PHYSIOLOGY,BIOCHEMISTRY, AND TOXICOLOGY Bacterial Communities within Digestive Tracts of Ground (Coleoptera: Carabidae)

1,2 1 3 JONATHAN G. LUNDGREN, R. MICHAEL LEHMAN, AND JOANNE CHEE-SANFORD

Ann. Entomol. Soc. Am. 100(2): 275Ð282 (2007) ABSTRACT We identiÞed the bacterial communities within the alimentary tracts of two granivorous ground beetles as a Þrst step in the exploration of bacteriaÐground symbioses. Terminal- restriction fragment length polymorphism analyses of bacterial rRNA extracted from the guts of Þeld-collected individuals of Harpalus pensylvanicus (DeGeer) and Anisodactylus sanctaecrucis (F.) (Coleoptera: Carabidae) revealed that gut-associated bacterial communities were of low diversity. Individuals from the same beetle species possessed similar bacterial community proÞles, but the two species exhibited unique proÞles. Bacterial 16S rRNA clone libraries constructed for the two beetle species showed that H. pensylvanicus had a more diverse community (six operational taxonomic units [OTUs]) compared with A. sanctaecrucis (three OTUs). Only one OTU, closely related to Hafnia alvei, was common between the two beetle species. Cloned partial 16S rRNA sequences for each OTU were most closely matched to the following cultivated bacteria: Serratia sp., Burkholderia fungorum, and H. alvei and Phenylbacterium sp., Caedibacter sp., Spiroplasma sp., Enterobacter strain B-14, and Weissella viridescens, representing the divisions Alpha-, Beta- and Gammaproteobacteria, Mollicutes, and Bacilli. Some, but not all of these organisms have been previously associated with . The identiÞcation of bacteria uniquely and consistently associated with these ground beetles provides the basis for further investigation of species-speciÞc functional roles.

KEY WORDS biological control, endosymbiont, granivore,

Mutualistic symbiotic associations with microorgan- bionts are manifested in the development, fecundity, isms are phylogenetically widespread in insects, and and survivorship of their insect hosts (Norris and the microbes serve an array of functions for their Baker 1967, Douglas 1998, Nakabachi and Ishikawa insect hosts, including nutritional services. Microbes 1999, Aksoy 2000). provide deÞcient nutrients, or building blocks and Many of the bacterial symbionts known to play a enzymes requisite for their biosynthesis, essential to role in insect nutrition belong to Enterobacteriaceae insect development and reproduction. SpeciÞc nutri- within the Gammaproteobacteria and less frequently ents produced by microbes include essential amino to other bacterial families (Douglas 1998, Moran and acids (Prosser and Douglas 1991, Douglas and Prosser Baumann 2000). These bacteria can live intracellularly 1992, Shigenobu et al. 2000, Gil et al. 2003), precursors within specialized tissues named bacteriocytes or bac- or enzymes essential to the generation of vitamins and teriotomes, or they can live extracellularly within the micronutrients (Wicker 1983, Nakabachi and Ishikawa gut or other tissues. Intracellular bacteria are often 1999, Aksoy 2000, Akman et al. 2002), and sterols (Norris obligate symbionts with reduced genomes that rely in et al. 1969, Wetzel et al. 1992, Morales-Ramos et al. 2000). part on their insect hosts for several gene products in Metabolic processes related to insect nutrition that are exchange for supplying their host with some nutri- assisted by mutualistic bacteria include the storage and tional function (Gil et al. 2003, Tamas and Andersson recycling of nitrogen (Potrikus 1981, Cochran 1985, 2003, Schaber et al. 2005). Many extracellular gut bac- Sasaki et al. 1996, Douglas 1998, Lauzon et al. 2000, Gil teria have been described previously (Ohkuma and et al. 2003), cellulose digestion (Breznak and Brune Kudo 1996, Harada et al. 1997, Dillon et al. 2002, Bro- 1994), sulfate assimilation (Douglas 1988, Shigenobu derick et al. 2004), and these species can be abundant et al. 2000, Gil et al. 2003), and augmentation of ex- within the guts of some insects (Cazemier et al. 1997). isting host metabolic processes (Heddi et al. 1993, Compared with intracellular species, the roles of ex- 1999). Ultimately, the effects of these nutritional sym- tracellular gut bacteria in host nutrition are not well understood and deserve additional study, particularly Mention of a proprietary product does not constitute an endorse- for economically important insect species. ment or a recommendation by the USDA for its use. Ground beetles (Coleoptera: Carabidae) are an im- 1 North Central Agricultural Research Laboratory, USDAÐARS, portant group of beneÞcial insects that consume a Brookings, SD 57006. 2 Corresponding author, e-mail: [email protected]. range of different foods. Most species are polyphagous 3 Invasive Weed Management Unit, USDAÐARS, Urbana, IL 61801. carnivores of , and this group has long been 276 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 100, no. 2 appreciated as a source of nonchemical pest manage- DNA from all gut samples was extracted using mod- ment of insect pests in cropland (Brust et al. 1986, iÞed bead beating procedures to optimize recovery of Lovei and Sunderland 1996, Lundgren 2005a). nucleic acids. Brießy, samples were washed once with ϫ Granivory is also widespread within Carabidae, oc- 1 phosphate-buffered saline (0.12 M KH2PO4 and curring most frequently within the tribes Harpalini 0.15 M NaCl, pH 8), and the biomass was resuspended and Zabrini (Zhavoronkova 1969, Zetto Brandmayr in fresh buffer. Cell lysis was accomplished using mix- 1990). Granivorous insects, including ground beetles, tures of zirconia/silica beads (0.1Ð2.5 mm), and lysis play an important role in shaping the density and efÞciencies were assessed microscopically. The sam- dispersion of weed communities within agricultural ples were shaken on a platform (MO BIO Laborato- systems (Cavers 1983, Crawley 2000). The bacterial ries, Inc., Carlsbad, CA) attached to a standard vortex symbionts that inßuence the feeding behavior and on high speed for 2 min. Additional cell disruption nutrition of ground beetles undoubtedly affect their procedures were applied as needed, including the utility as biological control agents of insect pests and addition of 750 ␮l of lysis buffer (0.1 M NaCl and 0.5 weed seed banks. M Tris, pH 8) or additional grinding with a sterile Harpalus pensylvanicus (DeGeer) and Anisodacty- stainless steel rod. Samples were split equally to re- lus sanctaecrucis (F.) are ground beetles that occur cover DNA either through chemical extraction or throughout much of North America (Bousquet and solid phase extraction by using commercially available Larochelle 1993), are commonly encountered in crop- columns according to manufacturerÕs instructions land (Kirk 1973, 1977; Brust et al. 1986; Ellsbury et al. (MO BIO Laboratories, Inc.) following cell lysis pro- 1998; Lundgren et al. 2006), and consume both insect cedures. For chemical extraction, an equal volume of pests (Brust et al. 1986) and weed seeds (Brust and phenol/chloroform/isoamyl alcohol (24:24:1) (Sigma- House 1988, Lundgren 2005b). Preliminary experi- Aldrich, St. Louis, MO) was added to the cell lysate, ments revealed that feeding A. sanctaecrucis and H. mixed well, and DNA from the recovered aqueous pensylvanicus diet-incorporated tetracycline hydro- fraction was precipitated overnight at Ϫ80ЊCin3M chloride and sorbic acid reduced consumption of sodium acetate, pH 5.2, and an equal volume of cold weed seeds (Chenopodium album L.) by 40% in the isopropanol (Ϫ4ЊC). The DNA pellet was washed laboratory relative to those fed diet without antibiot- once with 70% cold ethanol, air-dried, and resus- ics. We hypothesize that there is a microbial contri- pended in TE buffer (10 mM Tris and 1 mM EDTA, pH bution to seed digestion in these ground beetles. 8). The pool of DNA from each sample was quantiÞed Here, we report the Þrst in a series of articles on the using a NanoDrop ND-1000 spectrophotometer role of microbial endosymbionts in the nutrition of (NanoDrop Technologies, Wilmington, DE). DNA ground beetles. SpeciÞcally, we conducted analyses of quantities were in the range of 5Ð211 ng/␮l. gut DNA from individuals of A. sanctaecrucis and H. T-RFLP Analysis. T-RFLP analysis was applied to pensylvanicus by using terminal restriction fragment assess the complexity of the bacterial communities length polymorphism (T-RFLP) analyses of bacterial residing in A. sanctaecrucis and H. pensylvanicus rRNA to gauge the complexity of the gut bacterial guts, particularly noting the intra- and interspeciÞc communities and to evaluate both intra- and interspe- variation in the guts of these beetles. The 16S rRNA ciÞc variation of the gut communities on a small num- genes were ampliÞed using bacterial primers F27 ber of beetles. Also, we constructed 16S rRNA clone (5Ј-AGAGTTTGATCMTGGCTCAG-3Ј) labeled at the libraries to determine the identity of these gut bac- 5Ј end with 6-carboxyßuorescein and 1492R (5Ј-GGT teria. To our knowledge, there are no previous cul- TACCTTGTTACGACTT-3Ј) (Operon, Huntsville, AL). ture-independent studies of the gut bacterial commu- AmpliÞcation reactions were performed in 100-␮l re- nities from Carabidae. actions containing 50 ng of DNA, 2.5 U of TaKaRa Ex Taq (Takara Mirus Bio, Madison, WI), 10 ␮lof10ϫ Ex Taq buffer, 10 ␮l of dNTP mixture (2.5 mM each), 2.5 ␮g of T4 gene 32 protein (Roche Diagnostics, India- Materials and Methods napolis, IN), and 2 ␮l of each primer (40 pmol/␮l). Adults of H. pensylvanicus and A. sanctaecrucis were The samples were ampliÞed in a DNA Engine PTC-200 collected from organic farmland in Champaign, IL, Thermal Cycler (MJ Research, Waltham, MA) by us- during October 2004. Individuals were maintained in ing the following conditions: 94ЊC for 5 min, followed the laboratory before the experiment in unsterilized by 25 cycles consisting of 94ЊC for 1.5 min, 55ЊC for 1.5 soil (Fer-Til, GreenGro Products, Jackson, WI) and min, 72ЊC for 1.5 min, and a Þnal extension at 72ЊC for were fed cat food (IamÕs Original formula, The IamÕs 7 min. One sample from A. sanctaecrucis and three Company, Dayton, OH). samples of H. pensylvanicus required an additional DNA Extraction. A. sanctaecrucis (n ϭ 6; 17% male) nested reaction of 20 cycles by using 0.5Ð1 ␮lofthe and H. pensylvanicus (n ϭ 4; 50% male) adults were primary polymerase chain reaction (PCR) product collected, and the digestive tracts were dissected under the conditions described above. within 24 h. The entire alimentary canal, excluding the The correct size of the ampliÞed DNA product was Malpighian tubules, was dissected in a RingerÕs solu- conÞrmed by gel electrophoresis, and 70Ð95-␮l vol- tion (0.75 g of NaCl, 0.35 g of KCl, and 0.28 g of CaCl2 umes were cleaned using the QIAquick PCR puriÞ- in 1 liter of water). Guts were stored at Ϫ20ЊC for 48 h cation kit (QIAGEN, Valencia, CA) and eluted in 30 before DNA extraction. ␮l of buffer EB (QIAGEN). PuriÞed DNA was quan- March 2007 LUNDGREN ET AL.: GUT BACTERIA OF GROUND BEETLES 277 tiÞed spectrophotometrically, and Ϸ450 ng of each with products resolved on 4% Metaphor agarose sample was digested overnight in separate 12-␮l re- (Cambrex Corp., East Rutherford, NJ) gels. Plasmid actions with 10 U of HhaI (New England Biolabs, minipreps (Wizardplus Minipreps, Promega) were Beverly, MA) at 37ЊC. Fragments were analyzed at the performed on several representatives of each RFLP University of Illinois W. M. Keck Center for Compar- type, and sequencing reactions (2ϫ coverage) were ative and Functional Genomics (Urbana-Champaign, conducted using M13 F/R (sequencing performed at IL) with an ABI 377 genetic analyzer (Applied Bio- the Iowa State University Sequencing Facility, Ames, systems, Foster City, CA) by using a mixture of 3 ␮lof IA). Consensus sequences (Ϸ550 bases) were deter- digested PCR product and 0.5 ␮l of a 35-mer TET mined based on alignments and editing performed internal standard, along with Genescan-2500 5-car- with Bioedit 7.5 (http://www.mbio.ncsu.edu/BioEdit/ boxytetramethylrhodamine size standard (Applied page2.html). Clones representing the same RFLP pat- Biosystems). Fragments were analyzed using Gene- tern were grouped under a representative sequenced Scan Analysis version 2.1 (Applied Biosystems), and clone, and these groupings were further consolidated peaks above a threshold value of 80 ßuorescent units by considering all representative sequences Ͼ97% were determined to be above background relative to similar as the same (Speksnijder et al. 2001). Se- blank samples. quences representative of each clone grouping were Cloning and Sequencing. The T-RFLP analyses in- compared against the GenBank database by using dicated that the composition of gut microbial commu- BLASTn (Altschul et al. 1997) to determine the closest nities from both species was fairly simple and relatively database match. Chimeric sequences that were iden- consistent intraspeciÞcally but that species-speciÞc dif- tiÞed after screening with Chimera_Check version 2.7 ferences may be present. The DNA was pooled from RDP8.1 and Bellerophon (Huber et al. 2004) were the six A. sanctaecrucis and four H. pensylvanicus in- removed from further consideration. The coverages of dividuals used for the T-RFLP analysis. Two 16S rRNA our clone libraries were estimated using the following gene clone libraries were generated to identify the equation from Marchesi et al. (2001): populations within the gut microbiota representative clone coverage ϭ 1 Ϫ ͑n/N͒ ϫ 100 of each insect species. Based on these Þndings, we constructed two 16S rRNA gene clone libraries from where n is the number of clones only occurring once, the DNA extracts described above. and N is the total number of clones in the library. Partial (Ϸ560 bases) 16S rRNA gene sequences Unique 16S rRNA gene sequences representing were ampliÞed in a T-Gradient thermal cycler (Bio- the clones reported in Table 1 were deposited in metra, Goettingen, Germany) from the extracted GenBank under accession numbers EF154420Ð DNA by using oligonucleotide primers 338 F (5Ј-ACT EF154427, EF198466. CCT ACG GGA GGC AGC-3Ј) (Amann et al. 1990) and 907R (5Ј-CCG TCA ATT CMT TTR AGT TT-3Ј) Results (Lane et al. 1985) (Invitrogen, Carlsbad, CA) in 50-␮l reactions composed of 0.4 mg/liter bovine serum al- T-RFLP Analysis. T-RFLP proÞles revealed that the bumin (BSA) (Roche Diagnostics. Indianapolis, IN), bacterial communities existing in the beetle guts were ϫ 1 PCR buffer (Promega, Madison, WI), 2 mM MgCl2, not diverse and that proÞles were relatively similar 0.5 ␮M each primer, 1.25 U of TaqDNA polymerase among individuals of the same species (Fig. 1). Inter- (Promega), 0.2 mM each dNTP (Promega), 1 ␮lof speciÞc differences in the bacterial diversity within template DNA (Ϸ100 ng of DNA), and molecular the digestive systems of A. sanctaecrucis and H. pen- grade water (Promega). PCR ampliÞcation was per- sylvanicus were observable. Two fragments, 369 and formed using the following conditions: 94ЊC for 4 min; 379 bp, were similar between the two beetle species. 30 cycles of 94ЊC for 0.5 min, 55ЊC for 0.5 min, 72ЊC for Two operational taxonomic units (OTUs) could be 0.5 min, and a Þnal elongation at 72ЊC for 3 min. PCR identiÞed from A. sanctaecrucis, and six OTUs were products were examined with positive (Escherichia identiÞed from H. pensylvanicus. coli DNA) and negative controls (reagents only) in a Cloning and Sequencing. Forty-Þve of the 47 clones 1.2% agarose gel to conÞrm speciÞcity of the ampliÞ- from A. sanctaecrucis gave useable sequence informa- cation reactions. tion. These clones formed three groups that are closely PuriÞed PCR products (Wizard PCR prep; Pro- related (Ͼ99%) to previously cultivated organisms mega) were cloned into E. coli JM109 competent cells (Table 1). Seven clones were closely related to Burk- by using the pGEM-T Easy Vector System II (Pro- holderia species, and the remaining 38 clones were mega) per manufacturerÕs instructions. Inserts from afÞliated with the Enterobacteriaceae. The estimated randomly selected transformed colonies (47 for each coverage of this clone library was 100%. beetle species library) were reampliÞed using the All 47 clones from H. pensylvanicus gave useable same PCR protocol modiÞed by an initial lysing step sequence information. At the 97% level of sequence (15 min; 99ЊC). Clone sequences were screened by similarity, these clones formed six groups that are RFLP analysis with restriction enzymes RsaI (10 U) relatively closely related, with one exception, to pre- and MspI (10 U) (New England Biolabs), 1ϫ buffer viously cultivated organisms (Table 1). There were (New England Biolabs), 1 mg/ml BSA (Roche Diag- three groups with only a single representative, of nostics), molecular grade water (Promega), and 10 ␮l which two were Alphaproteobacteria. The third loner of DNA template in 20-␮l reactions at 37ЊC (90 min) was only 89% similar to a cultured database entry, 278 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 100, no. 2

Table 1. Bacterial residents of the digestive tracts of the seed-feeding ground beetles A. sanctaecrucis and H. pensylvanicus, based on the closest cultured matches to generated sequences of the 16S rRNA gene fragment

Clone relative Closest cultured database match Accession Clone no. Taxonomic afÞliation abundance (% similarity, Ϸ550 bases) no.a A. sanctaecrucis (45 clones) AS-13 7 B. fungorum (Ͼ99%) Betaproteobacteria AF215706 AS-25 23 H. alvei (Ͼ99%) Gammaproteobacteria (Enterobacteriaceae) AY572428 AS-41 15 Serratia sp. (Ͼ99%) Gammaproteobacteria (Enterobacteraceae) AF286869 H. pensylvanicus (47 clones) HP-1 1 Phenylobacterium koreense (97%) Alphaproteobacteria (Caulobacteraceae) AB166881 HP-6 1 Caedibacter caryophilus (92%) Alphaproteobacteria (Rickettsiales) X71837 HP-7 1 Spiroplasma sp. BIUS-1 (89%) Mollicutes AY189319 HP-10 5 Enterobacter sp. B-14 (100%) Gammaproteobacteria (Enterobacteriaceae) AJ639856 HP-43 11 W. viridescens (97%) Bacilli (Lactobacillales) M23040 AS-25 28 H. alvei (Ͼ99%) Gammaproteobacteria (Enterobacteriacae) AY572428

a Closest cultured match in Genbank.

Spiroplasma sp. BIUS-1. Eleven of the clones were that of A. sanctaecrucis (three OTUs). H. pensylvanicus genetically similar with another gram-positive entry, was represented by four taxonomic classes, compared Weisella viridescens. The remaining majority of clones with two for A. sanctaecrucis. Only one class, Gamma- were split between two groups (28 and Þve clones) of proteobacteria, was common between the two librar- Enterobacteriaceae. The estimated coverage of this ies, and this class was the dominant class in both clone library was 94%. species. Only one cloned OTU was shared between The clone library from H. pensylvanicus had a the two beetles, indicating a 13% similarity in the greater taxonomic richness (six OTUs) compared with species compositions between the two libraries (⌺

Fig. 1. T-RFLP proÞles for the gut communities of the granivorous ground beetles H. pensylvanicus and A. sanctaecrucis. Each proÞle represents an individual beetle gut, and ampliÞed 16S rRNA bacterial gene fragments were digested using the HhaI restriction enzyme. March 2007 LUNDGREN ET AL.: GUT BACTERIA OF GROUND BEETLES 279 shared OTU/⌺ total OTU). However, this dominant 25), Caedibacter sp. (HP-6), and Enterobacter B-14 OTU represented Ϸ50% of the total clones from each (HP-10). Caedibacter is a polyphyletic genus related library (23/45 for A. sanctaecrucis; 28/47 for H. pen- through their intracellular lifestyle within paramecia. sylvanicus). Congeners closely related to C. caryophilus tend to be intracellular symbionts of amoebas and paramecia Discussion (Beier et al. 2002). Although Hafnia alvei and Enter- obacter B-14 have not previously been found in asso- DNA-based analyses of the gut bacteria indicate the ciated with insects, many other Enterobactereaceae presence of beetle-speciÞc, indigenous bacterial com- are frequently associated with insect guts (Moran and munities. The T-RFLP proÞles revealed simple gut Baumann 2000, Delalibera et al. 2005, Dunn and Stabb communities that were relatively reproducible in in- 2005), so it is not surprising to Þnd these species as dividuals from the same beetle species, although only abundant bacteria within the alimentary canals of a small number of insects were analyzed in each spe- ground beetles. cies. Clonal analyses conÞrmed that the bacterial com- Based on our previous knowledge of the microor- munities within H. pensylvanicus were more diverse ganisms found within the guts of ground beetles, these than in A. sanctaecrucis, and both beetle species pos- gut bacteria could be serving many functions, such as sessed unique microorganisms. 16S rRNA clone librar- causing or preventing disease, degrading insecticides, ies showed that both beetle species share a single and directly or indirectly contributing to food diges- bacterium, identiÞed as H. alvei that was numerically tion. Many of the bacteria identiÞed here are potential dominant in both libraries (Table 1). An in silico digest pathogens, such as B. fungorum (Gerrits et al. 2005), H. of the T-RFLP proÞles supports the assertion that the alvei (Sakazaki 2005), and Serratia spp. (especially S. dominant peak (369 bp; Fig. 1) is likely H. alvei (J.C.-S., marcescens). At subpathogenic levels, S. marcescens unpublished data). It is remarkable that common soil may be important in regulating the dynamics of the bacteria were not well represented in the guts and that bacterial community within the insect gut. For exam- the bacterial communities present in the alimentary ple, within the Formosan termite, S. marcescens is a system of ground beetles were primarily made up of facultative anaerobe that aids in consuming oxygen at species that have been retrieved from and the periphery of the insect stomach, thereby main- plants. The sum of the observations indicates that taining a habitable gut for the strict anaerobes that these gut bacteria have a close association with the digest cellulose (Adams and Boopathy 2005). beetles and that they are not simply transient organ- Microbes associated with insects are known to func- isms consumed with the beetleÕs food. tion in degrading plant defensive chemicals and in- Many of the bacteria identiÞed from the ground secticides (Berenbaum 1988). Several bacterial spe- beetle guts are commonly associated with animals and cies present in guts are known to plants, and several of the species are related to culti- vated organisms or clones previously associated with catabolize aromatic hydrocarbons, which are com- insects. Burkholderia fungorum has only recently been monly found in insecticidal chemicals. Enterobacter named (Coenye et al. 2001), and in addition to its strain B-14 degrades chlorpyrifos and organophos- association with humans, it may have a symbiotic re- phate insecticides (Singh et al. 2004), and although it lationship with fungi (Coenye et al. 2001). Its conge- has not previously been found within insect guts, it is ner, Burkholderia cepacia has been isolated from the a candidate for catabolizing the insecticides fre- gut of a bee, Osmia bicornis Lividict (Mohr and Tebbe quently encountered by these beetles in agroecosys- 2005). Serratia marcescens is known to associate with tems. Also, B. fungorum (Bodour et al. 2003) and a a broad range of insects that include gypsy moths close genetic match to our H. alvei clone (Laramee et (Broderick et al. 2004), Þeld crickets (Adamo 2004), al. 2000) are constituents of polyaromatic hydrocar- house ßies (Cooke et al. 2003), termites (Adams and bon-degrading consortiums. Finally, Caulobactere- Boopathy 2005), locusts (Dillon et al. 2002), curcu- aceae are known to degrade aromatic compounds, lionids (McNeill et al. 2000), aphids (Saguez et al. which are often found in some pesticides. 2005), and apple maggots (Lauzon et al. 2003). Cau- Microbes are frequently associated with nutritional lobacteriaceae are frequently ingested by aquatic in- functions in insects, and the bacterial species found in sects (Thanabalu et al. 1992); the closest cloned se- ground beetle guts may assist in digestion. Many of the quence to ours was isolated from the gut of the Enterobacteriaceae, which constituted the majority of mealworm, Tenebrio molitor L. (Coleoptera: Tenebri- clones in our beetle guts, produce digestive enzymes, onidae) (Dunn and Stabb 2005). Spiroplasma spp. are and have a demonstrated role in insect nutrition. For common residents of insect guts and hemocoel (Klein example, Enterobacter spp. assist nitrogen recycling in et al. 2002, Williamson et al. 1998, Hurst et al. 2003, Rhagoletis pomonella (Walsh) by producing uricases Anbutsu and Fukatsu 2003). Although Weisella is oth- (Lauzon et al. 2000), and S. marcescens and H. alvei are erwise not frequently associated with insects, one sim- known to produce chitinases (Whitaker et al. 2004, ilar clone to our HP-47 (Weisella viridescens) was Ruiz-Sanchez et al. 2005), which may be useful in found in the guts of honey bees, Apis mellifera L. digesting insect prey or fungi. Given the original ob- (Mohr and Tebbe 2005). servations of reduced seed consumption in “cured” Three clones isolated in our study have not been ground beetles, this is a symbiotic function that merits previously associated with insects: Hafnia alvei (AS- additional exploration. 280 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 100, no. 2

Acknowledgments Brust, G. E., B. R. Stinner, and D. A. McCartney. 1986. Predator activity and predation in corn agroecosystems. We thank Lynn Connor, Debra Hartman, and Kendra Environ. Entomol. 15: 1017Ð1021. Jensen for assistance with the T-RFLP and cloning portions Cavers, P. B. 1983. Seed demography. Can. Entomol. 61: of the research, and Gabriella Carrasco and Juan Carlos Laso 3578Ð3590. for assisting with the feeding assays. We also thank Karen Cazemier, A. E., J.H.P. Hackstein, H.J.M. Op den Camp, J. Kester and Molly Hunter for comments on earlier drafts of Rosenberg, and C. van der Drift. 1997. Bacteria in the the article. intestinal tract of different species of arthropods. Mol. Ecol. 33: 189Ð197. Cochran, D. G. 1985. Nitrogen excretion in cockroaches. References Cited Annu. Rev. Entomol. 30: 29Ð49. Coenye, T., S. Laevens, A. Willems, M. Ohlen, W. Hannant, Adamo, S. A. 2004. Estimating disease resistance in insects: J.R.W. Govan, M. Gillis, E. Falsen, and P. Vandamme. phenoloxidase and lysozyme-like activity and disease re- 2001. Burkholderia fungorum sp. nov. and Burkholderia sistance in the cricket Gryllus texensis. J. Insect Physiol. caledonica sp. nov., two new species isolated from the 50: 209Ð216. environment, animals and human clinical samples. Int. J. Adams, L., and R. Boopathy. 2005. Isolation and character- Syst. Evol. Microbiol. 51: 1099Ð1107. ization of enteric bacteria from the hindgut of the For- Cooke, E. A., G. O’Neill, and M. Anderson. 2003. The sur- mosan termite. Bioresour. Technol. 96: 1592Ð1598. vival of ingested Serratia marcescens in house ßies (Musca Akman, L., A. Yamashita, H. Watanabe, K. Oshima, T. Shiba, domestica) after electrocution with electric ßy killers. M. Hattori, and S. Aksoy. 2002. Genome sequence of the Curr. Microbiol. 46: 151Ð153. endocellular obligate symbiont of tse tse ßies, Wiggles- Crawley, M. J. 2000. Seed predators and plant population worthia glossinidia. Nat. Genet. 32: 402Ð407. dynamics, pp. 167Ð182. In M. Fenner [ed.], Seeds: the Aksoy, S. 2000. TsetseÐa haven for microorganisms. Parasi- ecology of regeneration in plant communities. CABI Pub- tol. Today 16: 114Ð118. lishing, Oxon, United Kingdom. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Delalibera, I. J., J. Handelsman, and K. F. Raffa. 2005. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped Conrasts in cellulytic activities of gut microorganisms BLAST and PSI-BLAST: a new generation of protein between the wood borer, Saperda vestita (Coleoptera: database search programs. Nucleic Acids Res. 25: 3389Ð 3402. Cerambycidae), and the bark beetles, Ips pini and Den- Amann, R. I., L. Krumholz, and D. A. Stahl. 1990. Fluorescent- droctonus frontalis (Coleoptera: Curculionidae). Envi- oligonucleotide probing of whole cells for determinative, ron. Entomol. 34: 541Ð547. phylogenetic, and environmental studies in microbiology. J. Dillon, R. J., C. T. Vennard, and A. K. Charnley. 2002. A Bacteriol. 172: 762Ð770. note: gut bacteria produce components of a locust cohe- Anbutsu, H., and T. Fukatsu. 2003. Population dynamics of sion pheromone. J. Appl. Microbiol. 92: 759Ð763. male-killing and non-male-killing spiroplasmas in Dro- Douglas, A. E. 1988. Sulphate utilization in an aphid sym- sophila melanogaster. Appl. Environ. Microbiol. 69: 1428Ð biosis. Insect Biochem. 18: 599Ð605. 1434. Douglas, A. E. 1998. Nutritional interactions in insect-mi- Beier, C. L., M. Horn, R. Michel, M. Schweikert, H.-D. Gortz, crobial symbioses: aphids and their symbiotic bacteria and M. Wagner. 2002. The genus Caedibacter compro- Buchnera. Annu. Rev. Entomol. 43: 17Ð37. mises endosymbionts of Paramecium spp. related to the Douglas, A. E., and W. A. Prosser. 1992. Synthesis of the Rickettsiales (Alphaproteobacteria) and to Francisella essential amino acid tryptophan in the pea aphid tularensis (Gammaproteobacteria). Appl. Environ. Mi- (Acyrthosiphon pisum) symbiosis. J. Insect Physiol. 38: crobiol. 68: 6043Ð6050. 565Ð568. Berenbaum, M. R. 1988. Allelochemicals in insectÑmicrobeÐ Dunn, A. K., and E. V. Stabb. 2005. Culture-independent plant interactions; agents provacateurs in the coevolutionary characterization of the microbiota of the ant lion Myrme- arms race, pp. 97Ð123. In P. Barbosa and D. K. Letourneau leon mobilis (Neuroptera: Myrmeleontidae). Appl. Envi- [eds.], Novel aspects of insectÐplant interactions. Wiley, ron. Microbiol. 71: 8784Ð8794. New York. Ellsbury, M. M., J. E. Powell, F. Forcella, W. D. Woodson, Bodour, A. A., J.-M. Wang, M. L. Brusseau, and R. M. Maier. S. A. Clay, and W. E. Riedell. 1998. Diversity and dom- 2003. Temporal change in culturable phenanthrene de- inant species of ground beetle assemblages (Coleoptera: graders in response to long-term exposure to phenan- Carabidae) in crop rotation and chemical input systems threne in a soil column system. Environ. Microbiol. 5: for the northern great plains. Ann. Entomol. Soc. Am. 91: 888Ð895. 619Ð625. Bousquet, Y., and A. Larochelle. 1993. Catalogue of the Gerrits, G. P., C. Klaassen, T. Coenye, P. Vandamme, and J. F. Geadephaga (Coleoptera: Trachypachidae, Rhysodi- Meis. 2005. Burkholderia fungorum septicemia. Emerg. dae, Carabidae including ) of America Infect. Dis. 11: 1115Ð1117. north of Mexico. Mem. Entomol. Soc. Can. 167: 1Ð397. Gil, R., F. J. Silva, E. Zientz, F. Delmotte, F. Gonzalez- Breznak, J. A., and A. Brune. 1994. Role of microorganisms Candelas, A. Latorre, C. Rausell, J. Kamerbeek, J. Gadau, in the digestion of lignocellulose by termites. Annu. Rev. B. Holldobler, et al. 2003. The genome sequence of Entomol. 39: 453Ð487. Blochmannia floridanus: comparative analysis of reduced Broderick, N. A., K. F. Raffa, R. M. Goodman, and J. Han- genomes. Proc. Natl. Acad. Sci. U.S.A. 100: 9388Ð9393. delsman. 2004. Census of the bacterial community of Harada, H., H. Oyaizu, Y. Kosako, and H. Ishikawa. 1997. the gypsy moth larval midgut by using culturing and Erwinia aphidicola, a new species isolated from pea aphid, culture-independent methods. Appl. Environ. Microbiol. Acyrthosiphon pisum. J. Gen. Appl. Microbiol. 43: 349Ð 70: 293Ð300. 355. Brust, G. E., and G. J. House. 1988. Weed seed destruction Heddi, A., A. M. Grenier, C. Khatchadourian, H. Charles, and by arthropods and rodents in low-input soybean agro- P. Nardon. 1999. Four intracellular genomes direct wee- ecosystems. Am. J. Alternat. Agric. 3: 19Ð25. vil biology: nuclear, mitochondrial, principal endosym- March 2007 LUNDGREN ET AL.: GUT BACTERIA OF GROUND BEETLES 281

biont, and Wolbachia. Proc. Natl. Acad. Sci. U.S.A. 96: (Apoidea) at an oilseed rape Þeld. Environ. Microbiol. 8: 6814Ð6819. 258Ð272. Heddi, A., F. Lefebvre, and P. Nardon. 1993. Effect of Morales-Ramos, J. A., M. G. Rojas, H. Sittertz-Bhaktar, and G. endocytobiotic bacteria on mitochondrial enzymatic Saldana. 2000. Symbiotic relationship between Hypothen- activities in the weevil Sitophilus oryzae (Coleoptera: emus hampei (Coleoptera: Scolytidae) and Fusarium solani Curculionidae). Insect Biochem. Mol. Biol. 23: 403Ð (Monilales: Tuberculariaceae). Ann. Entomol. Soc. Am. 93: 411. 541Ð547. Huber, T., G. Faulkner, and P. Hugenholtz. 2004. Bellero- Moran, N. A., and P. Baumann. 2000. Bacterial endosymbi- phon: a program to detect chimeric sequences in multiple onts in animals. Curr. Opin. Microbiol. 3: 270Ð275. sequence alignments. Bioinformatics 20: 2317Ð2319. Nakabachi, A., and H. Ishikawa. 1999. Provision of riboßavin Hurst, G.D.D., H. Anbutsu, M. Kutsukake, and T. Fukatsu. to the host aphid, Acyrthosiphon pisum, by endosymbiotic 2003. Hidden from the host: Spiroplasma bacteria infect- bacteria, Buchnera. J. Insect Physiol. 45: 1Ð6. ing Drosophila do not cause an immune response, but are Norris, D. M., and J. K. Baker. 1967. Symbiosis: effects of a suppressed by ectopic immune activation. Insect Mol. mutualistic fungus upon the growth and reproduction of Biol. 12: 93Ð97. Xyleborus ferrugineus. Science (Wash., D.C.) 156: 1120Ð Kirk, V. M. 1973. Biology of a ground beetle, Harpalus pen- 1122. sylvanicus. Ann. Entomol. Soc. Am. 66: 513Ð518. Norris, D. M., J. M. Baker, and H. M. Chu. 1969. Symbiotic Kirk, V. M. 1977. Notes on the biology of Anisodactylus interrelationships between microbes and ambrosia bee- sanctaecrucis, a ground beetle of cropland. Ann. Entomol. tles. III. Ergosterol as the source of sterol to the insect. Soc. Am. 70: 596Ð598. Ann. Entomol. Soc. Am. 62: 413Ð414. Klein, M., Y. Braverman, A. Chizov-Ginzberg, A. Gol’berg, D. Ohkuma, M., and T. Kudo. 1996. Phylogenetic diversity of Blumberg, Y. Khanbegyan, and K. J. Hackett. 2002. In- the intestinal bacterial community in the termite Reticu- fectivity of beetle spiroplasmas for new host species. litermes speratus. Appl. Environ. Microbiol. 62: 461Ð468. BioControl 47: 427Ð433. Potrikus, C. J., and J. A. Breznak. 1981. Gut bacteria recycle Lane, D. J., B. Pace, G. J. Olsen, D. A. Stahl, M. L. Sogin, and uric acid nitrogen in termites: a strategy for nutrient N. R. Pace. 1985. Rapid-determination of 16S Ribosom- conservation. Proc. Natl. Acad. Sci. U.S.A. 78: 4601Ð4605. al-RNA sequences for phylogenetic analyses. Proc. Natl. Prosser, W. A., and A. E. Douglas. 1991. The aposymbiotic Acad. Sci. U.S.A. 82: 6955Ð6959. aphid: an analysis of chlortetracycline-treated pea aphid, Laramee, L., J. R. Lawrence, and C. W. Greer. 2000. Mo- Acyrthosiphon pisum. J. Insect Physiol. 37: 713Ð719. lecular analysis and development of 16S rRNA oligo- Ruiz-Sanchez, A., R. Cruz-Camarillo, R. Salcedo-Hernandez, nucleotide probes to characterize a diclofop-methyl- and J. E. Barboza-Corona. 2005. Chitinases from Serratia degrading bioÞlm consortium. Can. J. Microbiol. 46: marcescens Nima. Biotechnol. Lett. 27: 649Ð653. 133Ð142. Saguez, J., R. Hainez, A. Cherqui, O. Van Wuytswinkel, H. Lauzon, C. R., T. G. Bussert, R. E. Sjogren, and R. J. Prokopy. Jeanpierre, G. Lebon, N. Noiraud, A. Beaujean, L. Joua- 2003. Serratia marcescens as a bacterial pathogen of nin, J.-C. Laberche, et al. 2005. Unexpected effects of Rhagoletis pomonella ßies (Diptera: Tephritidae). Eur. J. chitinases on the peach-potato aphid (Myzus persicae Entomol. 100: 87Ð92. Sulzer) when delivered via transgenic potato plants (So- Lauzon, C. R., R. E. Sjogren, and R. J. Prokopy. 2000. En- lanum tuberosum Linne) and in vitro. Transgenic Res. 14: zymatic capabilities of bacteria associated with apple 57Ð67. maggot ßies: a postulated role in attraction. J. Chem. Ecol. 26: 953Ð967. Sakazaki, R. 2005. Genus XV. Hafnia Moller 1954, pp. 681Ð Lovei, G. L., and K. D. Sunderland. 1996. Ecology and be- 685. In D. J. Brenner, N. R. Krieg, and J. T. Staley [eds.], havior of ground beetles (Coleoptera: Carabidae). Annu. BergeyÕs manual of systematic bacteriology. Springer, Rev. Entomol. 41: 231Ð256. New York. Lundgren, J. G. 2005a. Ground beetle (Coleoptera: Cara- Sasaki, T., M. Kawamura, and H. Ishikawa. 1996. Nitrogen bidae) ecology: their function and diversity within nat- recycling in the brown planthopper, Nilaparvata lugens: ural and agricultural habitats. Am. Entomol. 51: 218. involvement of yeast-like endosymbionts in uric acid me- Lundgren, J. G. 2005b. Ground beetles as weed control tabolism. J. Insect Physiol. 42: 125Ð129. agents: the inßuence of farm management on granivory. Schaber, J., C. Rispe, J. Wernegreen, A. Buness, F. Delmotte, Am. Entomol. 51: 224Ð226. F. J. Silva, and A. Moya. 2005. Gene expression levels Lundgren, J. G., J. T. Shaw, E. R. Zaborski, and C. E. Eastman. inßuence amino acid usage and evolutionary rates in 2006. The inßuence of organic transition systems on ben- endosymbiotic bacteria. Gene 352: 109Ð117. eÞcial ground-dwelling arthropods and predation of in- Shigenobu, S., H. Watanabe, M. Hattori, Y. Sakaki, and H. sects and weed seeds. Renewable Agric. Food Syst. 21: Ishikawa. 2000. Genome sequence of the endocellular 227Ð237. bacterial symbiont of aphids Buchnera sp. APS. Nature Marchesi, J. R., A. J. Weightman, B. A. Cragg, R. J. Parkes, and (Lond.) 407: 81Ð86. J. C. Fry. 2001. Methanogen and bacterial diversity and Singh, B. K., A. Walker, J.A.W. Morgan, and D. J. Wright. distribution in deep gas hygdrate sediments from the 2004. Biodegradation of chlorpyrifos by Enterobacter Cascadia Margin as revealed by 16S rRNA molecular strain B-14 and its use in bioremediation of contaminated analysis. FEMS Microbiol. Ecol. 34: 221Ð228. soils. Appl. Environ. Microbiol. 70: 4855Ð4863. McNeill, M. R., B.I.P. Barratt, and A. A. Evans. 2000. Be- Speksnijder, A.G.C. L., G. A. Kowalchuk, S. DeJong, E. Kline, havioural acceptability of Sitona lepidus (Coleoptera: J. R. Stephen, and H. J. Laanbroek. 2001. Microvariation Curculionidae) to the parasitoid Microctonus aethiopoides artifacts introduced by PCR and cloning of closely related (Hymenoptera: Braconidae) using the pathogenic bac- 16S rRNA gene sequences. Appl. Environ. Microbiol. 67: terium Serratia marcascens Bizio. Biocontrol Sci. Technol. 469Ð472. 10: 205Ð213. Tamas, I., and S.G.E. Andersson. 2003. Comparative genomics Mohr, K. I., and C. C. Tebbe. 2005. Diversity and phylotype of insect endosymbionts, pp. 39Ð52. In K. Bourtzis and T. A. consistency of bacteria in the guts of three bee species Miller [eds.], Insect symbiosis. CRC, Boca Raton, FL. 282 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 100, no. 2

Thanabalu, T., J. Hindley, S. Brenner, S. Oei, and C. Berry. Williamson, D. L., R. F. Whitcomb, J. G. Tully, G. E. 1992. Expression of the mosquidocidal toxins of Bacillus Gasparich, D. L. Rose, P. Carle, J. M. Bove, K. J. Hackett, sphaericus and Bacillus thuringiensis subsp. isrealensis by J. R. Adams, R. B. Henegar, et al. 1998. Revised group recombinant Caulobacter crescentus, a vehicle for biolog- classiÞcation of the genus Spiroplasma. Int. J. Syst. Bac- ical control of aquatic insect larvae. Appl. Environ. Mi- teriol. 48: 1Ð12. crobiol. 58: 905Ð910. Zetto Brandmayr, T. 1990. Spermophagous (seed-eating) Wetzel, J. M., M. Ohnishi, T. Fujita, K. Nakanishi, Y. Naya, H. ground beetles: Þrst comparison of the diet and ecology Noda, and M. Sugiura. 1992. Diversity in steroidogenesis of the harpaline genera Harpalus and Ophonus (Col., of symbiotic microorganisms from planthoppers. J. Chem. Carabidae), pp. 307Ð316. In N. E. Stork [ed.], The role of Ecol. 18: 2083Ð2094. ground beetles in ecological and environmental studies. Whitaker, J. O., Jr., H. K. Dannelly, and D. A. Prentice. 2004. Intercept, Andover, United Kingdom. Chitinase in insectivorous bats. J. Mammal. 85: 15Ð18. Zhavoronkova, T. N. 1969. Certain structural peculiarities of Wicker, C. 1983. Differential vitamin and choline require- the Carabidae (Coleoptera) in relation to their feeding ments of symbiotic and aopsymbiotic S. oryzae (Co- habits. Entomol. Rev. 48: 462Ð471. leoptera: Curculionidae). Comp. Biochem. Physiol. 76A: 177Ð182. Received 7 June 2006; accepted 20 December 2006.