Comparison of Midgut Bacterial Diversity in Tropical Caterpillars (Lepidoptera: Saturniidae) Fed on Different Diets

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Comparison of Midgut Bacterial Diversity in Tropical Caterpillars(Lepidoptera: Saturniidae) Fed on Different DietsAuthor(s): Adrián A. Pinto-Tomás, Ana Sittenfeld, Lorena Uribe-Lorío, FelipeChavarría, Marielos Mora, Daniel H. Janzen, Robert M. Goodman, and Holly M.SimonSource: Environmental Entomology, 40(5):1111-1122. 2011.Published By: Entomological Society of AmericaDOI: http://dx.doi.org/10.1603/EN11083URL: http://www.bioone.org/doi/full/10.1603/EN11083

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INSECT-SYMBIONT INTERACTIONS

Comparison of Midgut Bacterial Diversity in Tropical Caterpillars(Lepidoptera: Saturniidae) Fed on Different Diets

ADRIAN A. PINTO-TOMAS,1,2,3 ANA SITTENFELD,4 LORENA URIBE-LORIO,4

FELIPE CHAVARRIA,5 MARIELOS MORA,4 DANIEL H. JANZEN,5,6

ROBERT M. GOODMAN,1,7 AND HOLLY M. SIMON1,8

Environ. Entomol. 40(5): 1111Ð1122 (2011); DOI: http://dx.doi.org/10.1603/EN11083

ABSTRACT As primary consumers of foliage, caterpillars play essential roles in shaping the trophicstructure of tropical forests. The caterpillar midgut is specialized in plant tissue processing; its pH isexceptionally alkaline and contains high concentrations of toxic compounds derived from the ingestedplant material (secondary compounds or allelochemicals) and from the insect itself. The midgut,therefore, represents an extreme environment for microbial life. Isolates from different bacterial taxahave been recovered from caterpillar midguts, but little is known about the impact of these micro-organisms on caterpillar biology. Our long-term goals are to identify midgut symbionts and toinvestigate their functions. As a Þrst step, different diet formulations were evaluated for rearing twospecies of tropical saturniid caterpillars. Using the polymerase chain reaction (PCR) with primershybridizingbroadly to sequences fromthebacterial domain, 16S rRNAgene librarieswereconstructedwith midgut DNA extracted from caterpillars reared on different diets. AmpliÞed rDNA restrictionanalysis indicated that bacterial sequences recovered from the midguts of caterpillars fed on foliagewere more diverse than those from caterpillars fed on artiÞcial diet. Sequences related to Methylo-bacterium sp., Bradyrhizobium sp., and Propionibacterium sp. were detected in all caterpillar librariesregardless of diet, but were not detected in a library constructed from the diet itself. Furthermore,libraries constructed with DNA recovered from surface-sterilized eggs indicated potential for verticaltransmission of midgut symbionts. Taken together, these results suggest that microorganisms associ-ated with the tropical caterpillar midgut may engage in symbiotic interactions with these ecologicallyimportant insects.

KEY WORDS midgut microbiota, tropical caterpillar, Rothschildia lebeau, insect-microbe interac-tions, Area de Conservacion Guanacaste

Insects perform key ecological functions in many ter-restrial ecosystems, including the recycling of organicmatter within forests (Schultz 2002). The establish-ment of mutualistic associations with microorganismsin their intestinal tract has been a major aspect of theevolution of many insect orders, such as Isoptera (ter-mites; Brune 1998, Leadbetter et al. 1999) andHemiptera (aphids; Baumann et al. 1995, Moran et al.2003). However, the presence and roles of microbial

mutualists have not been well documented in otherinsect groups, in particular, those feeding on foliage.Included in this group are caterpillars (Lepidopteralarvae), one of the main consumers of foliage in trop-ical forests (Stamp and Casey 1993).

Like other herbivores, caterpillars maintain di-gestive tract conditions to extract nutrients fromplant tissue while minimizing harmful effects fromplant secondary metabolites (Governor et al. 1997,Bernays and Janzen 1988). Digestive tract traitsinclude an exceptionally alkaline pH (Dow 1992),low oxygen tension, and highly reductive chemistry(Appel and Maines 1995). Therefore, the caterpillarmidgut may be considered as an extreme environ-ment for microbial life, given that it harbors poten-tially inhibitory substances derived from plant tis-sue and excreted by the insect itself (Russell andDunn 1991), has rapid food passage rates, and lacksspecialized diverticula typical of insects that houselarge populations of microorganisms (Johnson andBarbehenn 2000). In theory, these extreme gut con-ditions may also protect the caterpillar from patho-

1 Department of Plant Pathology, University of Wisconsin, Madi-son, WI 53706.

2 Departamento de Bioquõmica, Facultad de Medicina.3 Centro de Investigacion en Estructuras Microscopicas.4 Centro de Investigacion en Biologõa Celular y Molecular, Univer-

sidad de Costa Rica, San Pedro de Montes de Oca, Costa Rica.5 Area de Conservacion Guanacaste, Guanacaste, Costa Rica.6 Department of Biology, University of Pennsylvania, Philadelphia,

PA 19104.7 Gaylord Nelson Institute for Environmental Studies, University of

Wisconsin, Madison, WI 53706.8 Corresponding author, Division of Environmental & Biomolecu-

lar Systems, Oregon Health and Science University, Beaverton, OR97006, e-mail: [email protected].

0046-225X/11/1111Ð1122$04.00/0 � 2011 Entomological Society of America

gens and microorganisms on the foliage that couldout-compete the insect for ingested nutrients aswell as directly attack it. However, this protectionhas a potential tradeoff: the exclusion of beneÞcialbacteria that could contribute to caterpillar Þtness,as they do for many other animals on earth (Rubyet al. 2004). As an example, the midgut microbialcommunity of gypsy moth caterpillars [Lymantriadispar (L.), Lymantriidae] fed with different dietshas been described as relatively simple, with 15microbial phylotypes at its most complex and sevenphylotypes at its simplest (Broderick et al. 2004).Similarly, the intestinal microbiota ofHepialus gong-gaensis (Hepialidae) caterpillars was also found tobe relatively simple (Yu et al. 2008). Nevertheless,these and other studies (Sittenfeld et al. 2002, In-diragandhi et al. 2008) also serve to demonstratethat at least some microorganisms are capable ofsurviving in the extreme conditions of the caterpil-lar midgut. Further research is needed to elucidatewhether these microorganisms also have mutualisticassociations with the caterpillar that contains them.

Because of their ecological importance and remark-able diversity, tropical caterpillars have been studiedextensively in the Area de Conservacion Guanacaste(ACG) in northwestern Costa Rica. The feeding hab-its, host plants, parasitoids, development, and behaviorof hundreds of species have been well documented(Janzen 1981, 1984, 1993, 2003; Janzen and Gauld 1997;Janzen et al. 2009; Miller et al. 2006, 2007), but little isknown about their intestinal microbiota. Recent workdemonstrated that bacterial isolates from midguts andpupal material of two ACG species of large saturniidcaterpillars,Automeris zugana andRothschildia lebeau,produced a variety of enzymes with potential rele-vance to caterpillar digestive biology, including cel-lulases, xylanases, amylases, and chitinases (Pinto-Tomas et al. 2007). However, microorganisms thatcolonize and persist in the caterpillar midgut have notyet been identiÞed.

The general goal of this research is to identify po-tentially mutualistic bacteria that colonize the cater-pillar intestinal tract, distinguishing them from bacte-ria that are transient on ingested food. As a start, thediversity of midgut microorganisms from caterpillarsreared on a sterile food source to that from caterpillarsfed on their natural host plants was compared (as didBroderick et al. 2004). We hypothesized that bacteriarecovered consistently from midguts, irrespective ofdiet and species, were likely to have biologically rel-evant associations with their caterpillar host (referredto hereafter as “potential mutualists”). Saturniid cat-erpillars were used to test this hypothesis, becausetogether with the family Sphingidae, they representthe two major groups of large caterpillars in Neotro-pical forests (Janzen 1984), in terms of species rich-ness and impact of herbivory. In fact, 95% of all speciesof large ACG caterpillars are either saturniids or sph-ingids (Miller et al. 2006). Rothschildia lebeau, Cith-eronia lobesis, and Eacles imperialis were chosen forthis study because they are representative of hundredsof species of tropical saturniids (Bernays and Janzen

1988), they are readily available in ACG, both in thewild and as Þeld-maintained breeding colonies, andhave a conveniently large size.

Initially, different artiÞcial diet formulations wereevaluated for their impact on caterpillar growth anddevelopment. Then, bacteria from the midguts of cat-erpillars reared on artiÞcial and natural diets werecompared using a cultivation-independent approach.In this study, we consider gut-associated microorgan-isms those located in the gut lumen as well as thoseattached to the peritrophic membrane, the gut wall, orboth. Results suggest that particular bacterial speciespersisted in the extreme conditions of the caterpillarmidgut regardless of diet, and may, therefore, alsoform mutualistic associations with the insect.

Materials and Methods

Caterpillar Rearing. Three different species of Sat-urniidae were employed in this study:Citheronia lobe-sis (subfamily Ceratocampinae), Eacles imperialis(subfamily Ceratocampinae) and Rothschildia lebeau(subfamily Saturniinae). In all cases, eggs were ob-tained from adult females that had oviposited on thewalls of plastic bags at ACG rearing facilities in SectorSanta Rosa (tropical dry forest, 300-m elevation). Eggswere surface sterilized by immersion in 0.5% bleachfor 1 min, followed by three washes with sterile dis-tilled water. Surface sterilization was tested by rollingthe eggs over nutrient agar plates. The eggs weredecreed to be surface-sterile because there was nomicrobial growth on this general purpose culture me-dia. Eggs were placed in UV-sterilized 5.5-oz. cups(Bio-Serv, Frenchtown, NJ). First-instar caterpillarswere transferred to plastic containers for feeding onartiÞcial diet shortly after emergence, or placed in thewild on branches of the tree Spondias mombin L.(Anacardiaceae) and covered by mosquito netting asprotection against predators and parasites. The cater-pillar species employed in this study normally ovipositon S. mombin and developing caterpillars are usuallyfound on these trees as well. Caterpillars fed withartiÞcial diet were reared in individual compartments;Þrst in 32-cell rearing trays (Bio-Serv), after whichthey were transferred to individual 5.5-oz. cups at thethird stadium. After the Þnal molt, the very large (last)Þfth-instar caterpillars were transferred to 16-oz. cupsuntil pupation. To facilitate pupation, R. lebeau cat-erpillars in the prepupal stage (when caterpillars ceasefeeding and evacuate gut contents but do not molt)were provided with a sterile kimwipe (Kimberly-Clark, Neenah, WI) and a stick to spin their cocoon.For C. lobesis and E. imperialis caterpillars, prepupaewere transferred to 16-oz cups containing 10 g ofautoclaved soil to allow the insects to bury themselvesand pupate.

Eight different diet formulations were tested to as-sess caterpillar development on different foodsources. All diets were prepared aseptically from ster-ile ingredients. First, a commercial diet (Giant Silk-worm Moth Diet, Bio-Serv) was employed, herebyreferred to as “Bio-Serv diet”. Then the protocol de-

1112 ENVIRONMENTAL ENTOMOLOGY Vol. 40, no. 5

scribed by Riddiford (1985) to rear the extra-tropicalsaturniid caterpillar Hyalophora cecropia was fol-lowed, without the addition of antibiotics (this diet isreferred as “Standard Diet” throughout the text). Wealso evaluated six variations of the Standard Diet thatincluded 1) removing casein, 2) 10% casein, 3) 25%casein, 4) removing wheat germ, 5) 10% wheat germ,and 6) 25% wheat germ. Caterpillars were reared in asemiopen enclosure at the microbiology lab in theACG, hence exposing the insects to similar conditionsto those found in the wild with respect to temperature,humidity, and photoperiod. Feeding was performedevery 48 h in a laminar ßow hood to avoid microbialcontamination of the diet and of the caterpillars.Midgut Dissection and DNA Extraction for Micro-bial Diversity Analysis. Ten caterpillars from eachcombination of diet and species were dissected 16 dafter feeding was initiated. Before dissection, cat-erpillars were surface sterilized by brief immersionin 70% ethanol. Sterile dissecting scissors were usedto cut laterally behind the head capsule, and the gut(crop and midgut) was removed from the cuticlewith forceps and placed in dissection buffer (Ring-erÕs buffer, modiÞed from Cazemier et al. 1997: 47mM NaCl, 183 mM KCl, 10 mM Tris-HCl, pH 9.0)with 10% formaldehyde and 20 mM PPi (sodiumtetrapyrophosphate). Each set of ten midguts wascombined into one sample, incubated for 4 h andwashed three times with Ringers buffer alone toremove formaldehyde and PPi. Finally, the midgutpool was resuspended in 70% ethanol and stored at4�C until transported to the University of Wiscon-sin-Madison. There, total DNA was extracted asfollows: 0.2 g of homogenized sample was rehy-drated with PBS (phosphate buffered saline, 137mM NaCl, 2.7 mM KCl, 12 mM NaHPO4, 1.8 mMKH2PO4, pH 7.4), followed by the addition of 600 �lof Plant DNAzol (Invitrogen Life Tech., Grand Is-land, NY). The sample was transferred to a 2-ml,screw-cap microcentrifuge tube containing 0.6 mgof 0.1-mm-diameter zirconium-silica beads, 0.6 mgof 1.0-mm-diameter zirconium silica beads, and one2.5-mm-diameter silica bead (BioSpec ProductsInc., Bartlesville, OK). Bead-beating of the samplewas performed at 5.5 m/s for 30 s in a FastPrep FP120beadbeater (Bio101 Inc., LA Jolla, CA). The super-natant was recovered after centrifugation in a mi-crocentrifuge (Eppendorf, Hamburg, Germany),and 600 �l of Plant DNAzol reagent and 80 �l of 10%cetyltrimethylammonium bromide (CTAB)/5MNaCl solution were added. The sample was nextmixed and incubated for 1 h at 65�C, followed byextraction with equal volumes of phenol-chloro-form-isoamyl alcohol (25:24:1) and chloroform-iso-amyl alcohol (24:1). Precipitation and washes wereperformed according to the DNAzol manufacturerÕsprotocol. DNA pellets were resuspended in 70 �l ofTE buffer (pH 8.0) and stored at �80�C. The sameprocedure was used to extract microbial DNA from0.2 g of artiÞcial diet and from two pools of 10surface-sterilized R. lebeau eggs.

Polymerase Chain Reaction (PCR). Bacterial 16SrRNA genes were ampliÞed by PCR from total midgutDNA, artiÞcial diet and caterpillar egg DNA withuniversal bacterial primers 27 F (Giovannoni 1991)and 1492R (Lane 1991). PCR reaction mixtures werethe same as in Bintrim et al. (1997), except for theaddition of 0.2 �l of 50 mg/ml bovine serum albumin(Sigma-Aldrich Corp., St. Louis, MO) and 0.05% vol:vol Nonidet (Sigma-Aldrich Corp.) per 25 �l reaction.PCR was performed in a Mastercycler Gradient ma-chine (Eppendorff, Hamburg, Germany).Construction of 16S rRNA Gene Libraries. To se-

lectively eliminate chloroplast and caterpillar se-quences that also ampliÞed with the bacterial 16SrRNA gene primer set, PCR products obtained fromfoliage-fed caterpillars were digested with the enzymePvuII before library construction. There is a PvuIIrestriction site within most chloroplast rRNA genes,resulting in shorter DNA fragments upon restrictionwith the enzyme. Full-sized PCR products obtainedfrom ampliÞcation with 1) total midgut DNA, 2) DNAfrom standard diet alone, and 3) caterpillar egg DNAwere separated by electrophoresis in 1% TAE agarosegels. DNA fragments were then puriÞed using theWizard SV gel and PCR clean-up system (Promega,Madison, WI) following the manufacturerÕs instruc-tions. PuriÞed DNA was cloned into the pGEM-Tvector (Promega) by ligation according to the man-ufacturerÕs instructions and introduced into compe-tent Escherichia coli DH5� cells by electroporation.Amplified Ribosomal DNA Restriction Analysis(ARDRA). Vector primers SP6 and T7 were used toamplify DNA inserts from individual clones by usingthe PCR protocol described above. Using standardtechniques, PCR products were digested with two setsof different restriction enzymes, HhaI/SacI (Pro-mega) and Sau3AI/SacI (Promega) to generate twodifferent ARDRA Þngerprints for each clone. Diges-tion products were visualized by electrophoresis in 2%TAE agarose gels and ARDRA patterns were analyzedand grouped using the GelComparII software (Ap-plied Maths, Kortrijk, Belgium).Sequencing and Sequence Analysis. We selected

representative clones from each ARDRA pattern forsequencing, including at least one clone from each pat-tern. 16S rRNA genes were ampliÞed by PCR as de-scribedabove.PCRproductswerepuriÞedby treatmentwith Exonuclease I (Promega) and Shrimp AlkalinePhosphatase(Promega)accordingtothemanufacturerÕsinstructions. Sequencing reactions were performed us-ing vector primers SP6 and T7 and the BigDye reactionmix (Perkin-Elmer Corp., Foster City, CA) followingmanufacturerÕs instructions. Excess dye terminatorswere removed using CleanSEQ reaction (AgencourtBioscience, Beverly, MA) and loaded into an AppliedBiosystems 3700 automated DNA sequencing instru-ment at the UW-Madison Biotechnology Center. Se-lected clones representing dominant ARDRA patternswere also sequenced using internal primers 700 F and700R (Lane 1991). Nucleotide sequences were analyzedand manually edited using Sequencher 4.5 (Gene CodesCorporation, Ann Arbor, MI). Sequences were evalu-

October 2011 PINTO-TOMAS ET AL.: BACTERIAL DIVERSITY IN CATERPILLAR MIDGUT 1113

ated with the Chimera Check software (RDP database,[http://rdp8.cme.msu.edu/cgis/chimera.cgi]) to detectand remove chimeric artifacts. 16S rDNA sequenceswere compared with GenBankÕs nonredundant nucleo-tide database by using BLAST 2.0 (Altschul et al. 1997)and aligned with the MEGA 4.0 software program (Ta-muraetal. 2007).Sequencesweredeposited inGenBankunder accession numbers FJ593709ÐFJ593729.Estimation of Bacterial Diversity in CaterpillarMidguts.Operational taxonomic units (OTUs) were de-Þned as groups of clones with �98% nucleotide identitybetween16SrDNAsequences.EstimateS(R.K.Colwell,[http://viceroy.eeb.uconn.edu/estimates]) was em-ployed to calculate bacterial diversity indexes and gen-erate rarefaction curves following methods described byHughes and Bohannan (2004).Phylogenetic Analyses. Phylogenetic analyses were

conducted with MrBayes v. 3.0b4 (Ronquist andHuelsenbeck 2003). Each analysis consisted of fourindependent chains, one cold, and three incremen-tally heated (T � 0.2), starting from random trees, andwas run for Þve million generations with trees sampledevery 100 generations to calculate posterior probabil-ities for each branch. The majority rule consensus treewas calculated after removing the Þrst 500 trees cor-responding to the burn-in period, estimated accordingto the log-likelihood curve. The General Time Re-versible (GTR) model was used with priors for thesubstitution rates set to a ßat distribution.Statistical Analyses. One-way analysis of variance

(ANOVA) statistical tests were performed with SASsoftware, employing the GLM procedure (SAS Insti-tute Inc., Cary, NC). Statistical power calculationswere performed with the G*Power 3 software (Faul etal. 2007).

Results

Development of Three Species of Tropical Satur-niid Caterpillars on Artificial Diet and Host Plant. R.lebeau has been the subject of a number of recentstudies investigating the midgut microbiota of tropicalcaterpillars (Amaral-Zettler et al. 2005; Pinto-Tomaset al. 2007). The rearing of tropical saturniid caterpil-lars on artiÞcial diet has not previously been reported.We took advantage of the availability of a commer-cially formulated diet for the caterpillar of the com-mon North American saturniid, Hyalophora cecropiato develop a protocol for rearing R. lebeau. Followingthis protocol, R. lebeau caterpillars were successfullyreared from hatching to adulthood on the artiÞcial diet(referred to as “Bio-Serv diet” throughout the text, Fig.1). Subsequently, we reared R. lebeau caterpillars onH. cecropia diet without antibiotics (Riddiford 1985);this diet also supported caterpillar development to theadult stage and is referred to as “standard diet”throughout the text. To further investigate diet effectson caterpillar development and to select suitable dietcomponents to investigate the midgut microbiota, weevaluated R. lebeau caterpillars reared on nine differ-ent diets, including: Spondias mombin (a native hostplant); Bio-Serv diet; standard diet (complete except

for antibiotics); and six variations of the standard dietwith different nutrients omitted (or reduced). Devel-opment was signiÞcantly slowed in caterpillars rearedon diets with reduced nutrients (F � 6.79; df � 8, 99;P � �0.0001, n � 108; Fig. 2). However, the devel-opment of caterpillars reared on either the Bio-Servdiet or the standard diet was not statistically differentfrom that of caterpillars reared on foliage from S.mombin (F � 0.63; df � 2, 33 P � 0.54, n � 36).However, statistical power calculations indicated that,given the observed differences in caterpillar develop-ment, a sample size of 180 insects (60 per diet) wouldbe needed to reject the null hypothesis that develop-ment is statistically similar on these diets. Two otherspecies of saturniids, C. lobesis and E. imperialis,werealso successfully reared on both the Bio-Serv diet andthe standard diet. For these species, development wasgenerally similar on artiÞcial diets and on S. mombin,but body pigmentation was notably different (Fig. 1).These results were consistent with unpublished re-search on artiÞcial diet-raised caterpillars by DHJ inthe 1980s.Sex Differentiation inR. lebeau and C. lobesis. The

method described by Miller et al. (1977) for sex dif-ferentiation in four species of North American satur-niids was successfully applied to differentiate sex ofR.lebeau and C. lobesis caterpillars, but not to E. impe-rialis caterpillars. 100 fourth- and Þfth-instarR. lebeaucaterpillars in total were sorted according to the pres-ence (females) or absence (males) of the developingfemale genitalia, which is visible to the naked eye asfour white subsurface spheres on the ventral side ofthe eighth and ninth abdominal segments (see Fig.1H). Of 47 caterpillars that pupated, 30 yielded viableadults. We correctly predicted the sex of all 12 femalesand of 17 out of 18 males. A similar criterion wasapplied for sex differentiation in C. lobesis (Fig. 1H),but no genitalia were visible in E. imperialis caterpil-lars. Given our success in this study, this may be agenerally useful approach for sex determination intropical saturniids.Midgut Bacterial Diversity of Caterpillars RearedonDifferentDiets.We compared the midgut bacterialcommunity of R. lebeau and C. lobesis caterpillars fedon standard diet to those caterpillars fed on S. mombinfoliage. Four bacterial 16S rRNA gene libraries wereconstructed from caterpillar midgut DNA: one libraryfrom each species feeding on foliage and another li-brary from each species feeding on standard diet.Treatment with the restriction enzyme PvuII was in-cluded when constructing libraries from caterpillarsfed on foliage to eliminate chloroplast sequences thatwere consistently ampliÞed with the bacterial PCRprimers. AmpliÞed Ribosomal DNA Restriction Anal-ysis (ARDRA) employing two different combinationsof restriction enzymes was performed on 100 clonesfrom each library. Twenty-three different ARDRApatterns were observed in the library from C. lobesisfed on standard diet, 47 ARDRA patterns in the libraryfromC. lobesis fedwithS.mombin,34ARDRApatternsin the library fromR. lebeau fed with artiÞcial diet, and42 ARDRA patterns in the library from R. lebeau fed


with S. mombin. Representative clones from eachARDRA pattern were sequenced. For bacterial diver-sity analyses, Operational Taxonomic Units (OTUs)were deÞned as groups of clones whose 16S rDNAsequences shared �98% identity over at least 1,300nucleotides. After sequence analysis, several cloneswith dissimilar ARDRA patterns grouped within thesame OTU. A small number of ARDRA patterns ineach library were identiÞed as chimeric sequencesand removed from the analysis.

The 16S rRNA gene libraries constructed from cat-erpillars fed with S. mombin yielded signiÞcantlyhigher diversity indexes than libraries constructedfrom caterpillars fed with standard diet (Table 1).Furthermore, rarefaction curves indicated that librar-ies from foliage-fed caterpillars had signiÞcantlyhigher richness compared with the libraries from stan-

dard diet-fed caterpillars (95% conÞdence; Fig. 3).Moreover, the fact that rarefaction curves constructedwith data from caterpillars fed on standard dietreached plateaus suggested that the number of clonessampled was sufÞcient to provide an accurate estima-tion of midgut bacterial diversity. However, the slopesof the rarefaction curves constructed with data fromcaterpillars fed on S. mombin indicated that samplingwas insufÞcient to accurately estimate total bacterialspecies richness.

Results suggested that some similarities in midgutmicrobial composition were dependent upon dietsource, yet independent of caterpillar species. Forexample, libraries from R. lebeau and C. lobesis cater-pillars fed with standard diet had a higher �- to �-Pro-teobacteria ratio compared with caterpillars fed withS. mombin (Fig. 4). However, some differences de-

Fig. 1. Life cycle of the three tropical saturniid caterpillars fed artiÞcial (standard diet) and leaf (Spondias mombin) dietsin this study. A-C: Shown here are the wild-caught adult stages (moth, top) and the fourth-larval instar (bottom) forRothschildia lebeau(A),Citheronia lobesis(B), andEacles imperialis(C). D-F: Appearance of abnormally colored fourth instarcaterpillars when fed on standard diet. G. R. lebeau cocoons from caterpillar fed on standard diet (left panel) and leaves ofSpondias mombin (central panel). C. lobesis pupa from caterpillar fed on standard diet (right panel). H. Caterpillar sex in R.lebeau andC. lobesis determined by the absence (left panel, male) or presence (right panel, female) of the developing femalereproductive organs, which are visible as four white subsurface spheres on the ventral side of the eighth and ninth abdominalsegments (as indicated by black arrows on right).


tected in the midgut microbial community appearedtoberelated tocaterpillar species.For instance, clonesbelonging to the �-Proteobacteria subdivision formedthe dominant taxonomic group from libraries of C.lobesis, whereas �-Proteobacterial clones were pre-dominant in R. lebeau libraries, regardless of diet.These results suggest that both diet and caterpillarspecies affected midgut bacterial diversity.Identification of Potential Mutualist Phylotypes.

The taxonomicafÞliationofphylotypesdeterminedbyBLAST analysis is summarized in Table 2. Most of thesequences recovered from caterpillar midguts hadhigh sequence identity (�98% identity over at least1,300 nucleotides) with their closest sequencematches in Genbank. For caterpillars fed on standarddiet, the most frequently detected phylotype was re-lated to Bradyrhizobium sp. in R. lebeau (47% ofclones), and Serratia marcescens in C. lobesis (42% ofclones). The most frequently detected bacterial phy-lotype in caterpillars fed with S. mombin was related

to Methylobacterium sp. in both caterpillar species,corresponding to 17% of clones in C. lobesis and 26%of clones inR. lebeau. Interestingly, phylotypes relatedto Methylobacterium sp., Bradyrhizobium sp., Propi-onibacterium sp.,Escherichia coli, andAcinetobacter sp.were present in all libraries regardless of diet andcaterpillar species (see Table 2).

To ascertain whether the standard diet itself was asource of bacterial sequences, we constructed a 16SrRNA gene library with DNA extracted from freshlyprepared standard diet. Sequences related to the 16SrRNA gene ofAcinetobacter sp., Stenotrophomonas sp.,and Staphylococcus sp., which were also recoveredfrom the midgut libraries, were among those detectedamong 50 clones analyzed. However, other predom-inant phylotypes recovered from caterpillar midguts,such as those grouping with Bradyrhizobium, Serratia,andMethylobacterium,were not detected in this “con-trol library.”

Fig. 2. Development of R. lebeau caterpillars when fed different artiÞcial diets and S. mombin. Twelve caterpillars weretested with each diet. Data correspond to caterpillar weight two weeks after hatching. Diet modiÞcations include decreasingthe amount of wheat germ and casein. All results are shown as the mean � SEM (n� 12). Means labeled with different lettersare signiÞcantly different. See text for further details. Caterpillar stadia ranged from Þrst to third, according to the provideddiet, and weights ranged from 0.22 g to 1.53 g in the complete artiÞcial diets, from 0.02 g to 0.77 g in the nutrient deÞcientartiÞcial diets and from 0.24 g to 1.66 g in caterpillars fed with S. mombin.

Table 1. Diversity indexes of 16S rRNA gene libraries constructed from midguts of two species of saturniid caterpillars fed on differentdiets

Diversity indexaC. lobesis fed on

standard dietC. lobesis fed onS. mombin

R. lebeau fed onstandard diet

R. lebeau fed onS. mombin

Number of clonesb 98 94 89 95Richnessc 12 23 9 27Shannon index 1.70 � 0.01 2.71 � 0.01 1.51 � 0.01 2.71 � 0.01Shannon evenness 0.68 0.86 0.69 0.82FisherÕs � 3.58 � 0.63 9.71 � 1.59 2.49 � 0.48 12.58 � 2.04Simpson index 3.96 � 0.04 12.38 � 0.20 3.36 � 0.04 10.07 � 0.21

aDiversity indexes were calculated with EstimateS (http://viceroy.eeb.uconn.edu/estimates), employing 100 randomizations for each dataset.b Although 100 clones were analyzed from each library, those clones representing chimeric sequences were removed from diversity analysis.cOperational taxonomic units (OTUs) were deÞned as 16S rDNA sequences at least 98% identical over at least 1,300 bp.


To evaluate whether microorganisms were presentinside of R. lebeau eggs, we constructed two 16S rRNAgenelibrariesbyusingtotalDNAextractedfromsurface-sterilized eggs. Several of the dominant phylotypes de-tected in the midgut libraries were also recovered fromthe egg libraries, including Methylobacterium sp., Acin-etobacter sp.,Acidovorax sp., andSerratiamarcescens(Ta-ble2).Becauseof lackofavailableeggs,wewerenotabletorepeat thisexperiment for theothercaterpillar speciesemployed in this study.

We predicted that bacteria recovered consistentlyfrom caterpillar midguts, independent of diet and spe-cies, would be good candidates for bacterial mutual-ists. Based on this rationale, we constructed phyloge-netic trees with clones representing OTUs detected inthree or more libraries (Fig. 5). These included rep-resentatives from �, �, and � subdivisions of the Pro-teobacteria. Clones belonging to the �-Proteobacteria

subdivision formed four distinct clades (Fig. 5).Clones in two of those clades were most closely re-lated to Methylobacterium and Bradyrhizobium, twogenera known to include species capable of Þxingatmospheric nitrogen. The third clade comprised se-quences assigned to the genus Agrobacterium, andwere closely related to a potential microbial symbiontfound in the midgut of the gypsy moth caterpillar(Lymantria dispar; Broderick et al. 2004). Clones inthe fourth clade grouped with sequences in the genusCaulobacter, including the sequence from an intestinalsymbiont of beetle larvae (Zophobas morio, Tenebri-onidae). Clones related to Acidovorax sp. groupingwithin the �-Proteobacteria (Fig. 5), were detected inthree midgut libraries and in R. lebeau eggs. Thesesequences formed a clade with sequences of apparentmutualists recovered from the nephridia of two dif-ferent earthworm species (Schramm et al. 2003). Fi-

Number of clones sampled

Num

ber o

f OTU

s ob

serv

ed

8060402000

5

10

15

20

25

30

100

Fig. 3. Rarefaction curves for 16S rRNA gene libraries constructed from midguts of two species of saturniid caterpillarsfed on different diets. Error bars represent 95% conÞdence intervals for richness values. Curves were constructed usingEstimateS software and 100 randomizations per data set. Ð� Ð C. lobesis fed on artiÞcial diet; ÐEÐ C. lobesis fed on S. mombin;ÑŒ Ð R. lebeau fed on artiÞcial diet; Ð▫Ð R. lebeau fed on S. mombin.

0

10

20

30

40

50

60

70

80

α-Proteobact. β-Proteobact. γ-Proteobact. Actinobact. Firmicutes Bacteroidetes Acidobact. UnidentifiedOTUs

Num

ber o

f clo

nes

Taxonomic groups

Fig. 4. Distribution of clones according to eubacterial taxonomic groups in 16S rRNA gene libraries from two species ofsaturniid caterpillar midguts. Libraries were constructed from total microbial DNA extracted from the midguts of C. lobesisand R. lebeau caterpillars fed on either an artiÞcial diet or leaves from the natal host plant S. mombin. Clones were assignedto each group according to the current taxonomic classiÞcation of the Eubacteria. ÐC. lobesis fed on artiÞcial diet; C. lobesisfed on S. mombin; R. lebeau fed on artiÞcial diet; ÐR. lebeau fed on S. mombin.


nally, clones clustering in the �-Proteobacteria sub-division (Fig. 5) included sequences assigned to thegenera Enterobacter and Pseudomonas that alsogrouped together with potential midgut symbiontsrecovered from gypsy moth caterpillars (Broderick etal. 2004).

Discussion

As a Þrst step in distinguishing resident from tran-sient midgut microorganisms, a deÞned, sterile dietwas employed to rear the caterpillars under study.Reasoning that bacterial mutualists would be presentindependent of diet, the midgut microbiota from cat-erpillars fed on artiÞcial diet was compared with thatfrom caterpillars fed on leaves of their native hostplant. Both R. lebeau and C. lobesis caterpillars fed onS. mombin had a signiÞcantly more diverse bacterial

midgut community than did caterpillars fed on artiÞ-cial diet. Diet affected the composition of the bacterialcommunity detected in our 16S rRNA gene libraries.For example, a higher �- to �-proteobacterial ratio wasobserved in caterpillars fed on artiÞcial diet whencompared with caterpillars fed on S. mombin (Fig. 4).These results are in agreement with a study indicatingthat the microbial composition of midguts varied sub-stantially with diet in gypsy moth caterpillars (L. dis-par) (Broderick et al. 2004). Rarefaction analyses onclones recovered from 16S rRNA gene libraries indi-cated that the midgut microbiota of caterpillars fed onartiÞcial diet was relatively simple, consisting of aboutten different phylotypes (Fig. 3), in contrast to �20different phylotypes in the midgut microbiota of cat-erpillars fed on S. mombin. For comparison, there arean estimated 50 phylotypes reported from the intes-tine of Reticulitermes termites (Ohkuma and Kudo

Table 2. Bacterial phylotypes identified by culture-independent analysis of 16S rDNA sequences from midguts of two species ofsaturniid caterpillars fed on different diets

Operational Taxonomic Unit(OTUa)

Number of clones within corresponding OTUa in 16S rDNA libraries from:

C. lobesis onartiÞcial diet

C. lobesis onS. mombin

R. lebeau onartiÞcial diet

R. lebeau onS. mombin

R. lebeau eggs

Methylobacterium sp. 5 16 1 25 1Acinetobacter sp.b 8 7 23 3 10Bradyrhizobium sp. 9 5 42 3Escherichia coli 25 15 3 4Propionibacterium sp. 3 11 1 1Acidovorax sp. 3 8 1 1Flavobacterium sp. 2 6 3Staphylococcus sp.b 2 3 1Sphingomonas sp. 1 3 1Serratia marcescens 41 6 1Streptococcus sp. 1 2 2Ralstonia sp. 1 1 3Uncultured �-Proteobacterium 2 3Bacillus cereus/thuringiensis 1Uncultured Caulobactereaceae 1Corynebacterium sp. 1Bacillus subtilis 1Caulobacter sp. 2 3Enterococcus sp. 2Ochrobactrum sp. 5Stenotrophomonas sp.b 3 10Janthinobacterium sp. 2Sphingomonas mali 1Pantoea agglomerans 1 1Citrobacter freundii 1Enterobacter sp. 1 7Curtobacterium sp. 10Agrobacterium larrymoorei 8Rhizobium sp. 7Acidobacteriaceae bacterium 7Comamonas sp. 5Alpha-Proteobacterium (B. vestrisi) 2Curvibacter gracilis 2Sphingomonas pruni 1Brevundimonas nasdae 1Rhanella aquatilis 1Actinoplanes sp. 1Terrahaemophilus aromaticivorans 1Klebsiella sp. 5Unidentified clones NONE 1 (1 OTU) 2 (1 OTU) 4 (4 OTUs) NONE

aOTUs are deÞned as sequences at least 98% identical (over at least 1300bp). Sequences were queried in BLAST 2.0 (Altschul et al. 1997)to infer their taxonomic afÞliation. A taxonomic identiÞcation was assigned to OTUs that shared at least 98% identity with known organisms.b Sequences were also detected in 16S rRNA gene libraries constructed with DNA extracted directly from the artiÞcial diet employed in

caterpillar feeding.


1996) and 400 phylotypes reported from the humangut (Eckburg et al. 2005). Reduced complexity ofcaterpillar microbiota makes sense in light of the ex-treme conditions of the midgut environment.

To assist in the identiÞcation of potential caterpillarmutualists we analyzed a 16S rDNA clone library con-structed with DNA extracted from the artiÞcial dietitself. Sequences detected from this library repre-sented either contaminants of the diet or contami-nants of the PCR procedure (Tanner et al. 1998). Mostof the bacterial phylotypes identiÞed from homoge-nized caterpillar guts were not detected in the diet.Only sequences related to Acinetobacter spp. were

found both in the artiÞcial diet library as well as incaterpillar gut samples fed with artiÞcial diet and fo-liage; hence, suggesting they are not potential mutu-alists of these insects but rather contaminants. Addi-tionally, analysis of two 16S rDNA clone libraries fromsurface-sterilized R. lebeau eggs indicated that micro-organisms were contained within the eggs. As verticaltransmission from the adult female into its eggs is atypical mechanism of symbiont passage by insects(McFall-Ngai 2002), and given that caterpillars eattheir egg shell when hatching, it is possible that mi-croorganisms found in the midgut, but not in the dietitself, are vertically transmitted to the Þrst-instar cat-

AF390549 Clostridium sp. DQ196137 Clostridium perfringens

100

0.1

262EGG11 U31075 Klebsiella sp.

AY395007 Enterobacter sp. [Ld] AY941837 Enterobacter sp. 298EGG18 298EGG11 AJ853889 Enterobacter hormaechei 298EGG22 CLSM102 V00348 Escherichia coli

EF195341 Pseudomonas sp. [Px] AF378011 Pseudomonas putida AY395005 Pseudomonas putida [Ld] 262EGG9 AF094737 Pseudomonas putida

298EGG26 AY216797 Ralstonia sp.

RLSM19 AF098288 Ralstonia sp.

298EGG23 RLAD36 AY093698 Acidovorax sp. CLSM148

AJ543438 Acidovorax sp. [At] AJ543434 Acidovorax sp. [Ol]

AJ459874.1 Uncultured beetle bacterium 262EGG19 CLAD36 AE006011 Caulobacter crescentus

AE009348 Agrobacterium tumefaciens AY395023 Uncultured alpha proteobacterium [Ld]

RLSM1 RLSM91 Z30542 Agrobacterium larrymoorei CLAD24 AY904749 Bradyrhizobium elkanii CLAD19 RLSM60

AF459799 Methylobacterium sp [Tb] D32225 Methylobacterium mesophilicum

RLSM66 298EGG8

AY169421 Methylobacterium fujisawanense CLSM170 RLSM42

100100

100

58

8685

100

100

93

1009898

70

9278

100

95

99

100

100

100

100

10090

100

92

100

100

69

80

100

100

64

59

73

95

100

100

100

Fig. 5. Phylogeny of caterpillar midgut bacteria inferred from Bayesian analysis of 16S rRNA genes. Sequences recoveredfrom the midguts of two species of saturniid caterpillars are indicated in bold. The code for each sequence represents thecaterpillar species (CL � C. lobesis; RL � R. lebeau), the associated diet (AD � artiÞcial diet; SM � S. mombin), and theclone number. Sequences from libraries constructed with DNA extracted from surface-sterilizedR. lebeau eggs are designatedas “EGG” and are also indicated in bold. The codes for the EGG sequences correspond to the adult moth providing the eggs(e.g., 262 corresponds to moth 04-RL-262 and 298 corresponds to moth 04-RL-298) and the clone number. Sequences fromsymbionts previously isolated from terrestrial invertebrates include the initials of their hosts in brackets. These includecaterpillars such as Gypsy Moth larvae, (Lymantriadispar[Ld], Broderick et al. 2004) and Diamondback Moth larvae (Plutellaxylostella [Px], Indiragandhi et al. 2008), as well as ants (Tetraponera binghami [Tb], Van Borm et al. 2002) and earthworms(Aporrectodea tuberculata [At], andOctolasion lacteum, [Ol], Schramm et al. 2003). The numbers above branches representtheir Bayesian-calculated posterior probabilities.


erpillar. Our work identiÞed this possibility for mem-bers of the genera Methylobacterium and Acidovorax.

We identiÞed a number of potential bacterial mu-tualists based on their consistent association withthree species of saturniid caterpillar midguts, inde-pendent of diet and species (Fig. 5). Two of thesepotential symbionts are related to nitrogen-Þxing bac-teria in the generaMethylobacterium and Bradyrhizo-bium. The diet of caterpillars has been described asnitrogen deÞcient because the leaves of woody plantshave a much lower N:C ratio (ranging from 1:20Ð1:100;Scriber and Feeny 1979) than that typically requiredby insects (N:C ratio is around 1:10 in insect tissue and1:6 in the chitinous exoskeleton; Nardi et al. 2002).Rapid passage through the midgut may also reduce theability of caterpillars to extract nitrogenous com-pounds from their food. We assume that an associationwith nitrogen-Þxing bacteria may, therefore, be ben-eÞcial. Nitrogen-Þxing bacteria related to root-nodulesymbionts have been previously recovered from in-sect guts. Tetraponera ants were reported to harborclose relatives of Rhizobium sp. in a unique pouch-shaped organ at the junction of the midgut and theintestine (Van Borm et al. 2002). Moreover, the oc-currence of rhizobial bacteria was demonstrated inthe gut of the termiteNasutitermes nigriceps (Frohlichet al. 2007), which consumes a woody diet that is lowin nitrogen. While low oxygen concentrations in themidgut (Amaral-Zettler et al. 2005) is consistent withthe requirements for nitrogen Þxing activity, furtherresearch is necessary to determine whether nitrogenÞxation actually occurs there.

Another group of bacteria identiÞed as potentialmutualists may also be involved in nitrogen metabo-lism. Clones related to Acidovorax sp. group togetherwith symbiotic bacteria isolated from the nephridia ofearthworms (Schramm et al. 2003). These Acidovoraxsymbionts form a stable and host-speciÞc associationwith the earthworm. Given their location and theproteolytic characteristics typical of free-living Aci-dovorax species, they are proposed to play a role inprotein degradation to recycle nitrogen before ßuidsare excreted (Schramm et al. 2003). It is possible thatthese bacteria may play a similar role in caterpillarphysiology, but further research is needed to conÞrmthis possibility.

Another group of potential caterpillar mutualistsbelonged to the species Serratia marcescens. Clonesrelated to S. marcescens comprised the predominantphylotype detected in C. lobesis fed on artiÞcial diet,and were also present in libraries from C. lobesis fedon S. mombin. S. marcescens isolates were additionallyrecovered from the caterpillar midgut, eggs, and pupalmaterial ofR. lebeau (Pinto-Tomas et al. 2007), as wellas from midguts and pupae of another tropical Satur-niid, Automeris zugana (Sittenfeld et al. 2002). In thecurrent study, clones related to S. marcescens wererecovered from surface sterilized eggs of R. lebeau,though not from R. lebeau larvae or adults. Relatedsequences have been recovered from many arthropodspecies, including the gypsy moth (Broderick et al.2004). The association between S. marcescens and cat-

erpillars may in fact be ancient, given that the silk-worm [Bombyx mori (L.)] acquired the chitinase-coding chiA gene from Serratia by horizontal genetransfer, incorporating it into its own hormonally-regulated repertoire of chitinase genes (Daimon et al.2003). There are several possible roles for S. marc-escens in caterpillar biology, related to its ability tobreakdown chitin and Þx nitrogen (Gyaneshwar et al.2001). For example, it has been suggested that thechitinolytic activity of S. marcescens is key for its sym-biotic association with the sugarbeet root maggot, Tet-anopsmyopaeformis (Roder) (Diptera) (Iverson et al.1984). The bacterium is found in every developmentalstage of the insect and might have a role in debilitatingthe chitinous lining of the pupa, therefore facilitatingadult emergence (Iverson et al. 1984). Further re-search is needed to determine whether S. marcescensand other chitinolytic bacteria have roles in develop-ment, nutrient acquisition, or both in caterpillar phys-iology.

There is evidence that midgut microorganisms areimportant to caterpillar physiology. For example,Broderick et al. (2006) recently demonstrated thatBacillus thuringiensis, a commonly used biocontrolagent, is unable to kill gypsy moth caterpillars in theabsence of their normal midgut microbiota. Further-more, the addition of an Enterobacter symbiont thatnormally resides in the gypsy moth midgut restoredthe killing ability of B. thuringiensis. The gypsy mothenterobacterial symbiont appears to be closely re-lated, by 16S rRNA gene sequence analysis, to micro-organisms detected in the eggs of R. lebeau (Fig. 5),suggesting a potentially consistent association be-tween these two types of organisms.

In conclusion, our results suggest that two distinctfunctional groups, chitinolytic and nitrogen-Þxingbacteria, may have important roles in tropical cat-erpillar physiology. In addition to potentially mak-ing contributions to caterpillar nutrition and thereproductive cycle, midgut microbiota may also de-toxify plant defense compounds in the caterpillarfood source and provide protection from pathogens(Dillon and Dillon 2004). In return, the insect mayprovide its gut bacteria with a consistent foodsource, and may contribute to their dispersal in theenvironment. Furthermore, bacteria that are pres-ent in the caterpillar during transition to the adultstage may beneÞt from the protective pupal encase-ment during harsh periods, such as the dry season intropical dry forests. Thus, these results support thepossibility that some bacteria do persist in the ex-treme conditions of the caterpillar midgut andtherefore potentially have important roles in cater-pillar ecophysiology. Given that the PCR-based ap-proach used here allowed us to detect the presence,but not activity, of such potential mutualists, futureexperiments are needed to demonstrate the func-tional signiÞcance of these microorganisms withinthe caterpillar midgut. Further research is also nec-essary to determine their exact location within thecaterpillarÕs digestive system. Techniques such asßuorescence in situ hybridization and high resolu-


tion microscopy, for example, could be used to ad-dress these questions.

Acknowledgments

We want to express our deep gratitude to the personnel ofthe Area de Conservacion Guanacaste, Costa Rica, in par-ticular to Roger Blanco and Marõa Marta Chavarrõa for lo-gistic support, and to Marõa Garcõa, Adelina Morales, andRafael Cubillo for technical assistance in caterpillar rearingand dissections. We are grateful to S. Adams, E. Caldera, C.Currie, G. Suen, K. Grubbs, M. Poulsen, and K. Raffa forvaluable comments regarding previous versions of this man-uscript. This work was supported by National Science Foun-dation (NSF) grant MCB-0084222, grant 3-208-99 from CO-NICIT, San Jose, Costa Rica and by grant VI 801-99-506 fromVicerrectorõa de Investigacion, Universidad de Costa Ricaand facilitated by NSF grants DEB 9400829, DEB 9705072,and DEB 0515699 to D. H. Janzen.

References Cited

Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z.Zhang, W. Miller, and D. J. Lipman. 1997. GappedBLAST and PSI-BLAST: a new generation of proteindatabase search programs. Nucleic Acids Res. 25: 3389Ð3402.

Amaral-Zettler, L. A., A. B. Laatsch, E. Zettler, T. A. Nerad,J. Cole, F. Chavarria-Diaz, J. Diaz, D. H. Janzen, A.Sittenfeld, O. Mason, et al. 2005. A microbial observa-tory of caterpillars: isolation and molecular characteriza-tion of protists associated with the saturniid moth cater-pillarRothschildia lebeau. J. Eukaryot. Microbiol. 52: 107Ð115.

Appel, H.M., andL.W.Maines. 1995. The inßuence of hostplant on gut conditions of gypsy moth (Lymantria dispar)caterpillars. J. Insect Physiol. 41: 241Ð246.

Bintrim, S. B., T. J. Donohue, J. Handelsman, G. P. Roberts,and R. M. Goodman. 1997. Molecular phylogeny of Ar-chaea in soil. Proc. Natl. Acad. Sci. U.S.A. 94: 277Ð282.

Baumann, P., L. Baumann, C. Y. Lai, and D. Rouhbakhsh.1995. Genetics, physiology and evolutionary relation-ships of the genus Buchnera: intracellular symbionts ofaphids. Annu. Rev. Microbiol. 49: 55Ð94.

Bernays, E. A., and D. H. Janzen. 1988. Saturniid and sph-ingid caterpillars: two ways to eat leaves. Ecology 69:1153Ð1160.

Broderick, N. A., K. F. Raffa, R. M. Goodman, and J. Han-delsman. 2004. Census of the bacterial community ofthe gypsy moth larval midgut using culturing and culture-independent methods. Appl. Environ. Microbiol. 70: 293Ð300.

Broderick, N. A., K. F. Raffa, and J. Handelsman. 2006.Midgut bacteria required for Bacillus thuringiensis insec-ticidal activity. Proc. Natl. Acad. Sci. U.S.A. 103: 15196Ð15199.

Brune, A. 1998. Termite guts: the worldÕs smallest bioreac-tors. Trends Biotechnol. 16: 16Ð21.

Cazemier, A. E., J.H.P. Hackstein, H.J.M. Op den Camp, J.Rosenberg, and C. van der Drift. 1997. Bacteria in theintestinal tract of different species of arthropods. Micro-biol. Ecol. 33: 189Ð197.

Daimon, T., K. Hamada, K. Mita, K. Okano, M. Suzuki, M.Kobayashi, and T. Shimada. 2003. A Bombyx mory gene,BmChi-h, encodes a protein homologous to bacterial andbaculovirus chitinases. Insect Biochem. Mol. Biol. 33:749Ð759.

Dillon, R. J., and V. M. Dillon. 2004. The gut bacteria ofinsects: nonpathogenic interactions. Annu. Rev. Entomol.49: 71Ð92.

Dow, J.A. 1992. pH gradients in lepidopteran midgut. J. Exp.Biol. 172: 355Ð375.

Eckburg, P. B., E. M. Bik, C. N. Bernstein, E. Purdom, L.Dethlefsen, M. Sargent, S. R. Gill, K. E. Nelson, andD. A.Relman. 2005. Diversity of the human intestinal micro-bial ßora. Science 308: 1635Ð1638.

Faul, F., E. Erdfelder, A.-G. Lang, and A. Buchner. 2007.G*Power 3: a ßexible statistical power analysis programfor the social, behavioral, and biomedical sciences. Behav.Res. Methods 39: 175Ð191.

Frohlich, J., C. Koustiane, P. Kampfer, R. Rossello-Mora, M.Valens, M. Berchtold, T. Kuhnigk, H. Hertel, D. K. Ma-heshwari, andH.Konig. 2007. Occurrence of rhizobia inthe gut of the higher termite Nasutitermes nigriceps. Syst.Appl. Microbiol. 30: 68Ð74.

Giovannoni, S. J. 1991. The polymerase chain reaction, pp.177Ð201. In E. Stackebrandt and M. Goodfellow (eds.),Nucleic Acid Techniques in Bacterial Systematics. Wiley,New York.

Governor, H. L., J. C. Schultz, and H. M. Appel. 1997. Im-pact of dietary allelochemicals on gypsy moth (Lymantriadispar) caterpillars: importance of midgut alkalinity. J. In-sect Physiol. 43: 1169Ð1175.

Gyaneshwar, P., E. K. James, N. Mathan, P. M. Reddy, B.Reinhold-Hurek, and J. K. Ladha. 2001. Endophytic col-onization of rice by a diazotrophic strain of Serratiamarc-escens. J. Bacteriol. 183: 2634Ð2645.

Hughes, J., andB.J.M. Bohannan. 2004. Applications of eco-logical diversity statistics in microbial ecology, pp. 1321Ð1344. In G. A. Kowalchuk, F. J. de Bruijn, I. M. Head,A.D.L. Akkermans, and J. D. von Elsas, (eds.), MolecularMicrobial Ecology Manual, 2nd ed. Kluwer AcademicPublishers, Netherlands.

Indiragandhi, P., R. Anandham,M.Madhaiyan, and T.M. Sa.2008. Characterization of plant growth-promoting traitsof bacteria isolated from larval guts of diamondback mothPlutella xylostella (Lepidoptera: Plutellidae). Curr. Mi-crobiol. 56: 327Ð33.

Iverson, K. L., M. C. Bromel, A. W. Anderson, and T. P.Freeman. 1984. Bacterial symbionts in the sugar beetroot maggot, Tetanops myopaeformis (von Roder). Appl.Environ. Microbiol. 47: 22Ð27.

Janzen, D. H. 1981. Patterns of herbivory in a tropical de-ciduous forest. Biotropica 13: 271Ð282.

Janzen, D. H. 1984. Two ways to be a tropical big moth:Santa Rosa saturniids and sphingids. Oxf. Surv. Evol. Biol.1: 85Ð140.

Janzen, D. H. 1993. Caterpillar seasonality in a Costa Ricandry forest, pp. 448Ð477. In N. E. Stamp and T. E. Casey(eds.), Caterpillars: Ecological and evolutionary con-straints on foraging. Chapman & Hall, New York.

Janzen, D. H. 2003. How polyphagous are Costa Rican dryforest saturniid caterpillars? pp. 369Ð379. In Y. Basset, V.Novotny, S. E. Miller, and R. L. Kitching, (eds.), Arthro-pods of Tropical Forests. Spatio-Temporal Dynamics andResourceUse in theCanopy.CambridgeUniversityPress,Cambridge, United Kingdom.

Janzen, D. H. and I. D. Gauld. 1997. Patterns of use of largemoth caterpillars (Lepidoptera: Saturniidae and Sphin-gidae) by ichneumonid parasitoids (Hymenoptera) inCosta Rican dry forest, pp. 251Ð271. In A. D. Watt, N. E.Stork, and M. D. Hunter (eds.), Forests and insects. Chap-man & Hall, London, United Kingdom.

Janzen, D. H., W. Hallwachs, P. Blandin, J. M. Burns, J.Cadiou, I. Chacon, D. Dapkey, A. Deans, M. Epstein, B.


Espinoza, et. al. 2009. Integration of DNA barcodinginto an ongoing inventory of complex tropical biodiver-sity. Mol. Ecol. Resour. 9: 1Ð26.

Johnson, K. S., and R. V. Barbehenn. 2000. Oxygen levels inthe gut lumen of herbivorous insects. J. Insect Physiol. 46:897Ð903.

Lane, D. J. 1991. 16S/23S rRNA sequencing, pp. 115Ð175. InE. Stackebrandt and M. Goodfellow (eds.), Nucleic AcidTechniques in Bacterial Systematics. Wiley, New York.

Leadbetter, J. R., T. M. Schmidt, J. R. Graber, and J. A.Breznak. 1999. Acetogenesis from H2 plus CO2 by spi-rochetes from termite guts. Science 283: 686Ð689.

McFall-Ngai, M. J. 2002. Unseen forces: the inßuence ofbacteria on animal development. Dev. Biol. 242: 1Ð14.

Miller, T. A., W. J. Cooper, and J. W. Highfill. 1977. Deter-mination of sex in four species of giant silkworm mothlarvae (Saturniidae). J. Lepid. Soc. 31: 144Ð146.

Miller, J. C., D. H. Janzen, and W. Hallwachs. 2006. 100caterpillars: portraits from the Tropical Forests of CostaRica. Harvard University Press, Cambridge, United King-dom.

Miller, J. C., D. H. Janzen, and W. Hallwachs. 2007. 100butterßies and moths: portraits from the Tropical Forestsof Costa Rica. Harvard University Press, Cambridge,United Kingdom.

Moran, N. A., G. R. Plague, J. P. Sandstrom, and J. L.Wilcox.2003. A genomic perspective on nutrient provisioning bybacterial symbiontsof insects.Proc.Natl.Acad.Sci.U.S.A.100: 14543Ð14548.

Nardi, J. B., R. I. Mackie, and J. O. Dawson. 2002. Couldmicrobial symbionts of arthropod guts contribute signif-icantly to nitrogen Þxation in terrestrial ecosystems? J. In-sect Physiol. 48: 751Ð763.

Ohkuma, M., and T. Kudo. 1996. Phylogenetic diversity ofthe intestinal bacterial community in the termite Reticu-litermes speratus. Appl. Environ. Microbiol. 62: 461Ð468.

Pinto-Tomas, A. A., L. Uribe, J. Blanco, G. Fontecha, C.Rodrıguez, M. Mora, D. H. Janzen, F. Chavarrıa, J. Diaz,and A. Sittenfeld. 2007. Enzymatic activities of bacteriaisolated from the digestive tract of caterpillars and thepupal content of Automeris zugana and Rothschildia leb-eau (Lepidoptera: Saturniidae). J. Trop. Biol. 55: 401Ð415.

Riddiford, L. M. 1985. Hyalophora cecropia, pp. 335Ð343. InP. Singh and R. F. Moore (eds.), Handbook of InsectRearing vol. II, Elsevier, Amsterdam.

Ronquist, F., and J. P. Huelsenbeck. 2003. MrBayes 3:Bayesian phylogenetic inference under mixed models.Bioinformatics 19: 1572Ð1574.

Ruby, E., B. Henderson, and M. J. McFall-Ngai. 2004. Weget by with a little help from our (little) friends. Science303: 1305Ð1307.

Russell, V. W., and P. E. Dunn. 1991. Lysozyme in themidgut of Manduca sexta during metamorphosis. Arch.Insect. Biochem. Physiol. 17: 67Ð80.

Schramm, A., S. K. Davidson, J. A. Dodsworth, H. L. Drake,D. A. Stahl, and N. Dubilier. 2003. Acidovorax-like sym-bionts in the nephridia of earthworms. Environ. Micro-biol. 5: 804Ð809.

Schultz, J. C. 2002. How plants Þght dirty. Nature 416: 267.Scriber, J. M., and P. Feeny. 1979. Growth of herbivorous

caterpillars in relation to feeding specialization and to thegrowth form of their food plants. Ecology 60: 829Ð850.

Sittenfeld,A., L.Uribe-Lorıo,M.Mora,V.Nielsen,G.Arrietaand D. H. Janzen. 2002. Does a polyphagous caterpillarhave the same gut microbiota when feeding on differentspecies of food plants? Rev. Biol. Trop. 50: 547Ð560.

Stamp, N. E. and T. M. Casey (eds). 1993. Caterpillars: eco-logical and evolutionary constraints on foraging. Chap-man & Hall, New York.

Tamura, K., J.Dudley,M.Nei, and S.Kumar. 2007. MEGA4:molecular evolutionary genetics analysis (MEGA) soft-ware version 4.0. Mol. Biol. Evol. 24: 1596Ð1599.

Tanner, M. A., B. M. Goebel, M. A. Dojka, and N. R. Pace.1998. SpeciÞc ribosomal DNA sequences from diverseenvironmental settings correlate with experimental con-taminants. Appl. Environ. Microbiol. 64: 3110Ð3113.

Van Borm, S., A. Buschinger, J. J. Boomsma, and J. Billen.2002. Tetraponera ants have gut symbionts related to ni-trogen-Þxing root-nodule bacteria. Proc. R. Soc. Lond. B.269: 2023Ð2027.

Yu, H., Z. Wang, L. Liu, Y. Xia, Y. Cao, and Y. Yin. 2008.Analysis of the intestinal microßora inHepialus gonggaen-sis larvae using 16S rRNA sequences. Curr. Microbiol. 56:391Ð396.

Received 22 March 2011; accepted 23 June 2011.


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Comparison of Midgut Bacterial Diversity in Tropical Caterpillars (Lepidoptera: Saturniidae) Fed on Different Diets