INVERTEBRATE MICROBIOLOGY

Gut-Associated Bacteria Throughout the Life Cycleof the Bark Beetle Dendroctonus rhizophagus Thomasand Bright (Curculionidae: Scolytinae)and Their Cellulolytic Activities

Jesús Morales-Jiménez & Gerardo Zúñiga & Hugo C. Ramírez-Saad &

César Hernández-Rodríguez

Received: 4 October 2011 /Accepted: 13 December 2011 /Published online: 12 January 2012# Springer Science+Business Media, LLC 2012

Abstract Dendroctonus rhizophagus Thomas and Bright(Curculionidae: Scolytinae) is an endemic economically im-portant insect of the Sierra Madre Occidental in Mexico. Thisbark beetle has an atypical behavior within the genus becausejust one beetle couple colonizes and kills seedlings and youngtrees of 11 pine species. In this work, the bacteria associatedwith the Dendroctonus rhizophagus gut were analyzed byculture-dependent and culture-independent methods. Analysisof 16S rRNA sequences amplified directly from isolates of gutbacteria suggests that the bacterial community associated withDendroctonus rhizophagus, like that of other Dendroctonusspp. and Ips pini, is limited in number. Nine bacterial genera ofγ-Proteobacteria and Actinobacteria classes were detected inthe gut of Dendroctonus rhizophagus. Stenotrophomonas andRahnella genera were the most frequently found bacteria fromDendroctonus rhizophagus gut throughout their life cycle.

Stenotrophomonas maltophilia, Ponticoccus gilvus, andKocu-ria marina showed cellulolytic activity in vitro. Stenotropho-monas maltophilia, Rahnella aquatilis, Raoultella terrigena,Ponticoccus gilvus, andKocuria marina associated with larvaeor adults of Dendroctonus rhizophagus could be implicated innitrogen fixation and cellulose breakdown, important rolesassociated to insect development and fitness, especially underthe particularly difficult life conditions of this beetle.

Introduction

Microbial communities of many groups of insects have beenwidely studied [7, 11, 13, 36, 40]. In particular, spectacularexamples of species-level bacterial diversity have been foundin the gut, and complex associations have been recognizedbetween gut bacteria and insects [7, 27]. These interactions arediverse, ranging from antagonism and commensalism to mu-tualism and from obligate to facultative [17, 30]. In additiongut bacteria can contribute to insect development and survivalthrough synthesis of essential nutrients, nitrogen fixing, uricacid recycling, food digestion, pheromone production, andmetabolism of toxins [6, 20, 31, 38, 43]. They also come tohave important implications in fitness, niche diversification,and species diversification [30].

Bark beetles carry on their life cycle on nutritionally poorand unbalanced substrates as phloem, bark and wood [58].These substrates are rich in complex polysaccharides (e.g.,cellulose and hemicelluloses), but scarce in other nutrients(e.g., assimilable nitrogen). While associations betweenbark beetles and their gut bacteria have barely been studied,their environmental characteristics strongly suggest thatboth bacteria and yeasts may have important functional roles

J. Morales-Jiménez : C. Hernández-Rodríguez (*)Departamento de Microbiología,Escuela Nacional de Ciencias Biológicas,Instituto Politécnico Nacional,Prol. De Carpio y Plan de Ayala. Col. Sto. Tomas, Mexico City,Distrito Federal, CP 11340, Mexicoe-mail: chdez38@hotmail.com

G. ZúñigaDepartamento de Zoología,Escuela Nacional de Ciencias Biológicas,Instituto Politécnico Nacional,Prol. De Carpio y Plan de Ayala. Col. Sto. Tomas, Mexico City,Distrito Federal, CP 11340, Mexico

J. Morales-Jiménez :H. C. Ramírez-SaadLaboratorio de Ecología Molecular,Departamento de Sistemas Biológicos,Universidad Autónoma Metropolitana—Xochimilco,04960, México, Mexico

Microb Ecol (2012) 64:268–278DOI 10.1007/s00248-011-9999-0

in cellulose breakdown as well as provision of B vita-mins, sterols, and/or essential amino acids [32, 49]. An-other possible function of gut bacteria is to facilitate thedetoxification process [2] as bark beetles must toleratedefensive compounds present in the host tissues duringthe host colonization and insect development. Perhaps,this toxic environment exerts a selective pressure thatcould explain why bacterial communities that have beenstudied in bark beetles are less diverse compared to thosefound in other insects.

Dendroctonus rhizophagus Thomas and Bright (Cole-optera: Curculionidae: Scolytinae) is endemic of the SierraMadre Occidental in the northwest from Mexico, wherethese parasites kill trees of 11 pine species [34]. It has anatypical behavior as this species does not carry out massattacks as do other species of the genus. Usually, only onecouple colonizes the bottom of the stem of seedlings andyoung pine trees (< 3 m, 10 cm diameter) [12, 51].

The life cycle of this species is annual and synchronous andis regulated largely by conditions of temperature and humid-ity. The emergence, dispersal, and colonization ofDendrocto-nus rhizophagus occur in late July and early August, whileoviposition and larval development take place in July–April,and finally, pupation and imago maturation happen in May–July [19].Dendroctonus rhizophagus always kills their hosts,unlike other stem-colonizing bark beetle species such as theblack turpentine beetle Dendroctonus terebrans and the redturpentine beetle Dendroctonus valens, which, in their nativedistribution range, develop within large pine trees (> 5 m)without killing them. This condition probably strongly limitsthe presence of microorganisms in the gut of this speciesbecause they must survive in an environment poor in nutrientsand toxic due to the effect of tree monoterpenes. However,those gut-associated microorganisms that are able to surviveprobably harbor very effective and efficient metabolic func-tions key for the survival of their hosts. That is, both insectsand microbiota must overcome a quickly changing environ-ment due to the rapid degradation experienced by trees in ashort period immediately after the onset of colonization. Forthis reason, the aim of this work was to describe the gut-associated bacterial community of this bark beetle indifferent developmental stages by culture and culture-independent methods and to test the cellulolytic capaci-ties of cultured bacteria.

Material and Methods

Bark Beetle Collection

Larvae, pupae, and feed emerged adults from Dendroctonusrhizophagus were collected from two geographical locationsin the Sierra Madre Occidental, Mexico (Table 1). All sampleswere obtained manually, directly from galleries of infestedpine trees using fine forceps. They were then transported tothe laboratory in sterile vials containing sterile moist paper.Larva, pupa, and adult insects, after being disinfected super-ficially with 70% ethanol and submerged repeatedly in aphosphate buffer solution (PBS) to avoid external contamina-tion, were dissected under sterile conditions. In the case ofadults, the gut was extracted after removal of elytra, wings,and tergites to expose the insect abdomen. The individual gutswere transferred to a 1.5-ml microcentrifuge tube with 0.2 mlof PBS or culture medium.

Microbiology and Molecular Biology Techniques

Bacterial isolation and culturing were assessed with techni-ques previously described [35]. Bacterial viable counts wereperformed on single guts of at least five insects for each lifestage and sex. After scoring CFU values, single colonieswere selected, and pure cultures were stored at -70°C forfurther analysis.

Bacterial and gut metagenomic DNAs from five larva, pupa,and adult beetles were extracted following protocols describedby Hoffman et al. [25] and Morales-Jiménez et al. [35]. Thepure DNAs were stored at -70°C until they were used inmolecular techniques. RAPD fingerprints were generated frombacterial isolates in order to recognize bacterial related species.For this purpose, a single primer was used, and PCR conditionswere those described by Williams et al. [57]. 16S rRNA genesof bacterial isolates and the gut bacterial community wereamplified by using the primers and PCR conditions describedin Relman [44]. PCR products were purified using the QIA-quick PCR purification kit (Qiagen, Valencia, CA) and se-quenced in an ABI PRISM 310 genetic analyzer (AppliedBiosystems, Foster City, CA) using the same primers. 16SrRNA libraries of gut metagenomic DNA were generated byemploying purified PCR products cloned in Escherichia coliTop-10 cells (Invitrogen-Life Technologies, Carlsbad, CA)

Table 1 Dendroctonusrhizophagus samples usedin this study

Location in Mexico Locationcode

Latitude/longitude Host tree Insects (No.)

San Juanito, Bocoyna,Chihuahua

BCH2 27°92' N/107°60' W Pinus engelmannii Adults (20)

Pupae (20)

Eggs (300)

El Salto, Durango SD 23°50' N/105°22' W Pinus arizonica var cooperi Larvae (40)

Gut-Associated Bacteria of Dendroctonus rhizophagus 269

with pJET1.2/blunt (CloneJETTM PCR Cloning Kit; Fermen-tas, Glen Burnie,MD) according to themanufacturer's instruc-tions. Transformants were subjected to plasmid extraction bystandard methods [45], and a restriction analysis with EcoRIwas performed to detect insertions. Plasmidic DNA of eachclone of the 16S rRNA library was digested with HpaII andHhaI endonucleases to display RFLP patterns in electropho-resis at 3% high-performance agarose 1000 (GIBCO Labora-tories, Grand Island, NY). The plasmids of each RFLP patternwere extracted using a High Pure Plasmid Isolation Kit(Roche) and sequenced with the ABI PRISM 310 geneticanalyzer (Applied Biosystems, Foster City, CA) using pJET1.2 forward and pJET 1.2 reverse sequencing primers.

To know whether gut bacterial communities are differentamong sexes and insect life stages, a DGGE analysis wasperformed. DNA obtained from pools of five larva, pupa,and adult guts was used for PCR amplification of V3–V5 ofthe 16S rRNA gene. DGGE primers, PCR conditions, and thegeneral methodology of this technique were performed fol-lowingMuyzer et al. [37], while the silver stain procedure wasthat of Sanguinetti et al. [46]. Selected DGGE bands wereexcised from gels, reamplified with the same primers, andthen sequenced as described previously. Maximum likelihoodanalysis of DGGE band sequences was performed using theK80+I + G model (α00.434 for the gamma distribution; p-inv00.15; transition/transversion ratio00.93) with 1,000bootstrap replicates.

Phylogenetic Analyses

Clone sequences obtained were tested for chimera structuresusing Bellerophon (http://comp-bio.anu.edu.au/bellerophon/bellerophon.pl) [28], and chimeras were excluded from fur-ther analysis. Sequences from clones and isolated bacteriawere compared with the non-redundant GenBank library us-ing BLAST search [3]. A collection of taxonomically relatedsequences was obtained from the National Center for Biotech-nology Information (NCBI) Taxonomy Homepage (http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/).DNA sequences were aligned using CLUSTAL X [52], editedand confirmed visually in BIOEDIT [23].

Maximum likelihood analyses were performed usingPhyML [21] (http://atgc.lirmm.fr/phyml/). MODELTEST3.06 [41] was used to select appropriate models of sequenceevolution by the AIC model [42]. The GTR + I + G model(α00.459 for the gamma distribution; A00.237, C00.233,G00.317, T00.212; p-inv00.204) was selected for the treesearch. The confidence at each node was assessed by 1,000bootstrap replicates. Anabaena affinis was used as outgroup.The similitude percentages among sequences were calculatedusing MatGAT v. 2.01 software [9]. The limits for genus andspecies were set at 95% and 97%, respectively [47]. Due to thehigh similarity among 16S rRNA gene sequences of Pantoea

agglomerans, Klebsiella spp., Enterobacter spp., and Raoul-tella terrigena carbohydrate fermentation tests (D-melezitoseand L-Sorbose ), an API-20E bacterial identification test strip(bioMérieux, Marcy I'Etoile, France) was employed to con-firm the phylogenetic approach [24]. All sequences generatedin this study were deposited in the GenBank database, underthe accession numbers JN12146 through JN12175.

Isolation of Cellulolytic Microorganisms

Individual larva, pupa, and adult guts were placed in sterileEppendorf tubes containing 200 μl of PBS and processed asdescribed in previous sections. Serial ten-fold dilutions werespread on duplicate plates of Congo red agar (0.5-g l-1

K2HPO4, 0.25-g l-1 MgSO4, 1.88-g l-1 carboxymethyl cel-lulose, 0.2-g l-1 Congo red, 2-g l-1 gelatin, 100-ml l-1 soilextract, and 15-g l-1 agar). Plates were incubated in a growthchamber at 28°C for 3 to 5 days. The cellulolytic activity ofmicroorganisms was detected by a clear zone around thecolonies [50]. Pure cultures were obtained by multiple sub-sequent subculturing on Congo red agar. All strains withcellulolytic activities were grown in a mineral medium withcarboxymethyl cellulose as the sole carbon source (2.5-g l-1

NaCl, 7.0-g l-1 K2HPO4, 3.0-g l-1 KH2PO4, 0.1-g l-1

MgSO4, and 2.5-g l-1 Carboxymethyl cellulose). Colonycellulolytic activity was indexed as the diameter of thecellulolytic halo divided by the diameter of the colony. Atleast two measurements were taken for each colony type.

Results

Bacterial Species in Dendroctonus rhizophagus Larvae

Ten RFLP patterns were identified in all clones of 16S rRNAgene libraries usingHpaII andHhaI. A clone representing eachRFLP pattern was sequenced and identified by nucleotidesimilitude and the phylogenetic approach, and from these anal-yses, two to three bacterial genera were identified in the larvalgut by culture and culture-independent analyses. Phylogeneticanalysis of 16S rRNA revealed that the relative abundance ofRahnella aquatilis and Pseudomonas fluorescens clones was81% and 9%, respectively (Table 2). Meanwhile, the culturedpopulations of Rahnella aquatilis, Stenotrophomonas malto-philia, and Pseudomonas fluorescens were around 2.85×106±4.29 105 (98%), 3.75×104±6.61 103 (1%), and 2.8×103±5.65×102 (0.1%) UFC/gut (% of total culturable bacterialpopulation), respectively (Table 2 and Fig. 1).

Bacterial Species in Dendroctonus rhizophagus Pupae

The culture fraction of the microbial community associatedwith the pupa gut was dominated exclusively by Rahnella

270 J. Morales-Jiménez et al.

aquatilis, with a density around 1.95×103±1.02×103 UFC/gut, and in the unculturable fraction by Propionibacteriumsp. detected using DGGE.

Bacterial Species in Dendroctonus rhizophagus Adults

Culture-dependent and culture-independent analysis of adultguts revealed organisms affiliated with γ-Proteobacteria andActinobacteria (Table 2 and Fig. 1). Likewise, in larvae,

Rahnella aquatilis was the most abundant species (n025,96%) in the 16S rRNA library, but only one clone wasidentified as Klebsiella sp. The γ-Proteobacteria Rahnellaaquatilis, Raoultella terrigena, Stenotrophomonasmaltophilia, and Pseudomonas fluorescens as well as Actino-bacteria Ponticoccus gilvus and Kocuria marina were cul-tured (Table 2 and Fig. 1). The sequence of the 16S rRNAgene of the strain identified as Raoultella terrigena was clus-tered with sequences of Raoultella terrigena, Pantoea

Table 2 Bacterial taxa associated with guts of larvae, pupae, and adults of Dendroctonus rhizophagus in culture-dependent and culture-independent analyses

Identifiedbacteriaa

Detectionstrategiesb, c

Codes Insect stage

Larva Pupa Adult

Rahnellaaquatilis

Isolation 4-DR, 6-DR, 12-DR,DR-2A, PDR-D,PDR-4, PDR-1,PDR-H

2.85×106 ± 4.29 105 1.95×103 ± 1.02×103 2.2×106 ± 5.6×106

Library clones DR-12A, DRL-D6,DR-A3, DR-E12,DRL-F6, DRL-B3,DR-D9, DR-A4

0.8 NP 0.96

DGGE B-B, B-C + + +

Raoultellaterrigena

Isolation DR-E5 ND ND +

Library clones ND NP ND

DGGE ND ND ND

Stenotrophomonasmaltophilia

Isolation 1-DR, 2-DR, 3-DR 3.75×104 ± 6.61×103 ND 5.5×104 ± 3.13×104

Library clones ND NP ND

DGGE ND ND ND

Pseudomonasfluorescens

Isolation DR-E10 2.8×103 ± 5.65×102 ND 1.6×103 ± 0

Library clones DRL-1E, DRL-C11 0.1 NP ND

DGGE ND ND ND

Acinetobacterlowffii

Isolation DR-A6 NP NP 4.94 × 103 ± 1.8 × 102

Library clones ND NP ND

DGGE ND ND ND

Ponticoccusgilvus

Isolation 19-DR ND ND 2×103 ± 6.9×102

Library clones ND NP ND

DGGE ND ND ND

Kocuria marina Isolation DR-E1 ND ND +

Library clones ND NP ND

DGGE ND ND ND

Klebsiella sp. Isolation ND ND ND

Library clones DRL-1C 0.1 NP 0.04

DGGE ND ND ND

Propionibacteriumsp.

Isolation ND ND ND

Library clones ND NP ND

DGGE B-D + + +

NP not performed, ND not detecteda The limits for genus and species were 95% and 97%, respectively (Schloss and Handelsman, 2005)b The relative abundance was determined by viable count of cultured bacteria and clone proportions in 16S rRNA librariesc All attempts to construct a 16S rRNA library of pupa gut were unsuccessful. A possible explanation of this result could be the low bacterialdensities detected in the pupa stage

Gut-Associated Bacteria of Dendroctonus rhizophagus 271

Anabaena affinis (AF247591)

DR-E1 (JN712172)Kocuria marina (HM209736)Kocuria varians (NR029297)

Kocuria flava (EF602041)Microlunatus ginsengisoli (AB245389)

19-DR (JN712171)Ponticoccus gilvus (AM980985)Propionibacteriaceae bacteriumNML 02-0265 (EF599122)

2-DR (JN712148)3-DR (JN712147)1-DR (JN712146)

Stenotrophomonas maltophilia (FJ418173)Stenotrophomonas maltophilia (EF620448)

Stenotrophomonas dokdonensis (DQ178977)Xanthomonas campestris pv. zantedeschia (AY605124)

Pseudomonas azotoformans (D84009)DR-E10 (JN712168)DRL-1E (JN712169)DRL-C11 (JN712167)Pseudomonas fluorescens (68342549)

Pseudomonas extremoaustralis (AJ583501)Pseudomonas azelaica (AM088475)

Pseudomonas nitroreducens (D84022)Acinetobacter brisouii (DQ832256)

Acinetobacter rhizosphaerae (DQ536511)Acinetobacter johnsonii (EF114343)

Acinetobacter haemolyticus (AM184255)DR-A6 (JN712170)Acinetobacter lwoffii (DQ328322)

Pseudacinetobacter hongkongensis (AF543466)Klebsiella variicola At-22 (288887617)

DRL-1C (JN712165)Klebsiella trevisanii (AF129444)

Klebsiella planticola (AF181574)Pantoea agglomerans (EU304255)Raoultella terrigena isolate PSB15 (HQ242728)DR-E5 (JN712166)

Raoultella terrigena strain 84 (NR037085)

Klebsiella oxytoca (HM461887)DR-A4 (JN712164)Rahnella aquatilis (U90757)

PDR-D (JN712159)PDR-1 (JN712161)PDR-4 (JN712160)

12-DR (JN712158)DR-D9 (JN712162)6-DR (JN712157)4-DR (JN712156)PDR-H (JN712163)DR-E12 (JN712155)Uncultured bacterium clone spb28a3 (DQ321557)

DRL-2A (JN712154)DRL-B3 (JN712153)DRL-F6 (JN712151)DRL-D6 (JN712149)

DR-12A (JN712150)DR-A3 (JN712152)

584

775

559

616

997

246

989

569

593

763

763

980

811

799

831

1000

1000

969

999

1000

511

1000

900677

1000

948

774

588

999

980

1000

999

984

1000

1000

1000

952

0.1

Act

inob

acte

ria

-Pro

teob

acte

ria

272 J. Morales-Jiménez et al.

agglomerans, and Klebsiella spp., with similarities above99%. On the other hand, metabolic tests of this strain, suchas D-melezitose and L-sorbose fermentation and the API-20Etest, confirm that this strain belongs to the Raoultella terri-gena species. The populations of Rahnella aquatilis, Steno-trophomonas maltophilia, Pseudomonas fluorescens, andAcinetobacter lowffii were around 2.2×106±5.6×106 (97%),5.5×104±3.13×104 (2%), 1.6×103±0 (0.07%), and 4.94×103±1.8×102 (0.2%) UFC/gut (% of total culturable bacterialpopulation), respectively. The densities of Raoultella terrigenaandKocuria marinawere not determined because these strainswere isolated from enrichment cultures. The ActinobacteriaPonticoccus gilvuswas not found in culture-independent anal-ysis, but a population of approximately 2×103±6.9×102 UFC/gut was estimated.

Diversity of Bacterial Species by DGGE Analysis

DGGE analysis of bacterial communities associated withlarvae, pupae, and adults of Dendroctonus rhizophagus gutsshowed a low number of bands in both sexes and all threelife stages, an observation consistent with the number ofspecies detected by culture and culture-independent meth-ods. The comparison of band patterns among larvae, pupae,adults, and sexes in Dendroctonus rhizophagus showed thatthe communities are highly homogeneous (Figs. 2 and 3a,b). A total of ten bands in males, females, and larvae wererecognized, while only nine bands were present in pupae.The comparison of migration bands of DGGE analysisrevealed that both Stenotrophomonas maltophilia and Rah-nella aquatilis were broadly distributed in the alimentarycanal of Dendroctonus rhizophagus during all stages of thelife cycle. Band intensity of DGGE patterns suggests thatRahnella aquatilis was the most abundant bacteria in theDendroctonus rhizophagus gut in all life stages. Sequencesfrom some DGGE bands clustered with sequences of Rah-nella aquatilis and the genus Propionibacterium (Fig. 4)support the idea that this member of the family Enterobac-teriaceae is a major bacterium in the Dendroctonus rhizo-phagus gut. Furthermore, an intense DGGE band wasassociated with the 18S rRNA gene of insects in a BLASTanalysis (Fig. 3b).

Cellulolytic Bacteria

Stenotrophomonas maltophilia and Ponticoccus gilvusstrains with densities around 1,300±81.64 and 1,000±346.41 CFU/gut isolated from male guts showed cellulolytic

activity on plates of Congo red–cellulose medium (Fig. 5,Table 3). Populations of cellulolytic bacteria were muchlower than the total number of cultured bacteria (0.16–1.16×106 CFU/gut). Also, Kocuria marina showed cellulo-lytic activity, but this strain was isolated from an enrichmentculture in a mineral liquid media with CMC as the solecarbon source. The extracellular enzyme activity indexesof Ponticoccus gilvus, Stenotrophomonas maltophilia, andKocuria marina were approximately 8.2±2.45, 4.18±0.28,and 3.8±0.65, respectively.

Discussion

A total of nine bacterial taxa were found in the larva, pupa,and adult gut of Dendroctonus rhizophagus by culture-dependent and culture-independent methods. This bacterialdiversity is slightly lower than that observed in other barkbeetles [35, 54, 59]. The gut-associated bacterial communityin pupae was lower in number and diversity than in larvaeand adults (Table 2). In the pupa stage, only Rahnellaaquatilis was cultured, and Propionibacterium sp. wasdetected by DGGE. Similar results have been reported inthe pine engraver (Ips pini), where low bacteria densitieswere recorded in the pupa gut [16]. We think that such low

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Figure 2 Denaturing gradient gel electrophoresis of 16S rDNA PCRproducts obtained from field-collected guts of Dendroctonus valensand Dendroctonus rhizophagus. Adult female guts of Dendroctonusrhizophagus (lane 1 anterior midgut, lane 2 posterior midgut, lane 3hindgut), adult female guts of Dendroctonus valens (lane 4 anteriormidgut, lane 5 posterior midgut, lane 6 hindgut), adult male guts ofDendroctonus rhizophagus (lane 7 anterior midgut, lane 8 posteriormidgut, lane 9 hindgut), adult male guts of Dendroctonus valens (lane10 anterior midgut, lane 11 posterior midgut, lane 12 hindgut), larvaguts of Dendroctonus rhizophagus (lane 13), and larva guts of Den-droctonus valens (lane 14 anterior midgut, lane 14 posterior midgut,lane 16 hindgut)

�Figure 1 Maximum likelihood tree (-lnL011,257.67698) of bacterialcommunity associated with Dendroctonus rhizophagus gut. The 16SrRNA sequence of Anabaena affinis was used as outgroup. Scale barindicates 10% estimated sequence divergence. Bootstrap support val-ues are indicated for major nodes having values ≥50%

Gut-Associated Bacteria of Dendroctonus rhizophagus 273

bacterial densities in the pupa gut may be due to decreasedmetabolic activity during this developmental stage, althoughmorphological changes that the gut undergoes during theinsect metamorphosis could be an additional factor.

DGGE patterns of 16S RNA amplified from metage-nomic DNA from larvae, pupae, and adults showed fewdifferences in bands and no significant differences betweenthe number of bands displayed at different life stages. Theresults show that at all stages, Rahnella aquatilis is a wide-spread bacterium in the Dendroctonus rhizophagus gut,whereas the other bacteria were present only in some par-ticular stages. This situation was also observed in the bac-terial community associated with the Dendroctonus valensgut by DGGE [35]. However, DGGE profile comparisons ofgut-bacterial communities among the different developmentstages and between the sexes of these species showed thattheir total bacterial communities are similar (Figs. 2 and 3).

Kocuria marina, Ponticoccus gilvus, and Raoultella ter-rigena were exclusively found in the adult gut by culturingmethods. Meanwhile, the genus Klebsiella was recognizedonly by 16S rRNA libraries in larvae and adults. The cultur-able fraction of gut microbiota of Dendroctonus rhizophaguswas integrated by seven bacterial species. Similar results havebeen reported in the Dendroctonus frontalis, Dendroctonusvalens, and Dendroctonus micans gut, where 13, 14, and 7

culturable bacteria species were recognized, respectively [35,55, 59]. The scarce gut bacterial community is a commonfeature in all bark beetles studied, probably due mainlyto the antibacterial activity of some monoterpenes pres-ent in pine resin and/or limitation of nitrogen sources [2,53]. This tendency in bark beetles contrasts with the highbacterial diversity recorded in other insects, such aswood-boring beetles. For example, the Asian longhornedbeetle (Anoplophora glabripennis) and the emerald ashborer (Agrilus planipennis) harbor a total of 23 and 42bacterial genera in their gut, respectively. Curiously, bothbeetles develop in sapwood. The Asian longhorned bee-tle feeds on the cambium and phloem of maples, horse-chestnuts, poplars, willows, elms, mulberries, and blacklocusts. On the other hand, the emerald ash borer feedson the phloem of ash trees [48, 55]. Compared withother insects that feed on woody tissues, such as Retic-ulitermes speratus (Isoptera: Rhinotermitidae) that harboraround 268 phylotypes in 11 bacterial divisions [26, 39],the gut bacterial community of Dendroctonus rhizopha-gus appears to be extremely simple.

Full evidence from viable counts, DGGE, and relativeabundance of clones in 16S rDNA libraries suggest thatRahnella aquatilis is the dominant species in the Dendroc-tonus rhizophagus gut. The abundance of this nitrogen-

1 2 3 4 5 6

B

C

D

A

1 2 3 4 5 6 7 8 9 10 11

ba

Figure 3 Denaturing gradient gel electrophoresis of 16S rDNA PCRproducts obtained from field-collected guts of Dendroctonus rhizopha-gus. a Comigration comparison among adult male guts (lanes 1 and 3),adult female guts (lane 2), pupa guts (lane 4), larva guts (lane 5), larvaguts of Dendroctonus valens (lane 6), mix of DNA from bacteriaisolates of Dendroctonus rhizophagus (lane 7), and bacterial isolates(lanes 8 and 9, Stenotrophomonas maltophilia; lanes 10 and 11,

Rahnella aquatilis). Full black arrow, Stenotrophomonas maltophilia.Black arrowhead, Rahnella aquatilis. b DGGE for band sequencing.Larva guts (lane 1), pupa guts (lane 2), adult male guts (lanes 3 and 5),adult female guts (lane 4), and larva guts of Dendroctonus valens (lane6). The sequences of band A was clustered with 18S rRNA genes ofinsects, B and C with Rahnella aquatilis, and C with Propionibacte-rium sp.

274 J. Morales-Jiménez et al.

fixing bacterium suggests that the nitrogen-fixing processmust be a very important dietary supplement of assimilablenitrogen for the insect. Rahnella aquatilis seems to bewidespread in the gut of Dendroctonus species, althoughquantitative data are not available. It has also been isolatedfrom other studied species, including Dendroctonus valensand Dendroctonus frontalis. It has frequently been isolatedor detected in 16S rRNA libraries from larvae and adults ofDendroctonus valens [35], and it reached a frequency ofdetection of 12.8 % by DGGE [1]. Also, this bacterialspecies was the most common one detected in 16S rRNAlibraries from larval and adult Dendroctonus frontalis gut,although it could be isolated from larva gut only once [54].The evidence that this work and other studies cited suggeststhat Rahnella aquatilis might be recognized as a resident gutbacterium of Dendroctonus. Additionally, neither Rahnellaspp. has been detected in other insects feeding on wood andphloem, such as the Asian longhorned beetle and the pineengraver [15, 16, 48, 55]. However, Rahnella aquatilis hasbeen recognized as the dominant bacterium in Hepialus

gonggaensis (a moth) and Decticus verrucivorus (wart-bitercricket) [14, 60], and it has also been isolated from seeds,ectomycorrhizas, and sapwood sawdust of conifers [10, 18,29], suggesting that it could be a conifer endophytic bacte-rium. In this sense, in our laboratory, an attempt was madeto detect Rahnella species in healthy pine phloem, but nostrains were isolated (data not shown). Evidently, beyondthe clear Rahnella aquatilis–Dendroctonus relationship,more studies are necessary to determine the ecological statusof this nitrogen-fixing bacteria in the tree-bark beetleenvironment.

Rahnella aquatilis, Pseudomonas fluorescens, and Steno-trophomonas maltophilia γ-Proteobacteria were commonlyfound in larva and adult guts of Dendroctonus rhizophagus,suggesting that these bacteria are maintained during the meta-morphosis from larva to adult. The genus Pseudomonas hasbeen reported in adult guts ofDendroctonus frontalis and in thecerambycids Anoplophora glabripennis, Saperda vestita, Rha-gium inquisitor, and Leptura rubra [22, 48, 55]. Although therole of this bacterium in the gut of wood- and bark-inhabiting

Anabaena affinis (AF247591)

Propionibacterium granulosum (NR025276)

Propionibacterium acidipropionici (FN824489)

Propionibacterium avidum (NR025274)

B-D

Propionibacterium acnes (CP001977)

Propionibacterium australiense (NR025076)

Microlunatus ginsengisoli (AB245389)

Ponticoccus gilvus (AM980985)

Kocuria marina (HM209736)

Cellulosimicrobium cellulans (AY665978)

Acinetobacter lwoffii (DQ328322)

Pseudacinetobacter hongkongensis (AF543466)

Pseudomonas fluorescens (68342549)

Stenotrophomonas maltophilia (EF620448)

Rahnella aquatilis (U90757)

Uncultured bacterium clone spb28a3 (DQ321557)

B-C

B-B

Klebsiella planticola (AF181574)

Klebsiella oxytoca (HM461887)

Pantoea agglomerans (EU304255)

588

652

922

665

555

688

739

899

953

872

784

684

684

918

0.1

Act

inob

acte

ria

-Pro

teob

acte

ria

Figure 4 Maximum likelihoodtree (-lnL01,166.06911) ofsome DGGE bands of thebacterial community associatedwith Dendroctonusrhizophagus gut. The 16SrRNA sequence of Anabaenaaffinis was used as outgroup.Scale bar indicates 10%estimated sequence divergence.Bootstrap support values areindicated for major nodeshaving values ≥50%

Gut-Associated Bacteria of Dendroctonus rhizophagus 275

beetles is unknown, we hypothesize that it could be involved interpene transformation of plant resin compounds, due to itscapabilities to metabolize monoterpenes and phenolics com-pounds [5]. Stenotrophomonas maltophilia has been found inadults of Dendroctonus valens and Dendroctonus frontalis and

at all life stages of Ips pini [16, 35, 54]. This ubiquitousnitrogen-fixing bacterium, in association with other diazotrophsfound in Dendroctonus rhizophagus, such as Klebsiella sp.,Rahnella aquatilis, and Raoultella terrigena, may fix and con-centrate assimilable nitrogen for insect nutrition, as has beendemonstrated in other insects [4, 40]. Both insect larvae andadults exhibit acetylene reduction (data not shown), but theparticular contribution of each nitrogen-fixing bacterial speciesin the gut must be determined.

On the other hand, Stenotrophomonas maltophilia (γ-Proteobacteria), Ponticoccus gilvus, and Kocuria marina(Actinobacteria) isolated from the Dendroctonus rhizo-phagus gut were the bacterial isolates capable of degradingcarboxymethylcellulose in vitro. Neither Actinobacteria hasever been reported in other bark beetles or tree tissues. Al-though isolates of the genus Kocuria with cellulolytic activityhave been isolated from the gut of the termite Zootermopsisangusticollis [56], to our knowledge, no cellulolytic capacitieshave been reported in members of the Ponticoccus or Steno-trophomonas genera. The presence of cellulose-degrading bac-teria has been demonstrated in the gut of insects that feed onwoody tree tissues, such as wood-boring beetles, includingSaperda vestita and Agrilus planipennis [15, 55]. The cellulo-lytic bacteria obtained from theDendroctonus rhizophagus gutcould be involved in the degradation of cellulosic substratessuch as pine bark and phloem, enabling them to serve as acarbon source. On the other hand, other bacteria, includingStenotrophomonas maltophilia, Pseudomonas, and Acineto-bacter, could participate in the oxidation, fermentation, andhydrolysis of the cellulose and lignin derived aromatic products[33].

Cellulose and hemicellulose hydrolysis, anaerobic respi-ration, and nitrogen fixation take place in specialized dilatedhindguts of termites, but no equivalent morphological adap-tations in the bark beetle gut are observed [8]. Notwithstand-ing, we observed partial digested woody material in adultsthat recently colonized the pine trees and larva galleries ofDendroctonus rhizophagus. The gut of Dendroctonus spp. ishistologically and morphologically compartmentalized,probably with their particular physiochemical microenviron-ments for cellulose degradation or nitrogen fixation. Evi-dently, more studies must be performed in order torecognize the role of gut cellulolytic bacteria in the Den-droctonus spp. life cycle.

In this study, a characterization was made on the bacterialcommunity associated with the gut of larvae and adults ofDendroctonus rhizophagus, found by using culture andculture-independent techniques. This is the first reportconcerning the bacterial community within the gut of Den-droctonus rhizophagus. Several important bacteria wererecognized, including nitrogen fixing and cellulolyticguilds, and their roles in the context of the insect–microberelationship were discussed.

Figure 5 Cellulolytic activity of bacterial isolates from the gut of Den-droctonus rhizophagus in Congo red-CMC. a Ponticoccus gilvus 19 DR.b Stenotrophomonas maltophilia 2 DR. c Kocuria marina DRE1

Table 3 Identification and cellulolytic activity of bacteria isolatedfrom D. rhizophagus gut

Isolate Enzyme activity (indexa)

Stenotrophomonas maltophilia (2-DR) 8 ± 0.65

Kocuria marina (DR-E1) 4.18 ± 0.28

Ponticoccus gilvus (19-DR) 8.2 ± 2.45

a Enzyme activity was indexed as the diameter of the colony plus theclear zone around it divided by the diameter of the colony in Congored–cellulose medium

276 J. Morales-Jiménez et al.

Acknowledgments We would like to thank Félix Aguirre Garrido forthe technical assistance with DGGE. This work was supported bygrants SIP 20080688, 20090738, 20100430, and 20111068; IPN; andCONAFOR-CONACyT 69539. Jesús Morales-Jiménez would like tothank CONACyT, and PIFI-IPN for the scholarships.

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