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Survey and phylogenetic analysis of culturable microbes in the oral secretions of three bark beetle species

Survey and phylogenetic analysis of culturable microbes in the oral secretions of three bark beetle species
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  © 2009 The Authors Entomologia Experimentalis et Applicata  131 :138–147,2009 138 Journal compilation © 2009 The Netherlands Entomological Society DOI: 10.1111/j.1570-7458.2009.00844.x  BlackwellPublishingLtd Survey and phylogenetic analysis of culturable microbes in the oral secretions of three bark beetle species  Yasmin J. Cardoza 1,2 *, Archana Vasanthakumar  1 , Alonso Suazo 2  & Kenneth F. Raffa 1 1 Department of Entomology, University of Wisconsin–Madison, Madison, WI 53706, USA, and  2 Department of Entomology,  North Carolina State University, PO Box 7613, Raleigh, NC 27695-7613, USA Accepted: 26 January 2009 Key words: fungi, bacteria, symbiosis, microbial diversity, Dendroctonus rufipennis , Dendroctonus  ponderosae , Ips pini , Coleoptera, Curculionidae, Scolytinae, 16S rRNA sequence  Abstract In a recent study, we reported a previously undescribed behavior in which a bark beetle exuded oralsecretions containing bacteria that have antifungal properties, and hence defend their galleries againstpervasive antagonistic Hyphomycete fungi. Actinobacteria, a group known for their antibiotic pro-perties, were the most effective against fungi that invade the spruce beetle galleries. In the present study,we describe the isolation and identification of microorganisms from oral secretions of three bark beetles (Coleoptera: Curculionidae: Scolytinae): the spruce beetle, Dendroctonus rufipennis  Kirby, themountain pine beetle, Dendroctonus ponderosae  Hopkins, and the pine engraver, Ips pini  Say. Bacteriaisolated from these three species span the major bacterial classes α -, β -, and γ  -Proteobacteria, Firmi-cutes, Bacteroidetes, and Actinobacteria, except for D. ponderosae , which yielded no α -proteobacteriaor Bacteroidetes isolates. Spruce beetles and pine engraver beetles had similar numbers of α -proteo-bacteria isolates, but pine engravers yielded twice as many Bacteroidetes isolates as spruce beetles.In contrast, mountain pine beetles yielded more isolates in the β - and γ  -proteobacteria than sprucebeetles and pine engravers. The highest percentage of Actinobacteria was obtained from spruce beetles,followed by pine engravers and mountain pine beetles. All of the fungal isolates obtained from thethree beetle species were Ascomycetes. The greatest fungal diversity was obtained in spruce beetles,which had nine species, followed by pine engravers with five, and mountain pine beetles with one. Introduction Some bark beetle species (Coleoptera: Curculionidae:Scolytinae) have the ability to colonize living trees afterdepleting their defenses, but in so doing they render thesubcortical environment suitable for competitors inaddition to their own brood. For example, when sprucebeetles, Dendroctonus rufipennis  Kirby, colonize hostphloem, at least four fungal species invade their galleriesand may cause beetle mortality or reduce oviposition(Cardoza et al., 2006a). Three of these fungi are apparently opportunists, which cannot colonize live trees on theirown but rapidly exploit hosts colonized by spruce beetles,and all three fungi are antagonists that significantly reducespruce beetle survival, egg gallery construction, ovi-position, larval galleries, and adult survival (Cardoza et al.,2006a). The fourth, Leptographium abietinum  (Peck)Wingfield, is the most common ophiostomatoid fungusvectored by spruce beetles (Six & Bentz, 2003; Aukemaet al., 2005), and has been reported to assist beetles inovercoming host resistance (Solheim & Safranyik, 1997).This fungus had moderately negative effects on ovipositionand so may represent a context-dependent symbiont.Spruce beetles can counteract the effects of these fungiby exuding and smearing oral secretions, especially within galleries pervaded with opportunistic fungi (Cardozaet al., 2006a). Bacteria in these secretions inhibited thegrowth of three of the fungi (Cardoza et al., 2006a). Thus,beetles appear to use a combination of behavioralresponses and bacteria to protect their reproductivegalleries from saprophytic antagonistic and context-dependent mutualistic fungi (Cardoza et al., 2006a). These * Correspondence: Yasmin J. Cardoza, Department of Entomology, North Carolina State University, PO Box 7613, Raleigh, NC 27695-7613, USA. E-mail:   Microbes in bark beetle oral secretions 139 relationships show important evolutionary and ecologicalparallels with certain ant–fungal–bacterial relationships(Currie, 2001).Most knowledge of microorganisms associated withbark beetles involves fungi, such as the well-documentedassociations with ascomycete fungi in the genera Ophiostoma , Ceratocystis , and Ceratocystiopsis , and their Graphium  and Leptographium  anamorphs (asexual repro-ductive stages), which are almost exclusively vectored by bark beetles (Whitney & Farris, 1970; Paine & Hanlon,1994; Harrington, 2005). In contrast, very little is knownabout bacterial symbionts of bark beetles. For example,we do not know how widespread the occurrence of bacteriain oral secretions of bark beetles is, or how they may relateto bark beetle development, ecology, and management.The objectives of this project were to: (i) investigatethe presence of bacteria in the oral secretions of three bark beetle species (spruce beetle, mountain pine beetle, Dendroctonus ponderosae  Hopkins, and pine engraverbeetle, Ips pini  Say); (ii) study the diversity of culturablemicrobes (bacteria and fungi) present in the oral secretions;and (iii) analyze the phylogenetic associations of culturableoral bacteria of these three bark beetle species. Materials and methods Insects Adult spruce beetles were collected from naturally infestedtrees on the Kenai Peninsula (62 ° 31 ′ N, 150 ° 3 ′ W) of Alaska, USA. All pine engravers were reared in red pine, Pinus resinosa  Aiton (Pinaceae), logs harvested from thefield using the method of Raffa & Dahlsten (1995).Mountain pine beetle adults were obtained from theUSDA-FS in Fort Collins, CO, and Logan, UT, both in theUSA, where they were collected from naturally infestedlodgepole pine ( Pinus contorta  Douglas) trees. Spruce andmountain pine beetles were shipped to the ForestEntomology Laboratory at the University of Wisconsin–Madison, Madison, WI, USA. All insects were segregatedby gender, placed within 200-ml screw cap glass jars,provided with pieces of excised spruce phloem for foodand crumpled Kimwipes® (Kimberly Clark, Roswell, GA,USA) to absorb excess moisture and provide insulationand a surface for walking. The insects were then stored at4 ° C until needed for experiments. Isolation of bark beetle bacteria and fungi Mouthparts of six individual bark beetles (three male andthree female) of each species were streaked across a 10%Tryptic Soy Agar (TSA) medium plate (Difco™; Becton,Dickinson and Co., Sparks, MD, USA). To minimizecontamination from other beetle body parts, we confinedthe insects within a glass Pasteur pipette using two piecesof mesh material. A pipette bulb was then used to forceautoclaved H 2 O over the beetles’ bodies 20 times. Thisprocedure was repeated twice more using fresh sterilewater. Clean spruce beetles were held perpendicular to theTSA plate with a pair of flame sterilized stainless steelfeatherweight wide-tip forceps so only the beetles’ mouthparts were in direct contact with the medium plates. Thesecretions were then washed off the plate by rinsing with1 ml of autoclaved double-distilled H 2 O. Oral secretionswere collected off the plates using 1 ml of autoclaved water(stock). Water was pipetted over the streaked region repeatedly to maximize collection of the oral secretions. Serial dilutionsof 1/10 and 1/100 were made. Aliquots of 100 µ l from eachdilution were plated on 10% TSA to allow for the mostdiverse microbial growth. Growth was checked frequently,and pure cultures of individual macromorphologieswere obtained. Individual bacterial and fungal colonieswere obtained from oral secretions of the three beetlespecies and were maintained in pure culture on 50% TSA(bacteria) or malt extract agar (Difco™) (fungi) media. PCR amplification of the ribosomal RNA gene and sequencing  Pure cultures, determined based on uniform macro-morphology, representative of the various oral secretionmorphologies present in at least 66% of the cultures froma given insect species, were obtained by transferringindividual colonies to 10% TSA plates. Cultures of individualisolates were obtained in this manner from at least threespecimens per species and these isolates were used foridentification based on molecular analysis.For molecular identification, individual bacterial iso-lates were grown in 5 ml of 50% tryptic soy broth at 28 ° Cfor 2 days. Individual fungal isolates were grown in 50%malt extract broth at 28 ° C for 2 days. DNA was extractedfollowing the methods described in Broderick et al. (2004)and White et al. (1990). These methods were selectedbecause they are widely used for molecular identificationand phylogenetic studies. Identities of microorganismswere obtained by performing PCR amplification andsubsequent sequencing of the 16S ribosomal RNA gene(White et al., 1990; Lane, 1991; Broderick et al., 2004).Sequences were determined on an ABI 377 DNA sequencer(Applied Biosystems, Foster City, CA, USA) at the Univer-sity of Wisconsin–Madison Biotechnology Center.The sequences obtained with each of the primerswere assembled and cleaned using DNASTAR Seqman(Dnastar, Madison, WI, USA) and then subjected to aclosest known sequence match in GenBank (National Centerfor Biotechnology Information, US National Library of Medicine, Bethesda, MD, USA) using the nucleotide-nucleotide BLAST search mode.  140 Cardoza et al.  Table 1 Identity of bacterial isolates from the oral secretions of three bark beetle species: spruce beetle (SB), mountain pine beetle (MPB), and pine engraver (PE)HostIsolate 1 GenBank no.Closest matchSimilarity (%)SBSBOF-1EU476056  Methylobacterium  spec.1 000/1 010 (99)SBOSC-1EU476057  Novosphingobium  spec. 942/957 (98)SBOSE-1EU476058 Sphingomonas rhizogenes  901/901 (100)SBOSIEU476073 Chryseobacterium  spec. 778/778 (100)SBBac4EU476059 Bacillus pumilus  752/755 (99)SBOSWEU476065 Staphylococcus succinus  852/855 (99)SBOSXEU476066 Staphylococcus succinus  567/574 (98)SBOSB1EU476064 Staphylococcus waneri  950/955 (99)SBBac10EU476067 Staphylococcus hominis  731/733 (99)SBOSUEU476055 Burkholderia cepacea  904/910 (99)SBBac7EU476053  Alcaligenes faecalis  946/960 (98)SBBac5EU476054  Alcaligenes faecalis  889/897 (99)SBBac9EU476063 Serratia grimesii  607/610 (99)SBBac1EU476061 Pseudomonas graminis  452/456 (99)SBBac3EU476060 Pseudomonas graminis  646/653 (98)SBOSA-1EU476062 Pseudomonas graminis  935/948 (98)SBOSMEU476052  Arthrobacter   spec. 646/653 (98)SBBac11EU476051  Micrococcus luteus  577/579 (99)SBOSDEU476070 Streptomyces  spec. 742/746 (99) Phylogenetic analysis Voucher specimens of bacterial colonies were preserved inthe forest entomology laboratory in the Department of Entomology of the University of Wisconsin. Once thebacteria isolated from the three beetle species wereidentified, we determined the degree of (dis)similarity in the bacterial composition of their oral secretions.Sequences from each library were aligned in ARB (Ludwiget al., 2004) using the Ribosomal Database Project (RDP)7.1 phylogenetic tree as comparison (Hugenholtz, 2002).Alignments were exported from ARB in phylip  format.A phylogenetic tree was constructed in paup * (Swofford,2003) using parsimony methods with Heuristic search.The tree was subjected to bootstrap resampling (1 000replications). Reference sequences from GenBank were alsoincluded in the phylogenetic tree. Taxonomic descriptionswere verified based on the position of each aligned sequencein the phylogenetic tree. Nucleotide sequences analyzedin this study were deposited in GenBank and theiraccession numbers are provided (Table 1). Results Isolations from the oral secretions of the three beetlespecies yielded a diverse bacterial community. Bacterialcolonies could be observed on the culture plates as soon as24–48 h after spreading. We observed no differences in themorphology of microbes associated with beetle gender. Allbeetle species hosted bacteria similar to members of α -, β -,and γ  -Proteobacteria, Actinobacteria, Bacteroidetes, andFirmicutes, except for mountain pine beetle, which yieldedno α -Proteobacteria or Bacteroidetes isolates (Figure 1,Table 1). Approximately 90% of the sequences were at least97% identical to known sequences in the database.The placement of the 16S rRNA sequences in the boot-strapped phylogenetic tree (Figure 1) was similar to thatobtained when the sequences were added to an existingRDP tree in ARB. Many sequences from the three beetlespecies clustered in the tree. For instance, the Bacillus cluster in the tree contained sequences from all threebeetle species. Within the larger Bacillus  cluster, smallerclusters were formed according to species, among whichwere isolates similar to Bacillus pumilus  Meyer and Gottheil, Bacillus cereus  Frankland and Frankland,  Bacillus subtilis (Ehrenberg) Cohn,  Bacillus popillae  Dutky, and unidentified Bacillus  spp. All Bacillus  isolates from mountain pinebeetle, except one, clustered with B. subtilis  and B. popillae .The largest cluster in the tree was the Actinobacterialcluster, containing isolates similar to nine genera, of whichsequences similar to Streptomyces  were prominent amongspruce beetle and pine engraver isolates. Spruce beetleisolates, in particular, clustered with various Streptomyces spp. Among isolates similar to Pseudomonas  spp. in the γ  -Proteobacteria, all mountain pine beetle isolates clustered   Microbes in bark beetle oral secretions 141 SBOSQEU476077 Streptomyces  spec. 627/630 (99)SBOSHEU476072 Streptomyces  spec. 976/982 (99)SBOSLEU476076 Streptomyces diasticus  799/801 (99)SBOSJ-1EU476074 Streptomyces olivaceus  988/990 (99)SBOSKEU476075 Streptomyces costaricanus  758/760 (99)SBOSREU476078 Streptomyces albidoflavus  910/916 (99)SBOSGEU476071 Streptomyces albidoflavus  992/999 (99)SBOSABEU476069 Streptomyces albidoflavus  915/920 (99)SBBac2EU476068 Streptomyces somaliensis 1 392/1 398 (99)MPBMPBDPFF3aEU476018 Brevibacillus  spec. 738/783 (94)MPBA1EU476012 Bacillus subtilis  611/618 (98)MPBOSA51EU476016 Bacillus subtilis  997/1035 (96)MPBOSA26EU476015 Bacillus subtilis  676/703 (96)MPBA29EU476014 Bacillus subtilis 1 021/1 052 (97)MPBA15EU476013 Bacillus subtilis 1 041/1 063 (97)MPBDPFF10EU476017 Stenotrophomonas maltophila  258/276 (93)MPBDPMF2EU476011  Alcaligenes faecalis  651/657 (99)MPBDPFF9BEU476010  Alcaligenes faecalis  741/742 (99)MPBA40EU476009  Alcaligenes faecalis 1 088/1 133 (96)MPB13EU476019 Pantoea cedenensis 1 059/1 102 (96)MPBDPBA28EU476020 Pseudomonas fluorescence  965/998 (96)MPBA23EU476025 Pseudomonas  spec.1 029/1 053 (97)MPBOSA52BEU476024 Pseudomonas brenneri  838/873 (95)MPBA43EU476023 Pseudomonas  spec. 834/845 (98)MPBDPFF2EU476022 Pseudomonas  spec. 826/826 (100)MPBDPFF1aEU476021 Pseudomonas fluorescens  706/713 (99)MPBOSA52AEU476026  Microbacterium oxydans  955/968 (98)PEPEIPFFD21EU476043  Methylobacterium fujisawaense  836/842 (99)PEIPFFA5EU476042  Methylobacterium adhaesivumtype  725/736 (98)PEIPFFA6EU476036 Chryseobacterium proteolyticum  783/794 (98)PEIPFFB6TEU476037 Chryseobacterium proteolyticum  664/672 (98)PEIPFB29EU476041 Bacillus anthracis  803/803 (100)PEIPMFA22EU476039 Bacillus megaterium 1 070/1 092 (97)PEIPMFA27EU476040 Bacillus megaterium 1 007/1 029 (97)PEIP16EU476046 Paenibacillus agaridevorans  751/763 (98)PEIPFFB9EU476047 Stenotrophomonas dokdonensis  636/661 (96)PEIPFFB7EU476048 Stenotrophomonas maltophila  842/847 (99)PEBacIPD3EU476038  Alcaligenes faecalis  801/802 (99)PEIPFA24EU476044 Raoultella terrigena  723/734 (98)PEIPMFA26EU476045 Raoultella terrigena  801/802 (99)PEIPC15AEU476028  Micrococcus luteus  786/787 (99)PEIPFFC13EU476033 Kocuria rosea  979/986 (99)PEIPFFC18EU476030 Corynebacterium aurimucosum  595/603 (98)PEIPFFC19EU476029 Brevibacterium aureum  956/961 (99)PEIPFFC14EU476050  Arthrobacter agilis  806/822 (98)PEIPFFD20EU476034  Mycobacterium jacuzzii  757/774 (97)PEIP5AEU476035 Rathayibacter tritici  865/875 (98)PEIPMFB27EU476031 Curtobacterium flaccumfaciens  579/587 (98)PEIPMFB28EU476032 Curtobacterium flaccumfaciens  389/391 (99)PEIPFFC15EU476027  Micrococcus luteus  773/774 (99)PEIPFFC12EU476049 Streptomyces exfoliatus  694/697 (99) 1 All isolates sequenced were present in at least 66% of the cultures obtained from individual beetle species.HostIsolate 1 GenBank no.Closest matchSimilarity (%)  Table 1 Continued   142 Cardoza et al. Figure 1 Phylogenetic relationship inferred from partial 16S rRNA sequences of bacteria obtained from oral secretion of the bark beetles Dendroctonus rufipennis , Dendroctonus ponderosae , and Ips pini . The tree was constructed using a maximum parsimony method. Bootstrap values for 1 000 replicates are given at each branching point. Only bootstrap values >50% are provided. The first two or three letters of the isolate name indicate the beetle species they were isolated from: SB, spruce beetle ( D. rufipennis ), MPB, mountain pine beetle ( D. ponderosae ), and PE, pine engraver ( I. pini ). Sequences not preceded by these letters represent the taxon sequences most closely matching those obtained in our study.
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