The Olive Fly Endosymbiont, “Candidatus Erwinia dacicola,” … · 2009. 11. 4. · different nutrients during an insect’s life time. Second, Diptera are holometabolous, undergoing

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2009, p. 7097–7106 Vol. 75, No. 220099-2240/09/$12.00 doi:10.1128/AEM.00778-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The Olive Fly Endosymbiont, “Candidatus Erwinia dacicola,” Switchesfrom an Intracellular Existence to an Extracellular Existence

during Host Insect Development�†Anne M. Estes,1* David J. Hearn,1§ Judith L. Bronstein,1 and Elizabeth A. Pierson2‡

Department of Ecology and Evolutionary Biology1 and Division of Plant Pathology and Microbiology, Department ofPlant Sciences,2 University of Arizona, Tucson, Arizona

Received 6 April 2009/Accepted 14 September 2009

As polyphagous, holometabolous insects, tephritid fruit flies (Diptera: Tephritidae) provide a unique habitatfor endosymbiotic bacteria, especially those microbes associated with the digestive system. Here we examine theendosymbiont of the olive fly [Bactrocera oleae (Rossi) (Diptera: Tephritidae)], a tephritid of great economicimportance. “Candidatus Erwinia dacicola” was found in the digestive systems of all life stages of wild olive fliesfrom the southwestern United States. PCR and microscopy demonstrated that “Ca. Erwinia dacicola” residedintracellularly in the gastric ceca of the larval midgut but extracellularly in the lumen of the foregut andovipositor diverticulum of adult flies. “Ca. Erwinia dacicola” is one of the few nonpathogenic endosymbiontsthat transitions between intracellular and extracellular lifestyles during specific stages of the host’s life cycle.Another unique feature of the olive fly endosymbiont is that unlike obligate endosymbionts of monophagousinsects, “Ca. Erwinia dacicola” has a G�C nucleotide composition similar to those of closely related plant-pathogenic and free-living bacteria. These two characteristics of “Ca. Erwinia dacicola,” the ability to transi-tion between intracellular and extracellular lifestyles and a G�C nucleotide composition similar to those offree-living relatives, may facilitate survival in a changing environment during the development of a polypha-gous, holometabolous host. We propose that insect-bacterial symbioses should be classified based on theenvironment that the host provides to the endosymbiont (the endosymbiont environment).

Bacteria are diverse, abundant, and ubiquitous and havegreatly influenced the evolution of eukaryotic life (38, 39).Bacterial symbionts of eukaryotes are generally studied interms of benefiting or impairing their eukaryotic hosts (3, 7, 21,26). The constraints that the host’s life history and diet imposeon their endosymbionts have yet to be considered in depth.Insect-bacterium symbioses, both mutualistic and pathogenic,are ideal for examining host constraints on endosymbionts.The diversity of insect lifestyles and diets provides a continuumof habitats for endosymbionts, exhibiting different selectionpressures on insect-associated bacteria.

Tephritid fruit flies (Tephritidae: Diptera) provide a uniquehabitat for endosymbiotic bacteria, especially digestive-systemmicrobes. First, most tephritids feed on fleshy fruit (such asolives) as larvae, and as adults they are optimal foragers onpollen, bird feces, phylloplane bacteria (15, 52), homopteranhoneydew (44), and other food sources in the environment (15,18). Thus, digestive-tract endosymbionts may be exposed todifferent nutrients during an insect’s life time. Second, Dipteraare holometabolous, undergoing complete metamorphosis.

Vertically transmitted endosymbionts must survive the break-down and rebuilding of the host tissues with which the bacteriaassociate (22). We predict that, in order to survive in such achanging environment, the endosymbiont would have a ge-nome similar in size to those of free-living relatives, rather thana greatly reduced genome, as is seen in obligate nutritionalmutualists, such as Buchnera aphidicola.

The interaction between the olive fly, Bactrocera oleae, andits digestive-system bacterial associates has been studied formore than a century (5, 9, 17, 28, 29, 32, 33, 36, 52, 55, 56), yetseveral important questions remain unanswered. Do all olivefly life stages have the same species of bacterial endosymbiont?With which organs of the digestive system do the bacteriaassociate? Where are the bacteria located in relation to hosttissues (are they extracellular or intracellular)? How ubiqui-tous is the association between the olive fly and specific bac-teria? Are the same species of bacteria found in olive flies fromthe Old World also in flies that have recently infested theUnited States? Is the nucleotide composition of the olive flysymbiont more similar to those of its free-living relatives or tothose of obligate endosymbionts?

The purpose of this paper is to address the questions listedabove and to characterize the olive fly-bacterial symbiosis morethoroughly by using microscopy and culture-dependent and-independent techniques to examine all life stages of wild oliveflies from the United States. We provide a comprehensivedescription of the biology of the olive fly symbiosis throughouthost development, and we highlight the distinctive nature of ahighly variable endosymbiont environment provided by thehost and its consequences for bacterial genome evolution. Bet-ter understanding of tephritid endosymbionts may provide in-

* Corresponding author. Mailing address: Department of Ecologyand Evolutionary Biology, University of Arizona, 310 BioSciencesWest, 1041 E. Lowell St., Tucson, AZ 85721. Phone: (520) 621-3792.Fax: (520) 621-9190. E-mail: [email protected].

‡ Present address: Department of Horticultural Sciences, TexasA&M, College Station, TX 77843-2133.

§ Present address: Department of Biological Sciences, Towson Uni-versity, Smith Hall, 8000 York Rd., Baltimore, MD 21252.

† Supplemental material for this article may be found at http://www.aem.org/.

� Published ahead of print on 18 September 2009.

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sight into control methods for these significant agriculturalpest insects.

MATERIALS AND METHODS

Collection and sampling. In 2003, infected olives were collected, and a Tucsonlaboratory population (TLP) culture was established from emerged larvae. TheTLP culture was maintained on surface-sterilized olives. Adult B. oleae flies weretrapped in torula yeast-baited McPhail traps (as described in reference 8) in olivegroves of the National Clonal Germplasm Repository at Wolfskill ExperimentalOrchard in Winters (Yolo County), CA, and in Tucson (Pima County), AZ, in2005. Sixty adults (30 males and 30 females) were collected from the Winters site,and 40 adults (20 males and 20 females) were collected from Tucson (APHISpermit number 68318).

All olive fly life stages (eggs, third-instar larvae, and pupae) were collectedfrom the TLP and California populations and were examined for bacterialmicrobiota. Life stages were surface sterilized by vortexing for 15 s in a 1%sodium hypochlorite (bleach)–0.1% Triton X mixture and then rinsing twice withdistilled water. The rinse water was plated on Luria-Bertani medium (4) andused as a template for PCR with the universal prokaryote 16S rRNA primers 10Fand 1507R (42). No colonies or PCR products were observed (data not shown).TLP females were presented with “agar olives,” a half-sphere of 2% agarwrapped in Parafilm (Pechiney Plastic Packaging, Chicago, IL) as an ovipositionsubstrate. Eggs (24 h postoviposition) were aseptically removed from agar olives.Wandering third-instar larvae and pupae (48 h postpupation) were fixed in 95%ethanol.

Samples of different organs in larvae and 1-week-old TLP adults were alsotaken in order to determine in which tissues bacteria reside. Adult flies andthird-instar larvae were surface sterilized as described above and dissected in alaminar flow hood. Larval and adult organs were preserved immediately in 95%ethanol for DNA extraction, and PCR was performed using 16S rRNA primersthat amplified an Enterobacter sp. and “Candidatus Erwinia dacicola” as de-scribed below. Larval organs sampled included the mycetome (midgut gastricceca) (n � 10) and salivary glands (n � 10). Adult organs analyzed were theesophageal bulb (evagination of the foregut) (n � 10), crop (n � 10), rectal sac(n � 8), ovaries (n � 5), ovipositor (n � 5), and testes (n � 8).

In addition, olives were screened in order to determine if “Candidatus Erwiniadacicola” and the Enterobacter sp. were present in drupe tissues. Bacterial pres-ence or absence was determined across six different olive treatments: fresh oliveslacking larvae (unripe and ripe), rotten olives lacking larvae (unripe and ripe),and rotten olives with larvae (unripe and ripe). Twenty olives were used pertreatment. Samples of olive flesh were fixed in 95% ethanol for DNA extraction,followed by bacterium-specific amplification as described below.

Culture-dependent identification of bacterial associates. Male and femaleolive flies from the TLP were surface sterilized as described above, homogenizedin isolation buffer, and plated onto various media (4), including Luria broth,Kings medium B, nutrient broth agar, Erwinia-selective medium, Trypticase soybroth, and crushed olive mesocarp with agar. The rinse water was plated andchecked for bacterial growth. All cultures were incubated at 28°C and checkeddaily, for 10 days, for colonies. Emergent colonies were picked and streaked forsingle colonies on the same type of medium from which they were collected.Bacteria from single colonies were grown overnight in liquid medium and werestored in 40% glycerol at �80°C.

Culture-independent identification of bacterial associates. To identify bacte-ria present in the wild populations and within specific organs of olive flies in thelab population, surface-sterilized whole animals or fly tissues were snap-frozen inliquid nitrogen and homogenized using a disposable plastic pestle (BelArt Prod-ucts, Pequannock, NJ) in a 1.5-ml microcentrifuge tube. DNA from fly tissueswas extracted using the mouse tail protocol of the Qiagen (Valencia, CA)DNeasy kit. DNA from olive tissues was extracted using the DNeasy plant kit(Qiagen, Valencia, CA). Whole-fly DNA samples were amplified with the uni-versal prokaryote 16S rRNA primers 10F and 1507R (42) to generate a nearlycomplete (�1,489-bp) sequence. These PCR products were cloned using aTOPO-TA one-shot kit (Invitrogen, Carlsbad, CA) and were sequenced in bothdirections by the Genomic Analysis and Technology Core of the University ofArizona on an Applied Biosystems 3730XL sequencer. Sequences were manuallyedited (Sequencher; Gene Codes, Ann Arbor, MI). Edited sequences weresubjected to BLAST analysis (1) against the GenBank database (http://www.ncbi.nlm.nih.gov/) to estimate taxonomic placement for initial primer design andsubsequent phylogenetic analyses. Additional primers were designed from analignment of several enteric bacteria to selectively amplify the 16S rRNA of “Ca.Erwinia dacicola” and the Enterobacter sp., amplified using universal primers.Primers EdF1 (5�-CTAATACCGCATAACGTCTTCG-3�) and EntF1 (5�-CTA

ATACCGCATAACGTCGCAA-3�) were paired with primer 1507R to generatea �1,300-bp sequence. EntF1 and EntR2 (5�-GAGTAATCCGATTAACGCTTG-3�) were used for subsequent screening of flies (�400-bp sequence) for theEnterobacter sp. For additional resolution of bacterial identity, the ompA (outermembrane protein) primer set UF1ompA (5�-TTAACGGTGCGGCTGAGTTACAACG-3�)–UR2ompA (5�-ACACCTGGTACACTGGTGCTAAAC-3�) andthe recA (recombinase A) primer set UF2RecA (5�-CCTGACCGATCTTGTCACC-3�)–UR2RecA (5�-GGTAAAGGCTCCATCATGCG-3�), producing a400-bp fragment, were designed from an alignment of several enteric bacteria.The PCR program used was a 65-55 touchdown program: 94°C for 2 min,followed by 10 cycles of 94°C for 45 s, 65°C for 45 s, a decrease by 1°C to 55°C,and 72°C for 1.5 min. The 65-55 touchdown was followed by 21 cycles of 94°C for45 s, 55°C for 45 s, and 72°C for 1.5 min. Bacteria of each species were scored aspresent or absent. In samples for which no PCR product was generated, theuniversal prokaryote primers 10F and 1507R (42) were used to determine if anybacteria were present. The extracted DNA of a homogenized olive fly with the“Ca. Erwinia dacicola” symbiont was used as a positive-control template. Neg-ative controls lacked DNA. PCR products were cloned, sequenced, and used forphylogenetic analysis. To ensure that the DNA extraction was successful, primersfor the B. oleae adh gene (encoding alcohol dehydrogenase) (adhF2 [5�-GGCATACTCACCGATCCCAATGTAGA-3�] and adhR2 [5�-CATGAGTGGAATTGCCTCTAGCGT-3�]) were designed using an alignment of several insect adhgenes. Analysis of variance was used to determine if different sexes or popula-tions of olive flies had similar associated bacteria (StatView, version 5.0; SASInstitute, Cary, NC).

Identification of olive fly bacterial symbionts using phylogenetic analysis.Nucleotide sequences from related bacteria, including free-living and insectendosymbiotic taxa, were obtained from NCBI for each locus (16S rRNA, ompA,recA) (see Fig. S1 in the supplemental material). All sequences generated duringthis study have been deposited in GenBank, and phylogenetic trees have beendeposited in TreeBASE. Sequences were aligned separately for each locus and asa concatenated matrix using ClustalW (54) with default parameters. Codons ofrecA and ompA were translated to amino acids and converted back to nucleotidesafter amino acid alignment. All alignments were manually edited in Mesquite,version 2.01 (35). Nonalignable sections (i.e., regions of extensive small indels)were excluded from phylogenetic analysis (see Table S1 in the supplementalmaterial). Only coding regions of recA and ompA were included for these loci.ModelTest (48) was used to compare models of evolution for individual loci (seeTable S1 in the supplemental material), and the best model was chosen on thebasis of the Akaike information criterion. A Bayesian analysis using MrBayes,version 3.1 (25), was performed to infer topologies, branch lengths, and supportvalues for each locus and for the concatenated matrix. Table S1 in the supple-mental material lists the partitioning and nucleotide evolution models as imple-mented by MrBayes. Two Metropolis-coupled Markov chain Monte Carlo(MCMC) analyses were run with four chains each for 5 million generations, withsampling every 1,000 generations. Burn-in periods (see Table S1 in the supple-mental material) were estimated by graphing likelihood values and were definedby the generation when log-likelihoods appeared to converge to stationarity.All-compatible-partitions (allcompat) consensus trees were generated from post-burn-in MCMC samples. The taxa in clades I and II were selected as outgroupsand were used to root the tree (Fig. 1).

Identification of bacteria by FISH. Fluorescent in-situ hybridization (FISH)was used to confirm the identity of the bacteria present in the esophageal bulbsof adults and the cultured bacterial isolates. Cultured Enterobacter sp. cellsisolated from olive flies were grown overnight at 28°C, pelleted to removemedium, and washed twice in phosphate-buffered saline (PBS) at pH 7.0. Cellswere fixed in 4% paraformaldehyde for 30 min at 22°C and were washed twicewith PBS. Cells were dried on microscope slides (Fisherbrand Super Frost Plus;Fisher Scientific, Pittsburgh, PA) for FISH. Insect tissues fixed in paraformal-dehyde had high levels of autofluorescence. Therefore, a different fixation pro-tocol (19) was used. Flies were surface sterilized, and the esophageal bulb wasdissected as described above. Esophageal bulbs were fixed in Carnoy’s fixative for4 days, rinsed twice for 5 min with 100% ethanol, and transferred to 6% H2O2

in ethanol for 4 days at 22°C. Bulbs were rinsed in 100% ethanol, followed bythree washes, each 5 min long, in PBS plus 0.01% Tween. For hybridization,bulbs were rinsed in hybridization solution three times (for 5 min each time) andincubated overnight at 40°C in a mixture of the FISH probe, 20% formamide,and hybridization buffer. Samples were rinsed in 3 M sodium chloride and 0.3 Msodium citrate, followed by a PBS rinse. To protect the fluorescent samples frombleaching, a drop of Vectashield (Vector Labs, Burlingame, CA) was added.Samples were viewed under a Zeiss 510 Meta confocal microscope. Esophagealbulbs and cultured cells were both hybridized with a 6-carboxytetramethylrho-damine-labeled EUB338 (2) probe (data not shown), EdF1-Cy5, and EnF2-Cy3

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FIG. 1. Phylogenetic identification of B. oleae symbionts. The results of Bayesian analysis of the concatenated sequences of the recA (�870 bp),ompA (�900 bp), and 16S rRNA (�1,400 bp) genes are shown. Six groups (I to VI) that also occur in analyses of individual loci (see Table S2in the supplemental material) are identified. Group V is an unresolved grade in all analyses. Posterior probabilities above 0.85 appear above andto the left of the corresponding nodes. The six clades have different grayscale values, and taxa incertae sedis are on black branches, as they changeposition from locus to locus. Designations beginning with “i” represent cultured isolates; those beginning with “b” represent bacteria amplifiedfrom the insect host named; and those beginning with “C” represent bacteria of Candidatus status. Samples of bacteria from Bactrocera oleae arein boldface, with the life stage (l, larva; p, pupa) or adult gender (m, male; f, female) or organ (o, ovipositor [female]) indicated.

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(Fig. 2). To determine probe specificity, a range of formamide concentrationsfrom 0 to 30% was used for both probes on both bacterial cells and esophagealbulbs. The optimal formamide concentration for both probes was 20%. Negativecontrols in PBS were also examined.

Bacterial localization within host tissues. Larvae (third instar), 1-month-oldfed adults, and recently eclosed (�24 h), unfed adults were collected from theTLP in order to determine where bacteria were located within host tissues byusing transmission electron microscopy (TEM). Animals were surface sterilized,and the larval mycetomes and adult esophageal bulbs or ovipositors were col-lected. Samples were fixed in a mixture of 1.5% paraformaldehyde, 2% glutar-aldehyde, 1.5% acrolein, and 1% dimethyl sulfoxide in 0.08 M cacodylate (pH7.3) for 90 min at 20°C, rinsed for a total of 20 min in 0.08 M cacodylate (pH 7.3),postfixed in 2.0% OsO4 in 0.08 M cacodylate, and dehydrated with a standardethanol series (30 to 100%) (modified from reference 37). Prior to embedding,samples were transferred into two exchanges of 100% propylene oxide for 10 min

each and were embedded in Epon resin. Thick sections were cut on a LeicaUltramicrotome E and stained in 2% toluidine blue with 2% borax for basichistology. Thin sections (thickness, 60 to 90 nm) were placed on 100-�m-meshFormvar-coated copper grids. Sections from two animals per organ type wereviewed on a JEOL 100 CX II TEM. Microscopy was conducted at the Universityof Arizona’s University Spectroscopy and Imaging Facilities. TEM negativeswere scanned at 400 dpi using an Epson Perfection 2450 Photo scanner.

GC contents of representative loci. The G�C nucleotide composition of olivefly endosymbionts was determined from recA, ompA, and 16S rRNA sequences.The total G�C content of each gene was determined by dividing the number ofG�C nucleotides present by the total number of nucleotides in a given sequence.The G�C contents for the three loci were compared to the genome character-istics of the 32 sequenced genomes (free-living bacteria, plant pathogens, andobligate mutualists) used in our phylogenetic analyses. Regression analysis wasused in each comparison to determine if the G�C contents of these loci wererepresentative of the GC content of the whole genome (StatView, version 5.0;SAS Institute, Cary, NC). The mean and range of the G�C percentage for eachlocus were determined for the groups identified in the three-gene concatenatedphylogeny (Table 1).

Nucleotide sequence accession numbers. The sequences generated during thisstudy have been deposited in GenBank under accession numbers GQ478372 toGQ478388 and GQ487328. Phylogenetic trees have been deposited in Tree-BASE under study number S2481.

RESULTS

Specific bacteria were associated with olive fly populations.Bacteria from surface-sterilized wild olive flies were identifiedby using sequences generated by PCR amplification of 16SrRNA, ompA, and recA. Phylogenetic analyses identified fiveclades of bacteria and one unresolved grade that appeared inallcompat consensus trees of the ompA (see Fig. S1B in thesupplemental material), recA (see Fig. S1C in the supplementalmaterial), and concatenated (Fig. 1; see also Table S1 in thesupplemental material) analyses. Phylogenetic analyses of 16SrRNA alone (see Fig. S1A in the supplemental material) re-sulted in poorly resolved trees. Four bacterial species wererecovered from olive flies (Fig. 1).

The most abundant bacterial species was identified fromsequences obtained from whole animals (larvae, adults) andovipositors (Fig. 1, samples b1 to b8). This abundant specieshad high sequence similarity to the entire 16S rRNA (99%BLAST identity) of “Ca. Erwinia dacicola” from Italian oliveflies (9). Phylogenetic analyses of bacterial sequences amplifiedfrom adult and larval olive flies (Fig. 1, samples b1 to b8) and

FIG. 2. FISH of an adult esophageal bulb. (A) Whole mount of anadult esophageal bulb labeled with EdF1-Cy5, specific to “Ca. Erwiniadacicola.” Bar, 20 �m. (B) Higher magnification of “Ca. Erwinia daci-cola” cells found within the esophageal bulb and labeled with EdF1-Cy5. Bar, 2 �m.

TABLE 1. Percent G�C contents of recA, ompA, and 16S rRNA for species used in the phylogenetic analysisa

SpeciesMean (range) % G�C content

16S rRNA ompA recA

Clade I 54.5 (53.8–55.9) (n � 6) unb 60 (58.2–63.9) (n � 4)Clade II 54.5 (53.8–54.96) (n � 4) 65.5 (63–68) (n � 2) 59.2 (52.1–63.3) (n � 3)Clade III 55.0 (54.2–55.6) (n � 12) 52.7 (51.9–54.3) (n � 5) 52.6 (49.8–55.4) (n � 11)“Ca. Erwinia dacicola,” Italy 54.7 (n � 1) un un“Ca. Erwinia dacicola,”

southwestern UnitedStates

54.2 (n � 1) 51.9 (51.8–52.2) (n � 3) 53.6 (53.4–53.7) (n � 2)

Clade IV 54.5 (48.9–55.7) (n � 21) 54.6 (52.1–56.6) (n � 17) 56.1 (51.9–61.2) (n � 16)Enterobacter sp. 56.1 (cultured), 54.7 (uncultured) (54.6–54.9) (n � 3) 55.19 (n � 1) 56.1 (n � 1)Clade V 52 (36–55) (n � 26) 54.0 (14–57.2) (n � 16) 47.9 (19–57.8) (n � 15)Clade VI 54.8 (53.8–55.8) (n � 7) 52.1 (50.3–53.8) (n � 7) 50.6 (48.4–54.0) (n � 7)

a The G�C composition was determined for each species in the clades identified in the three-gene concatenated phylogeny. Shown here are the mean, range, andnumber of species (n) for each clade. Also included are the statistics for the Enterobacter sp. and “Ca. Erwinia dacicola” sequenced from U.S. flies (this study) and for“Ca. Erwinia dacicola” from Italian flies (9).

b un, unknown.


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previously characterized “Ca. Erwinia dacicola” placed thisbacterium within a subclade of clade III (Erwinia plus Pantoea)(posterior probability [PP] in concatenated analyses, 1.0). Phy-logenetic analyses using ompA (see Fig. S1B in the supplemen-tal material) and recA (see Fig. S1C in the supplemental ma-terial) revealed the monophyly of these samples and showedthem to be allied to other Erwinia species. In concatenatedanalyses, the “Ca. Erwinia dacicola” clade was sister to a cladewith plant pathogens Brenneria (Erwinia) rhapontici and Er-winia persicina (PP � 1.0) (Fig. 1). “Ca. Erwinia dacicola” isthus far unculturable (9; also data not shown).

The second most abundant bacterial species was identifiedfrom sequences obtained from larvae, pupae (Fig. 1, samplesb10 to b12), and one cultured isolate (Fig. 1, sample i1) fromthe abdomen of a wild male olive fly. Several isolates from bothlarvae and male flies had a round, smooth colony morphologyon Luria-Bertani (LB) and Erwinia-selective media. The cul-tured isolate and the 16S rRNA sequences amplified fromolive fly larvae and pupae form clade IV (Enterobacteriaceae)(Fig. 1). The cultured isolate is a member of an unsupportedsister clade that contains Enterobacter cancerogenus, Entero-bacter agglomerans, Enterobacter cloacae (from the midgut ofLeptinotarsa decemlineata), and a bacterium isolated from thecollembolan Folsomia candida (PP in concatenated analyses,1.0). Initial sequence similarity results (99% BLAST match)and phylogenetic analysis using 16S rRNA alone (see Fig. S1Ain the supplemental material) suggested that the Enterobactersp. isolate was closely related to Enterobacter cloacae or Entero-bacter agglomerans. The recA analysis (see Fig. S1C in thesupplemental material) groups the cultured bacterium withEnterobacter cancerogenus and Enterobacter cloacae (PP �0.95). Further resolution was not obtained using ompA alone(see Fig. S1B in the supplemental material).

Two other bacterial species (Fig. 1, samples b9 and b13)were amplified only once in adult flies. Sample b9, from a wildfemale fly, was closely related to the plant pathogen Citrobacterfreundii, although relationships were weak using ompA alone(see Fig. S1B in the supplemental material). Sample b13, froma wild California male fly, could not be identified using thetaxon sampling of this phylogeny. BLAST results showed 99%similarity to Raoultella ornithinolytica.

“Ca. Erwinia dacicola” and the Enterobacter sp. were asso-ciated with all olive fly life stages. PCR analysis revealed that“Ca. Erwinia dacicola” and the Enterobacter sp. were present inall life stages of wild olive flies surveyed, suggesting that theypersist through metamorphosis (data not shown). Sequenceanalysis revealed that “Ca. Erwinia dacicola” was present in94% of the females and 86% of the males. The sex of the fliesdid not influence the presence or absence of “Ca. Erwiniadacicola” (P � 0.2932; n � 113). In 9.8% of the adults, neither“Ca. Erwinia dacicola” nor the Enterobacter sp. was found.There were no flies with both bacteria. One male had only theEnterobacter sp. There was no difference in the frequency of“Ca. Erwinia dacicola” between individuals from Californiaand those from Arizona (P � 0.7213; n � 113). “Ca. Erwiniadacicola” and the Enterobacter sp. were not present in eitherripe or unripe olives without fly larvae. The rot tunnels that thelarvae made while feeding on the olive always contained “Ca.Erwinia dacicola” in ripe (n � 20) and unripe (n � 20) olives(data not shown).

“Ca. Erwinia dacicola” and the Enterobacter sp. are locatedwithin larvae and adults. “Ca. Erwinia dacicola” was consis-tently amplified from several structures of the B. oleae digestivetract. The endosymbiont was always present in the larval mid-gut (mycetome) (n � 10) and the evagination of the adultforegut (esophageal bulb) (n � 10). More than 70% of the 8rectal sacs and 10 crops sampled had “Ca. Erwinia dacicola”(data not shown). None of the sampled larval salivary glands(n � 10) or adult testes (n � 8) or ovaries (n � 5) had eitherbacterium. Ovipositors of four of the five females sampled had“Ca. Erwinia dacicola.” The fifth ovipositor had only theEnterobacter sp.

Endosymbionts are both intracellular and extracellular.Bacterial species present in adult esophageal bulbs were iden-tified using FISH probes specific to “Ca. Erwinia dacicola” andEnterobacter 16S rRNA. In adults, all bacterial cells in theesophageal bulb hybridized with the general enteric bacterialprobe EUB338 and the “Ca. Erwinia dacicola”-specific probeEdF1-Cy5 (Fig. 2A and B) but not with the Enterobacter-specific EnF2-Cy3 probe, demonstrating that these cells were“Ca. Erwinia dacicola.” The EnF2-Cy3 and EUB338 probeslabeled the cultured Enterobacter sp. cells. Both of these resultswere confirmed using PCR. No labeling was seen in negativecontrols, and autofluorescence was very low (data not shown).

TEM revealed bacteria that were intracellular residents oflarval cells (see Fig. 4A to D) (e.g., residing within the cellularmembrane of epithelial cells of the digestive tissue) and extra-cellular residents of adult tissues (e.g., residing in the digestivesystem lumen but outside the cellular membrane of host cells)(Fig. 2A and B and 3A to D). In the adult, bacteria within theesophageal bulb (Fig. 3A to D) and ovipositor diverticula (Fig.3E) had the classic rod-shaped morphology of an enteric bac-terium (M1 type cells) and were surrounded by a matrix, sug-gesting biofilm development. These bacteria were longitudinalnearest the host epithelium, creating a very densely packedarray of cells in cross section (Fig. 3B and E). In the esopha-geal bulb center, bacteria were more randomly oriented andmay have included cells dispersing from the biofilm, sincebacteria were observed in the stem connecting the esophagealbulb to the rest of the digestive system (data not shown). Otherstudies have noted bacteria dispersing from the esophagealbulb (9).

Although microscopy of the olive fly pupal stage was unsuc-cessful, recently eclosed (�24 h posteclosion) adults were ex-amined to determine the number of bacteria present immedi-ately after pupation. The esophageal bulbs of recently eclosedadults contained fewer than 10 bacterial cells closely associatedwith the bulb epithelium (Fig. 3C), and a few bacteria wereloosely associated with membranes. In contrast, bulbs of older,fed adults were filled with bacteria (Fig. 3A and B).

In larvae, bacterial cells were present within host cells (intra-cellular) (Fig. 4A to D), instead of inside the digestive systemlumen (extracellular), as in the adult esophageal bulb. Within hostcells, the endosymbiont exhibited two bacterial morphologies.The majority of bacteria in host cells had an amorphous morphol-ogy (M2-type cells) (Fig. 4A, B, and D) similar to that of otherinsect intracellular endosymbionts, such as Buchnera (41), “Can-didatus Carsonella” (53), and “Candidatus Ishikawaella” (24).The cytoplasm of the amorphous morphology was diffuse andgranular, with electron-dense inclusion bodies clustered at one


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region of the cell (Fig. 4D). A second bacterial morphology,circular in cross section with a denser cytoplasm, typical of entericbacteria and similar to what is seen in the adult stage (M1-typecells) (Fig. 4C), was rarely present. Although two distinct bacterialmorphologies were present, PCR confirmed that “Ca. Erwiniadacicola” was the only bacterium found in the mycetome.

The G�C compositions of recA, ompA, and 16S rRNA fromolive fly symbionts are similar to those of closely related,free-living species. The G�C nucleotide contents of recA,ompA, and 16S rRNA were correlated with the overall genomeG�C content (n � 32; R2 � 0.94, 0.97, and 0.70, respectively;P � 0.0001 in all cases), and the whole-genome G�C content

FIG. 3. Extracellular bacteria in lumen of adult esophageal bulb and ovipositor. (A) Cross section through the esophageal bulb of a 1-month-oldfed adult olive fly. A host nucleus (n) is identified in the epithelial tissue of the bulb that surrounds the bacteria. Extracellular bacteria (b) fill theesophageal bulb lumen. Bar, 80 �m. (B) Close-up of the esophageal bulb shown in panel A. Extracellular bacteria form a classic bacterial biofilm,with those closest to the host epithelia (e) seen in cross section. Bacteria farther away from the host epithelia are seen in longitudinal section,revealing a typical gram-negative rod shape. Endosymbionts are surrounded by an extracellular matrix. Bar, 5 �m. (C) Esophageal bulb of arecently eclosed, unfed adult olive fly. Arrows point to the three bacteria present in the esophageal bulb lumen. These bacteria are located nearthe host epithelium. Bar, 35 �m. (D) High (�190,000) magnification of the bacteria from panel C. Bar, 5 �m. (E) Cross section through the lumenof a female ovipositor showing seven diverticula filled with extracellular bacteria. Extracellular bacteria within one of the diverticula are indicated.The nuclei of two host cells next to the diverticulum are evident. These bacteria are similar in morphology to those seen in the adult esophagealbulb (panel B). Bar, 30 �m.

FIG. 4. Intracellular bacteria of larval mycetome. (A) Larval midgut showing a field of bacteria with the amorphous morphology (M2). Fourbacterial cells with the M1 morphology are indicated by arrowheads. Arrows point to a host cell membrane surrounding the bacteria. Bar, 5 �m.(B) Cell wall (arrow) of a gram-negative bacterium having the amorphous M2 morphology. Magnification, �140,000. Bar, 4 �m. (C) High-magnification (�100,000) inset from panel A of a bacterial cell having the M1 morphology. Note the host cell membrane surrounding the bacterialcell. Bar, 2.5 �m. (D) M2 cells shown at �72,000 magnification. Arrowhead points to electron-dense granules. Bar, 2 �m. Host mitochondria (m)are indicated in panels B, C, and D.


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is positively correlated with the genome size (n � 32; R2 �0.90; P � 0.0001) in the sequenced bacterial genomes used inour phylogenetic analysis. The G�C contents of recA, ompA,and 16S rRNA were correlated with the genome size (n � 32;R2 � 0.76, 0.9, and 0.63, respectively; P � 0.0001 in all cases).The G�C compositions of recA, ompA, and 16S rRNA of both“Ca. Erwinia dacicola” and the Enterobacter sp. associated witholive flies are similar to those of closely related, free-livingenteric bacteria (Table 1; see also Table S3 in the supplemen-tal material). The genome sizes of the bacteria, estimated fromthe equation of the regression line of all three loci, were similarto the genome sizes of their free-living enteric bacterial rela-tives (Table 1; see also Table S3 in the supplemental material).

DISCUSSION

“Ca. Erwinia dacicola” is a tightly associated symbiont ofthe olive fly. Culturable bacterial endosymbionts of the olive flywere first described by Petri in 1909 (47). A century later,Capuzzo et al. used molecular techniques to identify a specificuncultivable bacterium, “Candidatus Erwinia dacicola,” in Ital-ian adult olive flies (9). Other studies have identified transientgammaproteobacterial species acquired during feeding (5, 52),as well as the alphaproteobacteria Acetobacter tropicalis (29)and Asaia spp. (52), within the olive fly.

Our study found two bacteria, a culturable Enterobacter sp.and the unculturable “Ca. Erwinia dacicola,” present in all lifestages of wild olive flies, suggesting that these bacteria can bemaintained through changes in diet and host metamorphosis.Although other low-abundance species that our universal 16SrRNA primers do not amplify may be present, we focused onsymbionts consistently present at high density, not on all spe-cies within the olive fly digestive system.

“Candidatus Erwinia dacicola” is found both in the TLP andin 90% of wild flies surveyed. It is possible that the 10% of wildindividuals testing negative for “Ca. Erwinia dacicola” wererecently eclosed individuals with endosymbiont titers below thethreshold detectable by PCR. qPCR of other recently eclosedflies showed extremely low densities of “Ca. Erwinia dacicola”(data not shown), and TEM revealed fewer than a dozen bac-teria in the esophageal bulbs of recently eclosed flies (Fig. 3B),so PCR screens may underestimate endosymbiont presence.“Ca. Erwinia dacicola” has also been found within adult oliveflies in Italy (9, 52) and Greece (29). The high frequency ofassociation of “Ca. Erwinia dacicola” with the fly, its presencein different populations and in all life stages, its inability to becultured on standard microbiological media, its vertical trans-mission to offspring, and its ability to reside within larval hostcells suggest that this bacterium is a tightly associated endo-symbiont of olive flies (46). To our knowledge, intracellularendosymbionts have not been reported in other tephritids (30),and other olive fly-associated bacteria, such as Acetobactertropicalis, are exclusively extracellular (29, 52). An intracellularstage of “Ca. Erwinia dacicola” implies a more specific andlonger-term interaction with the host (26).

The Enterobacter sp. is only occasionally found in the oliveflies screened and is likely to be a nonspecific microbe. Entero-bacter spp. are common in insects, including the other tephrit-ids Rhagoletis completa (49), Rhagoletis pomonella (30), andBactrocera tau (C. S. Prabhakar, P. Sood, B. A. Padder, S. S.

Kanwar, V. Kapoor, P. K. Metha, and P. N. Sharma, unpub-lished data); cockroaches (12; also C. M. Gibson, unpublisheddata); fire ants (31); and cucumber beetles (D. R. Herndon andK. D. Spence, unpublished data). The Citrobacter sp. and sam-ple b13 were thought to be phylloplane bacteria acquired dur-ing feeding (5, 14), since they were PCR amplified only once inwild-caught adults.

Our phylogenetic analyses demonstrate that “Ca. Erwiniadacicola” is closely related to several insect-vectored, plant-pathogenic Erwinia species, such as Erwinia amylovora, Erwiniapyrifoliae, Erwinia tracheiphila, and Pantoea (Erwinia) stewartii.These pathogens persist primarily in association with theirplant hosts and insect vectors, with limited survival in the soil(50). These closely related plant pathogens differ from eachother and from “Ca. Erwinia dacicola” in the duration and theintimacy of their association with their insect versus planthosts. Erwinia amylovora and Erwinia pyrifoliae overwinter atcanker margins and in plant host buds, causing wilt (34, 51).Insect vectors are attracted to these bacterium-filled exudatesand transmit the pathogens to new infection sites (16, 23, 51).In contrast, Erwinia tracheiphila and Pantoea stewartii overwin-ter in beetle digestive systems, and disease incidence on hostplants is correlated with insect survival (11, 20, 40). “Ca. Er-winia dacicola” is found (via PCR) only in the rot tunnelswhere larvae are present, presumably deposited in the olivethrough regurgitation or defecation. Whether the bacterium ismetabolically active in rot or whether the host or symbiont isresponsible for the rot is unknown.

“Ca. Erwinia dacicola” transitions between an intracellularand an extracellular existence. During olive fly development,“Ca. Erwinia dacicola” resides intracellularly within larval mid-gut cells and extracellularly in the adult foregut. Other insectendosymbionts survive both intracellularly and extracellularly(10, 45); however, the ramifications of this transition for sym-biosis and for endosymbiont evolution have not been dis-cussed. The transition from an intracellular to an extracellularexistence within the digestive system during insect develop-ment could be essential to endosymbiont survival in a polypha-gous, holometabolous host. Prior to pupation, Diptera voidtheir digestive tracts, reducing the number of extracellularmicrobes (22). During metamorphosis, larval digestive systemtissues degrade, and adult tissues are assembled from larvalmidgut regenerative cells (27). Vertically transmitted endo-symbionts must have mechanisms for surviving metamorphosisand reestablishing themselves in the adult. Although the mech-anisms of endosymbiont survival during complete metamor-phosis are unknown, we hypothesize that bacterial cells presentin the regenerative cells recolonize the adult gut.

In adults, “Ca. Erwinia dacicola” resides in a bacterial bio-film in the digestive system lumen. Nutrient acquisition foropportunistic foragers, such as the adult olive fly, is oftenspatially and temporally patchy. The formation of bacterialbiofilms may provide resistance to periods of low nutrients andother stresses (13). Olive fly endosymbionts could not be re-moved from adult flies fed bleach and antibiotics (rifampin[rifampicin], streptomycin, tetracycline, and ampicillin) (datanot shown) once biofilms were established. The extracellularlifestyle could also be important for transmission to offspring.The presence of “Ca. Erwinia dacicola” in the ovipositor, butnot in the ovaries or testes, suggests that olive fly endosymbi-


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onts (47) are vertically transmitted via smearing as the eggpasses by the symbiont-rich ovipositor diverticulum (Fig. 3E)(52). Extracellular bacteria have more rigid and well-formedcell walls that may facilitate egg-smearing transmission.

The endosymbiont environment hypothesis and bacterialgenome characteristics. The olive fly, “Ca. Erwinia dacicola,”provides, to our knowledge, the first example of a tightly as-sociated symbiosis in a polyphagous, holometabolous insect.This study expands our knowledge of the types of environ-ments in which endosymbionts survive and leads us to proposethat symbioses be discussed in terms of the endosymbiontenvironment the host provides. In insects, the endosymbiontenvironment ranges from constant to variable based on at leastthree axes: the host diet (monophagous versus polyphagous),the host life cycle (hemimetabolous versus holometabolous),and the location of the endosymbiont within host tissues (in-tracellular versus extracellular). The most constant environ-ment for endosymbionts would be provided by monophagous,hemimetabolous insects, such as aphids and sharpshooters,where endosymbionts reside exclusively within bacteriomesand hosts feed exclusively on one food type. The most variableenvironment would be found in polyphagous, holometabolousinsects, such as the olive fly. Endosymbionts of polyphagous,holometabolous insects must survive changes in insect diet andchanges to tissues during complete metamorphosis. We sug-gest that the environment an insect endosymbiont experiencesmay greatly influence its genome composition, genome size,and evolution (A. M. Estes, unpublished data).

The nucleotide compositions and genome sizes of bacteriaare directly related to the degree of environmental variability,since bacteria quickly accumulate mutations and lose genesthat are not under selection (6, 43). Free-living bacteria expe-riencing a diversity of environmental conditions have moreG�C-rich genes and larger genomes than obligately intracel-lular endosymbionts (3, 6, 21). Thus, we predicted that endo-symbionts of polyphagous, holometabolous insects, such as theolive fly, would have genomes that are less A�T biased andlarger than those of obligate endosymbionts of monophagous,holometabolous insects, such as aphids. Indeed, the nucleotidecompositions of the three loci analyzed from “Ca. Erwiniadacicola” and the Enterobacter sp. were more similar to thosefor plant-pathogenic and free-living relatives than to those forobligately intracellular endosymbionts (Table 1; see also TableS3 in the supplemental material). The G�C nucleotide com-positions of the recA, ompA, and 16S rRNA genes were shownto be a good proxy for genome size, and the genome sizes ofboth bacteria were estimated to be similar to those of free-living and plant-pathogenic relatives.

The data presented here suggest that polyphagous, holo-metabolous insects associate with specific microbes. This studyof the olive fly endosymbiont, “Ca. Erwinia dacicola,” providesdetailed microscopy to show that the bacterium associates withdifferent host tissues and transitions between intracellular andextracellular stages during host development. This is one of thefew examples of an insect endosymbiont that makes such atransition. Examination of digestive-system endosymbionts ofother polyphagous, holometabolous insects will be essential tounderstanding the mechanisms that influence bacterial ge-nome structure and evolution.

ACKNOWLEDGMENTS

We thank Leland S. Pierson III for unlimited access to lab equip-ment and ancillary lab supplies; Elizabeth Arnold, Leland S. PiersonIII, the Bronstein lab, Monica Alvarez, Antonio Belcari, and threeanonymous reviewers for comments on the manuscript; Justin Clarkfor assisting with olive tissue samples; Hannah Burrack and the lab ofFrank Zalom at the University of California, Davis, for California olivefly samples; David Bentley and Carl Boswell for microscopy assistance;Phat Tran for assistance with FISH; and the Pierson and Bronsteinlabs at the University of Arizona, especially Krishna Maddula, forhelpful discussions.

This work was supported by a Doctoral Dissertation ImprovementGrant, DEB-0608480 (to J.L.B. and A.M.E.); an Integrative GraduateEducation and Research Traineeship, NSF-IGERT DGE 0114420,from the National Science Foundation (to A.M.E.); and a Grant-in-Aid from the Society for Integrative and Comparative Biology (toA.M.E.).

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The Olive Fly Endosymbiont, “Candidatus Erwinia dacicola,” … · 2009. 11. 4. · different nutrients during an insect’s life time. Second, Diptera are holometabolous, undergoing