTranscriptionalregulation of biomass-degrading enzymes in the filamentousfungus Trichoderma reeseirdquoThe Journal of Biological Chemistryvol 278 no 34 pp 31988ndash31997 2003
[41] G Vargas-Asensio A Pinto-Tomas B Rivera et al ldquoUncover-ing the cultivable microbial diversity of costa rican beetles andits ability to break down plant cell wall componentsrdquoPLoSONEvol 9 no 11 Article ID e113303 2014
[42] M P Coughlan ldquoThe properties of fungal and bacterial cel-lulases with comment on their production and applicationrdquoBiotechnology and Genetic Engineering Reviews vol 3 no 1 pp39ndash110 1985
[43] L R Lynd P J Weimer W H Van Zyl and I S PretoriusldquoMicrobial cellulose utilization fundamentals and biotechnol-ogyrdquoMicrobiology andMolecular Biology Reviews vol 66 no 3pp 506ndash577 2002
[44] S M Geib T R Filley P G Hatcher et al ldquoLignin degradationin wood-feeding insectsrdquo Proceedings of the National Academy
International Journal of Microbiology 11
of Sciences of the United States of America vol 105 no 35 pp12932ndash12937 2008
[45] H Alper and G Stephanopoulos ldquoEngineering for biofuelsexploiting innate microbial capacity or importing biosyntheticpotentialrdquo Nature Reviews Microbiology vol 7 no 10 pp 715ndash723 2009
[46] K Yaoi and Y Mitsuishi ldquoPurification characterizationcloning and expression of a novel xyloglucan-specific glycosi-dase oligoxyloglucan reducing end-specific cellobiohydrolaserdquoThe Journal of Biological Chemistry vol 277 no 50 pp 48276ndash48281 2002
[47] L Ayed N Assas S Sayadi and M Hamdi ldquoInvolvementof lignin peroxidase in the decolourization of black olivemill wastewaters by Geotrichum candidumrdquo Letters in AppliedMicrobiology vol 40 no 1 pp 7ndash11 2005
[48] Y Baba A Shimonaka J Koga H Kubota and T KonoldquoAlternative splicing produces two endoglucanases with oneor two carbohydrate-binding modules in Mucor circinelloidesrdquoJournal of Bacteriology vol 187 no 9 pp 3045ndash3051 2005
[49] M Dashtban H Schraft andW Qin ldquoFungal bioconversion oflignocellulosic residues opportunities amp perspectivesrdquo Interna-tional Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009
[50] D A Ribeiro J Cota T M Alvarez et al ldquoThe Penicilliumechinulatum secretome on sugar cane bagasserdquo PLoS ONE vol7 no 12 Article ID e50571 2012
[51] O Borokhov and S Rothenburger ldquoRapid dye decolorizationmethod for screening potential wood preservativesrdquo Appliedand Environmental Microbiology vol 66 no 12 pp 5457ndash54592000
[52] E Abadulla T Tzanov S Costa K-H Robra A Cavaco-Paulo and G M Gubitz ldquoDecolorization and detoxification oftextile dyes with a laccase from Trametes hirsutardquo Applied andEnvironmental Microbiology vol 66 no 8 pp 3357ndash3362 2000
[53] T J Dreaden J M Davis Z W de Beer et al ldquoPhylogenyof ambrosia beetle symbionts in the genus Raffaeleardquo FungalBiology vol 118 no 12 pp 970ndash978 2014
[54] T S Suryanarayanan T S Murali and G Venkatesan ldquoOccur-rence and distribution of fungal endophytes in tropical forestsacross a rainfall gradientrdquo Canadian Journal of Botany vol 80no 8 pp 818ndash826 2002
[55] A E Arnold and F Lutzoni ldquoDiversity and host range of foliarfungal endophytes are tropical leaves biodiversity hotspotsrdquoEcology vol 88 no 3 pp 541ndash549 2007
[56] J Heilmann-Clausen and L Boddy ldquoInhibition and stimulationeffects in communities of wood decay fungi exudates fromcolonized wood influence growth by other speciesrdquo MicrobialEcology vol 49 no 3 pp 399ndash406 2005
[57] A Kubartova E Ottosson A Dahlberg and J Stenlid ldquoPat-terns of fungal communities among and within decaying logsrevealed by 454 sequencingrdquo Molecular Ecology vol 21 no 18pp 4514ndash4532 2012
[58] L Prewitt Y Kang M L Kakumanu andMWilliams ldquoFungaland bacterial community succession differs for three woodtypes during decay in a forest soilrdquo Microbial Ecology vol 68no 2 pp 212ndash221 2014
[59] N M Reid S L Addison L J Macdonald and G Lloyd-JonesldquoBiodiversity of active and inactive bacteria in the gut floraof wood-feeding Huhu beetle larvae (Prionoplus reticularis)rdquoApplied and Environmental Microbiology vol 77 no 19 pp7000ndash7006 2011
[60] CC Lee R E Kibblewhite-AccinelliM R Smith KWagschalW J Orts and DW SWong ldquoCloning of Bacillus licheniformisxylanase gene and characterization of recombinant enzymerdquoCurrent Microbiology vol 57 no 4 pp 301ndash305 2008
[61] T L Rhoads A T Mikell Jr and M H Eley ldquoInvestigation ofthe lignin-degrading activity of Serratia marcescens biochem-ical screening and ultrastructural evidencerdquo Canadian Journalof Microbiology vol 41 no 7 pp 592ndash600 1995
[62] T Nagy K Emami C M G A Fontes L M A FerreiraD R Humphry and H J Gilbert ldquoThe membrane-bound120572-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-O-methyl-D-glucuronoxylooligosaccharides but not 4-O-methyl-D-glucuronoxylanrdquo Journal of Bacteriology vol 184 no 17 pp4925ndash4929 2002
[63] J Weslien L B Djupstrom M Schroeder and O WidenfalkldquoLong-term priority effects among insects and fungi colonizingdecaying woodrdquo The Journal of Animal Ecology vol 80 no 6pp 1155ndash1162 2011
[64] R T Jones L G Sanchez and N Fierer ldquoA cross-taxon analysisof insect-associated bacterial diversityrdquo PLoS ONE vol 8 no 4Article ID e61218 2013
[65] T Lemke U Stingl M Egert M W Friedrich and A BruneldquoPhysicochemical conditions and microbial activities in thehighly alkaline gut of the humus-feeding larva of Pachnodaephippiata (Coleoptera Scarabaeidae)rdquo Applied and Environ-mental Microbiology vol 69 no 11 pp 6650ndash6658 2003
[66] S M Geib M D M Jimenez-Gasco J E Carlson M Tienand K Hoover ldquoEffect of host tree species on cellulase activityand bacterial community composition in the gut of larval Asianlonghorned beetlerdquo Environmental Entomology vol 38 no 3pp 686ndash699 2009
[67] A E Cazemier J C Verdoes F A G Reubsaet J H PHackstein C van der Drift and H J M Op den CampldquoPromicromonospora pachnodae sp nov a member of the(hemi)cellulolytic hindgut flora of larvae of the scarab beetlePachnoda marginatardquo Antonie van Leeuwenhoek vol 83 no 2pp 135ndash148 2003
[68] F A Genta R J Dillon W R Terra and C Ferreira ldquoPotentialrole for gut microbiota in cell wall digestion and glucosidedetoxification in Tenebrio molitor larvaerdquo Journal of InsectPhysiology vol 52 no 6 pp 593ndash601 2006
[69] D-C Oh J J Scott C R Currie and J Clardy ldquoMycangimycina polyene peroxide from a mutualist Streptomyces sprdquo OrganicLetters vol 11 no 3 pp 633ndash636 2009
Scarabaeidae)rdquo Applied and Environmental Microbiology vol69 no 11 pp 6659ndash6668 2003
[12] R N Coulson ldquoPopulation dynamics of bark beetlesrdquo AnnualReview of Entomology vol 24 no 1 pp 417ndash447 1979
[13] P D Schloss I Delalibera Jr J Handelsman and K F RaffaldquoBacteria associated with the guts of two wood-boring beetlesanoplophora glabripennis and Saperda vestita (Cerambycidae)rdquoEnvironmental Entomology vol 35 no 3 pp 625ndash629 2006
[14] A Vasanthakumar J O Handelsman P D Schloss L S Bauerand K F Raffa ldquoGut microbiota of an invasive subcorticalbeetle Agrilus planipennis Fairmaire across various life stagesrdquoEnvironmental Entomology vol 37 no 5 pp 1344ndash1353 2008
[15] J Morales-Jimenez G Zuniga H C Ramırez-Saad and CHernandez-Rodrıguez ldquoGut-associated bacteria throughoutthe life cycle of the bark beetle Dendroctonus rhizophagusThomas and Bright (Curculionidae Scolytinae) and their cellu-lolytic activitiesrdquoMicrobial Ecology vol 64 no 1 pp 268ndash2782012
[16] R Kumar S Singh and O V Singh ldquoBioconversion of lig-nocellulosic biomass biochemical and molecular perspectivesrdquoJournal of IndustrialMicrobiology and Biotechnology vol 35 no5 pp 377ndash391 2008
[17] C Sanchez ldquoLignocellulosic residues biodegradation and bio-conversion by fungirdquo Biotechnology Advances vol 27 no 2 pp185ndash194 2009
[18] E D Scully S M Geib K Hoover et al ldquoMetagenomicprofiling reveals lignocellulose degrading system in a microbialcommunity associated with a wood-feeding beetlerdquo PLoS ONEvol 8 no 9 Article ID e73827 2013
[19] T L Erwin ldquoTropical forest their richness in Coleoptera andother arthropod speciesrdquoThe Coleopterists Bulletin vol 36 no1 pp 74ndash75 1982
[20] Y Basset L Cizek P Cuenoud et al ldquoArthropod diversity in atropical forestrdquo Science vol 338 no 6113 pp 1481ndash1484 2012
[21] A Solis Escarabajos de Costa Rica Las Familias Mas ComunesHeredia Costa Rica Editorial Inbio 1999
[22] D Borror and R White A Field Guide to the Insects HoughtonMifflin Company New York NY USA 1987
[23] M Egert U Stingl L D Bruun B Pommerenke A Bruneand M W Friedrich ldquoStructure and topology of microbialcommunities in the major gut compartments of Melolonthamelolontha larvae (Coleoptera Scarabaeidae)rdquo Applied andEnvironmentalMicrobiology vol 71 no 8 pp 4556ndash4566 2005
[24] S-O Suh J V McHugh D D Pollock and M Blackwell ldquoThebeetle gut a hyperdiverse source of novel yeastsrdquo MycologicalResearch vol 109 no 3 pp 261ndash265 2005
[25] J J Scott D-C Oh M C Yuceer K D Klepzig J Clardy andC R Currie ldquoBacterial protection of beetle-fungusmutualismrdquoScience vol 322 no 5898 p 63 2008
[26] J A Ceja-Navarro N H Nguyen U Karaoz et al ldquoCom-partmentalized microbial composition oxygen gradients andnitrogen fixation in the gut of Odontotaenius disjunctusrdquo TheISME Journal vol 8 no 1 pp 6ndash18 2014
[27] J B Nardi C M Bee L A Miller N H Nguyen S-O Suhand M Blackwell ldquoCommunities of microbes that inhabit thechanging hindgut landscape of a subsocial beetlerdquo ArthropodStructure and Development vol 35 no 1 pp 57ndash68 2006
[28] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982
[29] B RMVyas andH PMolitoris ldquoInvolvement of an extracellu-lar H2O2-dependent ligninolytic activity of the white rot fungus
Pleurotus ostreatus in the decolorization of Remazol brilliantblue Rrdquo Applied and Environmental Microbiology vol 61 no 11pp 3919ndash3927 1995
[30] J-D Bok D A Yernool and D E Eveleigh ldquoPurificationcharacterization and molecular analysis of thermostable cellu-lases CelA andCelB fromThermotoga neapolitanardquoApplied andEnvironmentalMicrobiology vol 64 no 12 pp 4774ndash4781 1998
[31] K M G Machado D R Matheus and V L R BononildquoLigninolytic enzymes production and Remazol Brilliant BlueR decolorization by tropical Brazilian basidiomycetes fungirdquoBrazilian Journal of Microbiology vol 36 no 3 pp 246ndash2522005
[32] G T Howard and B A White ldquoMolecular cloning and expres-sion of cellulase genes fromRuminococcus albus 8 in Escherichiacoli bacteriophage 120582rdquo Applied and Environmental Microbiologyvol 54 no 7 pp 1752ndash1755 1988
[33] N L Glass and G C Donaldson ldquoDevelopment of primersets designed for use with the PCR to amplify conserved genesfrom filamentous ascomycetesrdquo Applied and EnvironmentalMicrobiology vol 61 no 4 pp 1323ndash1330 1995
[34] D Lane ldquo16S23S rRNA sequencingrdquo inNucleic Acid Techniquesin Bacterial Systematics E Stachebrandt and M GoodfellowEds Wiley Chichester UK 1991
[35] Q Wang G M Garrity J M Tiedje and J R Cole ldquoNaıveBayesian classifier for rapid assignment of rRNA sequencesinto the new bacterial taxonomyrdquo Applied and EnvironmentalMicrobiology vol 73 no 16 pp 5261ndash5267 2007
[36] J Oksanen G Blanchet R Kindt et al Vegan CommunityEcology Package 2014
[37] P L Buttigieg and A Ramette ldquoA guide to statistical analysisin microbial ecology a community-focused living review ofmultivariate data analysesrdquo FEMSMicrobiology Ecology vol 90no 3 pp 543ndash550 2014
[38] DMartinez RM Berka B Henrissat et al ldquoGenome sequenc-ing and analysis of the biomass-degrading fungus Trichodermareesei (syn Hypocrea jecorina)rdquo Nature Biotechnology vol 26no 5 pp 553ndash560 2008
[39] E D Scully K Hoover J E Carlson M Tien and S M GeibldquoMidgut transcriptome profiling of Anoplophora glabripennisa lignocellulose degrading cerambycid beetlerdquo BMC Genomicsvol 14 article 850 2013
[40] P K Foreman D Brown L Dankmeyer et al ldquoTranscriptionalregulation of biomass-degrading enzymes in the filamentousfungus Trichoderma reeseirdquoThe Journal of Biological Chemistryvol 278 no 34 pp 31988ndash31997 2003
[41] G Vargas-Asensio A Pinto-Tomas B Rivera et al ldquoUncover-ing the cultivable microbial diversity of costa rican beetles andits ability to break down plant cell wall componentsrdquoPLoSONEvol 9 no 11 Article ID e113303 2014
[42] M P Coughlan ldquoThe properties of fungal and bacterial cel-lulases with comment on their production and applicationrdquoBiotechnology and Genetic Engineering Reviews vol 3 no 1 pp39ndash110 1985
[43] L R Lynd P J Weimer W H Van Zyl and I S PretoriusldquoMicrobial cellulose utilization fundamentals and biotechnol-ogyrdquoMicrobiology andMolecular Biology Reviews vol 66 no 3pp 506ndash577 2002
[44] S M Geib T R Filley P G Hatcher et al ldquoLignin degradationin wood-feeding insectsrdquo Proceedings of the National Academy
International Journal of Microbiology 11
of Sciences of the United States of America vol 105 no 35 pp12932ndash12937 2008
[45] H Alper and G Stephanopoulos ldquoEngineering for biofuelsexploiting innate microbial capacity or importing biosyntheticpotentialrdquo Nature Reviews Microbiology vol 7 no 10 pp 715ndash723 2009
[46] K Yaoi and Y Mitsuishi ldquoPurification characterizationcloning and expression of a novel xyloglucan-specific glycosi-dase oligoxyloglucan reducing end-specific cellobiohydrolaserdquoThe Journal of Biological Chemistry vol 277 no 50 pp 48276ndash48281 2002
[47] L Ayed N Assas S Sayadi and M Hamdi ldquoInvolvementof lignin peroxidase in the decolourization of black olivemill wastewaters by Geotrichum candidumrdquo Letters in AppliedMicrobiology vol 40 no 1 pp 7ndash11 2005
[48] Y Baba A Shimonaka J Koga H Kubota and T KonoldquoAlternative splicing produces two endoglucanases with oneor two carbohydrate-binding modules in Mucor circinelloidesrdquoJournal of Bacteriology vol 187 no 9 pp 3045ndash3051 2005
[49] M Dashtban H Schraft andW Qin ldquoFungal bioconversion oflignocellulosic residues opportunities amp perspectivesrdquo Interna-tional Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009
[50] D A Ribeiro J Cota T M Alvarez et al ldquoThe Penicilliumechinulatum secretome on sugar cane bagasserdquo PLoS ONE vol7 no 12 Article ID e50571 2012
[51] O Borokhov and S Rothenburger ldquoRapid dye decolorizationmethod for screening potential wood preservativesrdquo Appliedand Environmental Microbiology vol 66 no 12 pp 5457ndash54592000
[52] E Abadulla T Tzanov S Costa K-H Robra A Cavaco-Paulo and G M Gubitz ldquoDecolorization and detoxification oftextile dyes with a laccase from Trametes hirsutardquo Applied andEnvironmental Microbiology vol 66 no 8 pp 3357ndash3362 2000
[53] T J Dreaden J M Davis Z W de Beer et al ldquoPhylogenyof ambrosia beetle symbionts in the genus Raffaeleardquo FungalBiology vol 118 no 12 pp 970ndash978 2014
[54] T S Suryanarayanan T S Murali and G Venkatesan ldquoOccur-rence and distribution of fungal endophytes in tropical forestsacross a rainfall gradientrdquo Canadian Journal of Botany vol 80no 8 pp 818ndash826 2002
[55] A E Arnold and F Lutzoni ldquoDiversity and host range of foliarfungal endophytes are tropical leaves biodiversity hotspotsrdquoEcology vol 88 no 3 pp 541ndash549 2007
[56] J Heilmann-Clausen and L Boddy ldquoInhibition and stimulationeffects in communities of wood decay fungi exudates fromcolonized wood influence growth by other speciesrdquo MicrobialEcology vol 49 no 3 pp 399ndash406 2005
[57] A Kubartova E Ottosson A Dahlberg and J Stenlid ldquoPat-terns of fungal communities among and within decaying logsrevealed by 454 sequencingrdquo Molecular Ecology vol 21 no 18pp 4514ndash4532 2012
[58] L Prewitt Y Kang M L Kakumanu andMWilliams ldquoFungaland bacterial community succession differs for three woodtypes during decay in a forest soilrdquo Microbial Ecology vol 68no 2 pp 212ndash221 2014
[59] N M Reid S L Addison L J Macdonald and G Lloyd-JonesldquoBiodiversity of active and inactive bacteria in the gut floraof wood-feeding Huhu beetle larvae (Prionoplus reticularis)rdquoApplied and Environmental Microbiology vol 77 no 19 pp7000ndash7006 2011
[60] CC Lee R E Kibblewhite-AccinelliM R Smith KWagschalW J Orts and DW SWong ldquoCloning of Bacillus licheniformisxylanase gene and characterization of recombinant enzymerdquoCurrent Microbiology vol 57 no 4 pp 301ndash305 2008
[61] T L Rhoads A T Mikell Jr and M H Eley ldquoInvestigation ofthe lignin-degrading activity of Serratia marcescens biochem-ical screening and ultrastructural evidencerdquo Canadian Journalof Microbiology vol 41 no 7 pp 592ndash600 1995
[62] T Nagy K Emami C M G A Fontes L M A FerreiraD R Humphry and H J Gilbert ldquoThe membrane-bound120572-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-O-methyl-D-glucuronoxylooligosaccharides but not 4-O-methyl-D-glucuronoxylanrdquo Journal of Bacteriology vol 184 no 17 pp4925ndash4929 2002
[63] J Weslien L B Djupstrom M Schroeder and O WidenfalkldquoLong-term priority effects among insects and fungi colonizingdecaying woodrdquo The Journal of Animal Ecology vol 80 no 6pp 1155ndash1162 2011
[64] R T Jones L G Sanchez and N Fierer ldquoA cross-taxon analysisof insect-associated bacterial diversityrdquo PLoS ONE vol 8 no 4Article ID e61218 2013
[65] T Lemke U Stingl M Egert M W Friedrich and A BruneldquoPhysicochemical conditions and microbial activities in thehighly alkaline gut of the humus-feeding larva of Pachnodaephippiata (Coleoptera Scarabaeidae)rdquo Applied and Environ-mental Microbiology vol 69 no 11 pp 6650ndash6658 2003
[66] S M Geib M D M Jimenez-Gasco J E Carlson M Tienand K Hoover ldquoEffect of host tree species on cellulase activityand bacterial community composition in the gut of larval Asianlonghorned beetlerdquo Environmental Entomology vol 38 no 3pp 686ndash699 2009
[67] A E Cazemier J C Verdoes F A G Reubsaet J H PHackstein C van der Drift and H J M Op den CampldquoPromicromonospora pachnodae sp nov a member of the(hemi)cellulolytic hindgut flora of larvae of the scarab beetlePachnoda marginatardquo Antonie van Leeuwenhoek vol 83 no 2pp 135ndash148 2003
[68] F A Genta R J Dillon W R Terra and C Ferreira ldquoPotentialrole for gut microbiota in cell wall digestion and glucosidedetoxification in Tenebrio molitor larvaerdquo Journal of InsectPhysiology vol 52 no 6 pp 593ndash601 2006
[69] D-C Oh J J Scott C R Currie and J Clardy ldquoMycangimycina polyene peroxide from a mutualist Streptomyces sprdquo OrganicLetters vol 11 no 3 pp 633ndash636 2009
of Sciences of the United States of America vol 105 no 35 pp12932ndash12937 2008
[45] H Alper and G Stephanopoulos ldquoEngineering for biofuelsexploiting innate microbial capacity or importing biosyntheticpotentialrdquo Nature Reviews Microbiology vol 7 no 10 pp 715ndash723 2009
[46] K Yaoi and Y Mitsuishi ldquoPurification characterizationcloning and expression of a novel xyloglucan-specific glycosi-dase oligoxyloglucan reducing end-specific cellobiohydrolaserdquoThe Journal of Biological Chemistry vol 277 no 50 pp 48276ndash48281 2002
[47] L Ayed N Assas S Sayadi and M Hamdi ldquoInvolvementof lignin peroxidase in the decolourization of black olivemill wastewaters by Geotrichum candidumrdquo Letters in AppliedMicrobiology vol 40 no 1 pp 7ndash11 2005
[48] Y Baba A Shimonaka J Koga H Kubota and T KonoldquoAlternative splicing produces two endoglucanases with oneor two carbohydrate-binding modules in Mucor circinelloidesrdquoJournal of Bacteriology vol 187 no 9 pp 3045ndash3051 2005
[49] M Dashtban H Schraft andW Qin ldquoFungal bioconversion oflignocellulosic residues opportunities amp perspectivesrdquo Interna-tional Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009
[50] D A Ribeiro J Cota T M Alvarez et al ldquoThe Penicilliumechinulatum secretome on sugar cane bagasserdquo PLoS ONE vol7 no 12 Article ID e50571 2012
[51] O Borokhov and S Rothenburger ldquoRapid dye decolorizationmethod for screening potential wood preservativesrdquo Appliedand Environmental Microbiology vol 66 no 12 pp 5457ndash54592000
[52] E Abadulla T Tzanov S Costa K-H Robra A Cavaco-Paulo and G M Gubitz ldquoDecolorization and detoxification oftextile dyes with a laccase from Trametes hirsutardquo Applied andEnvironmental Microbiology vol 66 no 8 pp 3357ndash3362 2000
[53] T J Dreaden J M Davis Z W de Beer et al ldquoPhylogenyof ambrosia beetle symbionts in the genus Raffaeleardquo FungalBiology vol 118 no 12 pp 970ndash978 2014
[54] T S Suryanarayanan T S Murali and G Venkatesan ldquoOccur-rence and distribution of fungal endophytes in tropical forestsacross a rainfall gradientrdquo Canadian Journal of Botany vol 80no 8 pp 818ndash826 2002
[55] A E Arnold and F Lutzoni ldquoDiversity and host range of foliarfungal endophytes are tropical leaves biodiversity hotspotsrdquoEcology vol 88 no 3 pp 541ndash549 2007
[56] J Heilmann-Clausen and L Boddy ldquoInhibition and stimulationeffects in communities of wood decay fungi exudates fromcolonized wood influence growth by other speciesrdquo MicrobialEcology vol 49 no 3 pp 399ndash406 2005
[57] A Kubartova E Ottosson A Dahlberg and J Stenlid ldquoPat-terns of fungal communities among and within decaying logsrevealed by 454 sequencingrdquo Molecular Ecology vol 21 no 18pp 4514ndash4532 2012
[58] L Prewitt Y Kang M L Kakumanu andMWilliams ldquoFungaland bacterial community succession differs for three woodtypes during decay in a forest soilrdquo Microbial Ecology vol 68no 2 pp 212ndash221 2014
[59] N M Reid S L Addison L J Macdonald and G Lloyd-JonesldquoBiodiversity of active and inactive bacteria in the gut floraof wood-feeding Huhu beetle larvae (Prionoplus reticularis)rdquoApplied and Environmental Microbiology vol 77 no 19 pp7000ndash7006 2011
[60] CC Lee R E Kibblewhite-AccinelliM R Smith KWagschalW J Orts and DW SWong ldquoCloning of Bacillus licheniformisxylanase gene and characterization of recombinant enzymerdquoCurrent Microbiology vol 57 no 4 pp 301ndash305 2008
[61] T L Rhoads A T Mikell Jr and M H Eley ldquoInvestigation ofthe lignin-degrading activity of Serratia marcescens biochem-ical screening and ultrastructural evidencerdquo Canadian Journalof Microbiology vol 41 no 7 pp 592ndash600 1995
[62] T Nagy K Emami C M G A Fontes L M A FerreiraD R Humphry and H J Gilbert ldquoThe membrane-bound120572-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-O-methyl-D-glucuronoxylooligosaccharides but not 4-O-methyl-D-glucuronoxylanrdquo Journal of Bacteriology vol 184 no 17 pp4925ndash4929 2002
[63] J Weslien L B Djupstrom M Schroeder and O WidenfalkldquoLong-term priority effects among insects and fungi colonizingdecaying woodrdquo The Journal of Animal Ecology vol 80 no 6pp 1155ndash1162 2011
[64] R T Jones L G Sanchez and N Fierer ldquoA cross-taxon analysisof insect-associated bacterial diversityrdquo PLoS ONE vol 8 no 4Article ID e61218 2013
[65] T Lemke U Stingl M Egert M W Friedrich and A BruneldquoPhysicochemical conditions and microbial activities in thehighly alkaline gut of the humus-feeding larva of Pachnodaephippiata (Coleoptera Scarabaeidae)rdquo Applied and Environ-mental Microbiology vol 69 no 11 pp 6650ndash6658 2003
[66] S M Geib M D M Jimenez-Gasco J E Carlson M Tienand K Hoover ldquoEffect of host tree species on cellulase activityand bacterial community composition in the gut of larval Asianlonghorned beetlerdquo Environmental Entomology vol 38 no 3pp 686ndash699 2009
[67] A E Cazemier J C Verdoes F A G Reubsaet J H PHackstein C van der Drift and H J M Op den CampldquoPromicromonospora pachnodae sp nov a member of the(hemi)cellulolytic hindgut flora of larvae of the scarab beetlePachnoda marginatardquo Antonie van Leeuwenhoek vol 83 no 2pp 135ndash148 2003
[68] F A Genta R J Dillon W R Terra and C Ferreira ldquoPotentialrole for gut microbiota in cell wall digestion and glucosidedetoxification in Tenebrio molitor larvaerdquo Journal of InsectPhysiology vol 52 no 6 pp 593ndash601 2006
[69] D-C Oh J J Scott C R Currie and J Clardy ldquoMycangimycina polyene peroxide from a mutualist Streptomyces sprdquo OrganicLetters vol 11 no 3 pp 633ndash636 2009
