Rickettsia Symbionts Cause Parthenogenetic Reproduction in ...negatively affect some aspects of host ﬁtness, causing reduc-tions in body weight, fecundity, and longevity in the pea

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2010, p. 2589–2599 Vol. 76, No. 80099-2240/10/$12.00 doi:10.1128/AEM.03154-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Rickettsia Symbionts Cause Parthenogenetic Reproduction in theParasitoid Wasp Pnigalio soemius (Hymenoptera: Eulophidae)�

M. Giorgini,1* U. Bernardo,1 M. M. Monti,1 A. G. Nappo,1 and M. Gebiola1,2

Istituto per la Protezione delle Piante, CNR, Portici (NA), Italy 80055,1 and Dipartimento di Entomologia e Zoologia Agraria,Universita di Napoli “Federico II,” Portici (NA), Italy 800552

Received 31 December 2009/Accepted 8 February 2010

Bacteria in the genus Rickettsia are intracellular symbionts of disparate groups of organisms. Some Rickettsiastrains infect vertebrate animals and plants, where they cause diseases, but most strains are verticallyinherited symbionts of invertebrates. In insects Rickettsia symbionts are known to have diverse effects on hostsranging from influencing host fitness to manipulating reproduction. Here we provide evidence that a Rickettsiasymbiont causes thelytokous parthenogenesis (in which mothers produce only daughters from unfertilizedeggs) in a parasitoid wasp, Pnigalio soemius (Hymenoptera: Eulophidae). Feeding antibiotics to thelytokousfemale wasps resulted in production of progeny that were almost all males. Cloning and sequencing of afragment of the 16S rRNA gene amplified with universal primers, diagnostic PCR screening of symbiontlineages associated with manipulation of reproduction, and fluorescence in situ hybridization (FISH) revealedthat Rickettsia is always associated with thelytokous P. soemius and that no other bacteria that manipulatereproduction are present. Molecular analyses and FISH showed that Rickettsia is distributed in the reproduc-tive tissues and is transovarially transmitted from mothers to offspring. Comparison of antibiotic-treatedfemales and untreated females showed that infection had no cost. Phylogenetic analyses of 16S rRNA and gltAgene sequences placed the symbiont of P. soemius in the bellii group and indicated that there have been twoseparate origins of the parthenogenesis-inducing phenotype in the genus Rickettsia. A possible route forevolution of induction of parthenogenesis in the two distantly related Rickettsia lineages is discussed.

The genus Rickettsia contains a group of obligate intracellu-lar symbionts of eukaryotic cells and belongs to the familyRickettsiaceae in the order Rickettsiales of the Alphaproteobac-teria (58, 90). Many species have medical importance as theyare pathogens of humans and other vertebrates; pathogenicRickettsia species infect their hosts through blood-feeding ar-thropods, including lice, fleas, ticks, and mites (51, 80). Inaddition to Rickettsia species that cause infectious diseases invertebrates, symbiotic species have been found in disparategroups of organisms, including arthropods, annelids, amoebae,hydrozoa, and plants (53). Rickettsia appears to be especiallycommon in arthropods, having been found in a wide range oftaxa in the classes Entognatha (springtails), Insecta (booklice,lice, bugs, leafhoppers, aphids, whiteflies, fleas, flies, lacewings,moths, beetles, and wasps), and Acarina (ticks and mites) (86).However, in most cases, the effect of Rickettsia on the inver-tebrate host has not been established yet. In general, Rickettsiabacteria are facultative symbionts, but in the booklouseLiposcelis bostrychophila the association is strictly obligate andRickettsia has an essential role in oocyte development (54, 92).Facultative symbiotic Rickettsia strains have been reported tonegatively affect some aspects of host fitness, causing reduc-tions in body weight, fecundity, and longevity in the pea aphid(16, 60, 64), reductions in viability in some blood-feeding ar-thropod vectors (5, 46), and increased susceptibility to insec-ticides in the sweet potato whitefly (41). There is also evidencethat Rickettsia has positive effects on host fitness, such as a

larger body size in infected leeches (40) and a possible role inthe oogenesis of a bark beetle (93). Finally, facultative symbi-otic rickettsiae can be reproductive parasites of insects. Rick-ettsia strains are the causal agents of male killing (infectedmale embryos die) in some ladybird (79, 88) and buprestidleaf-mining (42) beetles. They are also the cause of thelytokousparthenogenesis (in which mothers produce only daughtersfrom unfertilized eggs) in a parasitoid wasp (32). Both kinds ofreproductive manipulation bias the host sex ratio toward fe-males and favor the spread of the transovarially inherited Rick-ettsia strains in the infected populations. In general, Rickettsiais transmitted primarily vertically to host progeny, but inpathogenic species there is concomitant horizontal transmis-sion via intermediate vertebrate hosts, which plays an impor-tant role in maintaining the infection in populations of blood-feeding arthropods (53, 57). An exception is Rickettsiaprowazekii, the epidemic typhus agent, which spreads only viahorizontal transmission in louse host populations (5). Only oneRickettsia is known to be a plant pathogen, and leafhopperstransfer this pathogen horizontally between plants (20). Thefact that Rickettsia can be transmitted horizontally and thenperpetuated vertically in host descendants has probably beenone of the most important factors determining the enormousdiversity of Rickettsia symbiotic associations. This point hasbeen emphasized by analyses that have revealed considerableincongruence between Rickettsia and host phylogenies, indicat-ing that horizontal transfer has occurred multiple times overevolutionary timescales (53, 54, 86).

In addition to Rickettsia, diverse heritable bacteria are knownto manipulate host reproduction to enhance their transmission inarthropods (12, 23). Wolbachia (order Rickettsiales, familyAnaplasmataceae), a close relative of Rickettsia (90), is able to

* Corresponding author. Mailing address: Istituto per la Protezionedelle Piante, CNR, via Universita 133, Portici (NA), Italy 80055.Phone: 390817753658. Fax: 390817758122. E-mail: [email protected].

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induce all known forms of manipulation of reproduction, includ-ing cytoplasmic incompatibility, feminization of genetic males,male killing, and parthenogenesis (68). Previously, only Car-dinium (Sphingobacteria) has been shown to cause a similar rangeof reproductive phenotypes, except for male killing (35). Theemerging diversity of Rickettsia associated with arthropods (53,86), combined with evidence that it can manipulate host repro-duction in more than one way, suggests that this symbiont mayalso be a master manipulator.

In the Hymenoptera, the dominant mode of reproduction isarrhenotoky; that is, diploid females develop from fertilizedeggs, and haploid males develop from unfertilized eggs (76).However, thelytokous parthenogenesis is common, and insome lineages, like the superfamilies Chalcidoidea andCynipoidea, it is strongly associated with Wolbachia or Car-dinium infection (33, 35). Parthenogenesis-inducing (PI) bac-teria cause restoration of diploidy in unfertilized haploid eggs,which results in female offspring (28, 50, 69). PI Wolbachia andPI Cardinium also occur in other groups of haplodiploid ar-thropods, such as mites (82), scale insects (56), and thrips (4).Previously, the only example of PI caused by Rickettsia wasfound in the eulophid parasitoid wasp Neochrysocharis formosa(1, 32). Besides PI bacteria, uniparental (thelytokous) repro-duction in haplodiploid arthropods can also be caused by fem-inizing bacteria that are able to interact with the host sexdetermination system and force the development of genotypicmales toward functional phenotypic females. To date, onlyCardinium has been reported to be a causal agent of femini-zation in haplodiploid arthropods, and only two examples areknown: a mite in which Cardinium causes haploid male em-bryos to develop as haploid females (18, 83) and a parasitoidwasp in which diploid males are converted to females (27).

In this paper, thelytokous reproduction in a parasitoid wasp,Pnigalio soemius (Hymenoptera: Eulophidae), was studied.This wasp, which is probably a complex of cryptic species (8),is a solitary ectoparasitoid that attacks larvae of many leaf-miner insect species in the orders Coleoptera, Diptera, Hyme-noptera, and Lepidoptera (48), some of which are pests ofagricultural crops (37, 61). Female P. soemius wasps paralyzehost larvae by injection of venom and subsequently lay a singleegg next to the host inside a leaf mine; then the parasitoid larvaeats the killed host (7). Commonly, P. soemius reproducesbiparentally, and the occurrence of thelytoky has not beenreported previously. The aims of this study were to determinewhether symbiotic bacteria are involved in manipulating thereproduction of P. soemius and then to determine the taxonomicaffiliation and phenotype of the manipulators of reproductiondiscovered. By using antibiotic treatments and karyological anal-ysis of the insect studied, molecular and phylogenetic character-ization of the symbiotic bacteria, and detection of intracellularsymbionts by means of fluorescence in situ hybridization, it wasdemonstrated that a PI Rickettsia causes thelytokous reproduc-tion in P. soemius.

MATERIALS AND METHODS

Study insect. A thelytokous population of P. soemius was obtained from larvaeof the leafminer Trypeta artemisiae (Diptera: Tephritidae) collected in the fieldfrom plants of the common wormwood (Artemisia vulgaris) in Usseaux, Torino,Italy. A culture of the parasitoid was generated using a single female. As it wasdifficult to rear the natural host in the laboratory, an alternative host, the

leafminer Cosmopterix pulchrimella (Lepidoptera: Cosmopterigidae), was usedand maintained on wall pellitory (Parietaria diffusa) plants as described previ-ously (9). Due to high parasitoid mortality during larval development and torapid degradation of the host larvae stung by the female wasp, it was difficult toestablish a permanent colony of thelytokous P. soemius by allowing wasp larvaeto develop naturally in leaf mines with single host larvae. Consequently, anartificial rearing system (8) was used, in which several mature host larvae wereprovided to a single parasitoid during development. Although more time-con-suming, this system allowed us to significantly increase production of offspring.Briefly, inside leaf mines mature host larvae were exposed to oviposition bysingle parasitoids for 24 h, after which the mines were dissected and eggs weresingly transferred on a glass slide kept in a humid petri dish. A new host larva,killed by freezing it at �20°C for 3 min (killing was necessary as the parasitoidlarvae can eat only immobilized hosts), was added near each egg on the slide.Freshly killed larvae were added daily until the parasitoid pupated. Insects werereared at 25 � 1°C with a relative humidity of 60% � 10% using a photoperiodconsisting of 12 h of light and 12 h of darkness. To date, after 20 generations oflaboratory rearing, male progeny have never been obtained.

Antibiotic treatment. In order to determine if bacterial symbionts were in-volved in parthenogenetic reproduction in P. soemius, the sex ratio of the prog-eny produced by antibiotic-treated adult females was assessed. Ten newlyemerged adult females were individually fed honey containing rifampin (20mg/ml) for 24 h in a glass vial and then allowed to oviposit for their entire lifespan as described above. After the initial 24-h treatment with the antibiotic,adults were fed daily using honey streaks until they died. The effects of antibiotictreatment on fecundity and fertility were also examined in order to assess ifRickettsia influences host fitness. The following characteristics were determinedfor treated and untreated (fed only honey) females: number of eggs laid, per-centage of unhatched eggs, percentage of mortality for the progeny duringdevelopment from larvae to pupae, and number and sex of the adult progeny. Totest for antibiotic toxicity, the longevity of the treated females was compared tothat of the control females. The experimental conditions were 25 � 1°C, arelative humidity of 60% � 10%, and a photoperiod consisting of 12 h of lightand 12 h of darkness. Biological data that satisfied conditions of normality andhomoscedasticity, both untransformed and after appropriate transformation,were subjected to a one-way analysis of variance (ANOVA). When the assump-tions of the ANOVA were violated and could not be met by data transformation,the nonparametric Kruskal-Wallis test was used after having controlled for thedata distribution to have the same shape.

Gene sequencing and PCR detection of bacterial symbionts. Soon after col-lection all specimens were surface sterilized in 70% ethanol and rinsed five timesin sterile water before they were stored in 70% ethanol at �20°C. DNA wasextracted from two samples: a pool of five adult P. soemius females and a poolof 15 eggs laid by different females and collected soon after oviposition. ForDNA isolation, the protocol of Walsh et al. (81) was used, with a few modifica-tions. Briefly, the two samples were homogenized in DNA extraction bufferconsisting of 150 �l Chelex and 25 �l proteinase K (20 mg/ml) for the adultsample and 75 �l Chelex and 15 �l proteinase K (20 mg/ml) for the egg sample.Each sample was incubated for 1 h at 55°C and for 8 min at 100°C and centri-fuged at 13,000 rpm for 3 min.

To detect bacterial symbionts, the 16S rRNA gene was amplified using uni-versal primers 27F (5�-AGAGTTTGATCMTGGCTCAG-3�) and 1513R (5�-ACGGYTACCTTGTTACGACTT-3�) (87), cloned, and sequenced. PCRs wereperformed with 40-�l mixtures containing 1� buffer (Promega), each de-oxynucleoside triphosphate at a concentration of 0.2 mM, 3 U of GoTaq DNApolymerase (Promega), and each primer at a concentration of 300 nM. After aninitial denaturation for 3 min at 96°C, the temperature profile consisted of 94°Cfor 30 s, 52°C for 1 min, and 72°C for 2 min for 35 cycles, followed by a finalextension for 7 min at 72°C. Five microliters of each reaction mixture waschecked on a 1% agarose gel, and samples that yielded amplicons of the expectedsize (�1,500 bp) were precipitated, ligated into the pGEM-T Easy plasmidvector (Promega), and cloned into Escherichia coli TOP10 competent cells (In-vitrogen) according to the manufacturer’s instructions. Transformants werescreened by performing PCR with universal M13 vector primers, and inserts ofthe expected size (�1,800 bp) were amplified by nested PCR with universalbacterial primers 341f (5�-CCTACGGGAGGCAGCAG-3�) and 907r (5�-CCGTCAATTCMTTTGAGTTT-3�) (45). The PCR thermal profile was initial dena-turation for 3 min at 96°C, 30 cycles of 94°C for 45 s, 52°C for 50 s, and 72°C for50 s, and then final extension for 5 min at 72°C. All 500- to 600-bp ampliconswere directly sequenced with the 341f primer. A 154-bp fragment was obtained,and it included the hypervariable region V3, which has been proven to be usefulfor studying the composition of bacterial communities (15, 99). As almost allsequences (42 of 44 clones) corresponded to Rickettsia sequences and the other

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two were sequences of bacteria not associated with manipulation of reproduc-tion, 15 clones were chosen from the clones identified as Rickettsia (6 clones fromthe adult sample and 9 clones from the egg sample) and were sequenced with the27F and 1513R primers in order to characterize the Rickettsia strain. Further-more, a fragment of the citrate synthase gltA gene was sequenced. Rickettsia-specific primers CS409d and CS1273r (59) were used to amplify 864 bp of thegltA gene from the egg sample. Two more sequences were obtained from theadult female sample by using primers CS409d (59) and RicCS-AR (60), whichamplified a 600-bp fragment. The PCR conditions used for the gltA gene were thesame as those described above for amplification of the 1,500-bp fragment of the16S rRNA gene, except that the annealing temperature was 48°C; the ampliconsobtained were directly sequenced.

Sequencing was performed with an ABI Prism Big Dye terminator cyclesequencing kit and an ABI Prism 310 genetic analyzer (Applied Biosystems,Foster City, CA) at the Dipartimento di Biologia Vegetale, University of Napoli“Federico II,” Italy, sequencing facility. Sequences were assembled using Seq-Man in the Lasergene software package (DNASTAR, Madison, WI) and werecompared with known sequences in the GenBank database by using BLASTsearches (www.ncbi.nlm.nih.gov/blast) (3).

Finally, the possibility that the bacterial symbionts known to cause thelytokousreproduction in arthropods, including Cardinium, Wolbachia, and Rickettsia,were present was checked by performing diagnostic PCR both for the samplesused for sequencing and for 10 single P. soemius adult females. The following setsof primers were used: primers CLOf and CLOr1 (85) and primers ChF and ChR(96) for the Cardinium 16S rRNA gene, primers ftsZf1 and ftsZr1 (89) for theWolbachia cell cycle ftsZ gene, primers 16Swolb76-99f and 16Swolb1012-994r forthe Wolbachia 16S rRNA gene (49), and primers Rb-F and Rb-R (29) for theRickettsia 16S rRNA gene. As only Rickettsia was found in P. soemius females,the infection status of 10 adult males produced by antibiotic-treated females waschecked using only primers Rb-F and Rb-R. To confirm that negative results inPCR analyses were not due to poor-quality extracts, PCRs were performed for allsamples with primers ITS2F and ITS2R (14). These primers amplify �500 bp ofthe internal transcribed spacer 2 region of Hymenoptera. Therefore, only posi-tive samples were considered to assess the infection status.

Molecular phylogenetic analysis. DNA sequences of the 16S rRNA and gltAgenes (1,388 bp and 721 bp, respectively) were aligned with Rickettsia sequencesavailable in the GenBank database. Multiple alignments were constructed withthe ClustalW method of MegAlign in the Lasergene software package(DNASTAR, Madison WI). Phylogenies were reconstructed using maximumparsimony (MP) and maximum likelihood (ML) methods as implemented inPAUP 4.0b10 (71). The Rickettsia symbiont of the ciliate Diophrys appendiculata(75) was chosen as the outgroup for the analysis based on the 16S rRNA gene.A Wolbachia strain was chosen as the outgroup for the analysis based on the gltAgene. In the MP analysis, heuristic searches with tree bisection reconnection(TBR) branch swapping, the random addition sequence option, and Maxtrees setto increase without limits were performed. The evolutionary models used in theML analysis were provided by MODELTEST 3.7 (55). Based on both hierarchi-cal likelihood ratio test (hLRT) and Akaike (AIC) criteria, the models chosenwere K81 for the 16S rRNA gene sequences and K81uf�G (Kimura three-parameter model with unequal base frequencies and an estimated gamma shapeparameter) for the gltA sequences. All sites were weighted equally. Bootstrapsupport was evaluated by using 1,000 replicates in the MP analysis and 100replicates in the ML analysis.

Fluorescence microscopy. Localization of Rickettsia in the host’s reproductivetissues was studied using fluorescence in situ hybridization (FISH). The Rickettsiaprobe RickPn-Cy3 (�5-Cy3-TCCACGTCGCCGTATTGC-3�) was designed us-ing the 16S rRNA sequences of the P. soemius symbiont. Probe specificity waschecked using the Ribosomal Database Project “probe match” analysis tool(http://rdp.cme.msu.edu). Eggs were collected shortly after the oviposition. Ova-ries were extracted from adult females in a drop of phosphate-buffered saline(PBS) using a stereomicroscope. Eggs and ovaries were subjected to analysis bythe whole-mount FISH method described by Sakurai et al. (60), with slightmodifications. Eggs were dechorionated in 50% commercial bleach in PBS for 15min and then washed in PBS to remove the bleach. Samples were fixed overnightin a 4% paraformaldehyde neutral buffered solution, decolorized in 6% H2O2 inethanol for 2 h, and hybridized overnight. The hybridization buffer (20 mMTris-HCl [pH 8.0], 0.9 M NaCl, 0.01% SDS, 30% formamide) contained 10pmol/ml of fluorescent probe. Stained samples were observed with a ZeissAxiophot 2 epifluorescence microscope. The specificity of the signals observedwas verified using the following controls: no-probe control, RNase-digestedcontrol, and Rickettsia-free samples from an uninfected bisexual strain of P.soemius. Nuclei of the host cells were counterstained with 4�,6�-diamidino-2-phenylindole (DAPI) (0.4 �g/ml) in mounting medium. Double FISH was per-

formed with eggs of thelytokous P. soemius using RickPn-Cy3 and the universalprobe EUB338-Fluorescein (5�-fluorescein-GCTGCCTCCCGTAGGAGT-3�).

Determination of the wasp karyotype. In order to determine if the Rickettsiaphenotype resulted from induction of parthenogenesis or feminization, we ana-lyzed chromosome sets of individual female and male wasps produced by un-treated and antibiotic-treated adult females of P. soemius, respectively. If induc-tion of parthenogenesis occurred, diploid females and haploid males should havebeen found, as PI bacteria cause restoration of diploidy in unfertilized haploideggs (69). If feminization occurred, no difference in the level of ploidy betweenfemales and males should have been found (27, 83). Metaphase chromosomeswere obtained from single 4-day-old larvae (10 larvae for each sex) processedusing the scraping and air drying method described by Baldanza et al. (6), withthe following modifications. Each larva was nicked between the head and thethorax in 1.0 ml 0.1% colchicine in Shen solution (0.9 g NaCl, 0.042 g KCl, and0.025 g CaCl2 in 100 ml distilled water) and incubated in a 1.5-ml tube at roomtemperature for 30 min. After incubation, the colchicine solution containing thedissected larva was centrifuged at 1,300 rpm for 10 min. The supernatant wasremoved, 1.0 ml of a hypotonic solution (0.5% sodium citrate) was added, andafter 20 min the tube was centrifuged again at 1,300 rpm for 10 min. Thesupernatant was removed, and 0.4 ml of fixative (glacial acetic acid-methanol,1:3) was added. After 30 min of incubation at room temperature, the tissues werebroken by sucking and pushing them repeatedly with a pipettor equipped with a0.2-ml tip. The volume of fixative was adjusted to 1 ml, and the sample waspelleted by centrifugation at 1,300 rpm for 10 min. The supernatant was re-moved, 1.0 ml of fixative was added, and centrifugation at 1,300 rpm for 10 minwas performed again. Eventually, the pellet, suspended in 30 �l fixative, wasdropped on a slide, air dried, and stained with Giemsa stain (5% in phosphatebuffer, pH 6.8) for 30 min. Chromosomes were classified as described by Levanet al. (43).

Nucleotide sequence accession numbers. The sequences of the Rickettsia sym-biont from the thelytokous population of P. soemius have been deposited in theGenBank database under accession numbers EU881494 to EU881508 (1,388-bpsequences) and GU121643 to GU121669 (154-bp sequences) for the 16S rRNAgene and GU559856 for the gltA gene.

RESULTS

Effect of antibiotic treatment. Nine of 10 rifampin-treated P.soemius females produced only male offspring, and 97.5% ofthe offspring of treated females were males. Only one treatedmother produced progeny consisting of both sexes; it produced2 sons and 1 daughter, and the latter originated from the firstoviposited egg. The single F1 female was fed only honey (noantibiotic treatment), and its progeny were 3 males. In con-trast, untreated females produced only female offspring. Thenumbers of viable adult offspring produced by antibiotic-treated females (mean � standard error [SE], 4.0 � 1.11 off-spring; n � 10) were significantly lower than the numbers ofviable adult offspring produced by untreated females (7.7 �1.51 offspring; n � 10) (F � 4.97; df � 1; P � 0.039; ANOVA).There was not a significant difference (F � 1.89; df � 1; P �0.185; ANOVA) in fecundity (number of eggs laid) betweentreated females (mean � SE, 23.0 � 4.33 eggs) and untreatedfemales (30.7 � 3.55 eggs) or in the mortality of the progenythat occurred during embryonic and larval development. In-deed, the percentages of unhatched eggs were 17.57% �2.37% (mean � SE) for the eggs laid by treated females and14.94% � 2.59% for the eggs laid by untreated females (F �0.56; df � 1; P � 0.46; ANOVA). The percentages of mortalityfor the progeny during development from larvae to pupae were80.1% � 2.41% (mean � SE) for treated females and 68.5% �6.24% for untreated females (df � 1; P � 0.15; Kruskal-Wallistest). No negative effect of rifampin on the longevity of treatedfemales (mean � SE, 29.9 � 2.37 days; n � 10) compared tountreated females (32.9 � 3.42 days; n � 10) (F � 0.52; df �1; P � 0.480; ANOVA) was detected.

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Gene sequencing and PCR detection of bacterial symbionts.Using PCR, approximately 1,500 bp of the 16S rRNA gene wasamplified and cloned for a pool of adult females and a pool ofeggs of the thelytokous P. soemius. Forty-four positive cloneswere obtained (20 clones from the adult sample and 24 clonesfrom the egg sample) and amplified by nested PCR to se-quence a shorter fragment that included the hypervariable V3region. The 154-bp sequences obtained were checked againstsequences available in the GenBank database, and 42 of the 44clones showed the highest level of similarity to Rickettsia. Thesequences of the two remaining clones, which were retrievedfrom the adult sample, showed the highest levels of similarityto Methylobacterium sp. and Serratia proteamaculans. There isno evidence that these bacteria are involved in any type ofreproductive manipulation, and they have been reported to beenvironmental bacteria, bacteria associated with digestive or-gans, or pathogens of insects (30, 39, 73, 74). These two bac-teria were considered organisms that are not involved in in-duction of P. soemius parthenogenesis and were not includedin further analyses.

In order to characterize the Rickettsia obtained from P.soemius, 15 clones (6 clones from the adult sample and 9 clonesfrom the egg sample) were completely sequenced. Each of the1,388-bp sequences obtained was subjected to a BLAST searchand showed the highest level of sequence similarity (99%) toRickettsia bellii. The 15 sequences of the P. soemius symbiontexhibited high levels of similarity to one another (range, 99.4%to 100%; average, 99.8%). Also, a fragment of the gltA genewas amplified and directly sequenced. A 721-bp sequence wasobtained from the egg sample, and two 500-bp sequences wereobtained from the adult female sample. These sequences ex-hibited 100% nucleotide similarity to one another and exhib-ited the highest level of similarity (97%) to R. bellii.

Diagnostic PCR was used to screen the pool of adult fe-males, the pool of eggs, and 10 single adult females for infec-tion by known inducers of parthenogenesis of arthropods(Rickettsia, Wolbachia, and Cardinium). Only evidence of in-fection with Rickettsia was found. PCR analysis of adult malesproduced by antibiotic-treated females revealed that 9 of 10insects were still infected by Rickettsia.

Molecular phylogenetic analysis. MP analysis of the 16SrRNA gene showed that the sequences of 15 Rickettsia clonesinfecting thelytokous P. soemius form a well-supported lineagein the clade which includes R. bellii and symbionts of non-blood-feeding arthropods (Fig. 1A). MP analysis of gltA genesequences produced substantially the same result, placing theP. soemius symbiont in the clade that includes R. bellii (Fig.1B). The tree topologies produced by ML analyses of 16SrRNA and gltA gene sequences (data not shown) did not differconsistently and were congruent where nodes were highly sup-ported with trees produced by MP analyses.

Fluorescence microscopy. The distribution of Rickettsia inthe reproductive tissues of thelytokous P. soemius was studiedusing FISH analysis (Fig. 2 and 3). Inside the ovary, denseclusters of bacteria were observed in the germarium, in thegerm line cells, in the nurse cells, and in the developing oocytes(Fig. 2). In the oocytes, bacteria were concentrated at theposterior pole during mid to late oogenesis (Fig. 2G), but atthe end of oocyte development bacteria were also distributedin the ooplasm (Fig. 2H). Freshly laid eggs appeared to be

heavily infected with Rickettsia bacteria that were distributedthroughout the ooplasm, following a posterior-anterior gradi-ent; bacteria were still highly concentrated at the posteriorpole and sparse in the anterior area (Fig. 3A and B). Duringembryogenesis, at the syncytial stage Rickettsia bacteria local-ized at opposite poles of the mitotic spindles in dividing nucleiand were more concentrated in the posterior portion of theembryo, where they formed dense aggregates around nuclei ofthe pole cells, which are the germ line precursor cells (Fig. 3Cand D). Negative controls (no-probe, RNase-digested, andRickettsia-free sample controls) did not display signals, con-firming the specificity of the signals detected (data not shown).Simultaneous probing with the P. soemius symbiont-specificprobe RickPn-Cy3 and the universal bacterial probe EUB338-Fluorescein did not reveal bacteria other than Rickettsia (Fig.4D to F). Eggs oviposited by antibiotic-treated female waspsappeared to be still infected by Rickettsia, but the bacterialdensity was considerably lower than that in eggs laid by un-treated females (Fig. 4B).

Determination of the wasp karyotype. In the metaphaseplates examined (50 plates for each sex), females had a diploidcomplement consisting of 12 chromosomes, including fivemetacentric pairs and one acrocentric pair, while the malekaryotype was a haploid complement consisting of 6 chromo-somes (Fig. 5).

DISCUSSION

Bacteria in the genus Rickettsia are common intracellularsymbionts of disparate groups of organisms, yet the effects ofinfection with Rickettsia on invertebrate hosts are not knownfor most interactions (53, 86). Here, a Rickettsia that causesthelytokous parthenogenesis in the parasitoid wasp P. soemiuswas discovered.

Feeding antibiotics to uniparental females resulted in pro-duction of almost entirely male progeny, and the single F1

female produced by treated females produced F2 males exclu-sively (without additional antibiotic treatment). These resultsare consistent with the hypothesis that the Rickettsia symbiontis involved in determining the wasp reproductive phenotype.The infection status of thelytokous females was investigated bycloning and sequencing a portion of the bacterial 16S rRNAgene amplified with universal primers and by diagnostic PCRscreening using primers specific for Rickettsia, Wolbachia, andCardinium. Rickettsia was always detected in P. soemius sam-ples, whereas no evidence of other bacteria that manipulatereproduction was found. Sequencing of PCR amplicons recov-ered Rickettsia 16S rRNA and gltA gene sequences for adultfemales, as well as for parthenogenetic eggs. Furthermore,FISH revealed that this symbiont is distributed in the cells ofthe ovaries, as well as in eggs. FISH also showed that there wasno bacterium other than Rickettsia in the eggs. We concludedthat Rickettsia is the causal agent of thelytokous reproductionof P. soemius and is vertically transmitted from mothers tooffspring.

Two kinds of bacterium-induced manipulation of reproduc-tion leading to all female progeny and uniparental reproduc-tion of infected host populations have been found previously inhaplodiploid arthropods, namely, thelytokous parthenogenesis(development of unfertilized eggs into females occurs through



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restoration of diploidy) (33) and feminization (genotypic malesdevelop as functional phenotypic females due to interaction ofbacteria with their host’s sex determination system). Two ex-amples of feminization are known in haplodiploid arthropods,in which haploid male embryos (83) and diploid male embryos(27) develop as functional haploid and diploid females, respec-

tively. Analysis of the ploidy level in P. soemius revealed thatfemales are diploid and males induced by antibiotic treatmentare haploid, suggesting that the Rickettsia phenotype reflectsgenuine induction of thelytokous parthenogenesis. AnotherRickettsia has been shown to induce thelytokous reproductionin a different eulophid wasp, N. formosa (1), suggesting that

FIG. 1. Phylogenetic position of the symbiont of thelytokous P. soemius in the genus Rickettsia. (A) One of the 14 most parsimonious trees of16S rRNA gene sequences. The tree is rooted with the sequence of the Diophrys ciliate symbiont. The aligned final data set consisted of 1,362characters (with gaps treated as a fifth base), 94 of which were informative for parsimony. The tree length was 365 (consistency index [CI], 0.764;retention index [RI], 0.810). (B) One of the two most parsimonious trees of gltA gene sequences. The tree is rooted with the sequence of aWolbachia strain. The aligned final data set consisted of 712 characters (with gaps treated as a fifth base), 143 of which were informative forparsimony. The tree length was 555 (CI, 0.746; RI, 0.721). The numbers above the branches are bootstrap percentages based on 1,000 replicates(values less than 50% are not shown). Rickettsiae that have not been formally named are indicated by the name of the host followed by “symbiont.”The GenBank accession number is indicated after each Rickettsia name. Major groups of the genus Rickettsia described by Gillespie et al. (26) andWeinert et al. (86) are indicated on the right. Symbionts associated with manipulation of reproduction are indicated by PI for induction ofparthenogenesis and by MK for male killing.



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Rickettsia may routinely utilize PI as a mechanism to spread inhost populations.

Although feeding antibiotics to infected females inducedproduction of progeny that were almost all male, 90% of the

screened males produced by treated mothers were infected, asdetermined by PCR. The occurrence of Rickettsia in males wasconfirmed by FISH analysis of freshly laid eggs produced byantibiotic-treated females (putative male eggs); however, in

FIG. 2. Distribution of Rickettsia (bright red) in the ovary of thelytokous P. soemius. (A and B) Distribution of bacteria inside three ovarioles.(C and D) Bacteria infecting the germarium and the germ line cells. (E and F) Rickettsia localized around nuclei of the nurse cells. (G) Rickettsialocalized in the nurse cells and concentrated at the posterior pole of the developing oocyte. DAPI-stained nuclei are blue. (H) Posterior-anteriorgradient of bacteria in a developed oocyte. The posterior pole is on the right. (A, C, E, G, and H) Fluorescence channel. (B, D, and F) Overlayof fluorescence and bright-field channels. Autofluorescence of ooplasm and chorion in developed oocytes is shown in panels A, B, and H. Bars,50 �m.



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these eggs the bacterial density appeared to be much lowerthan that in the eggs laid by untreated females. This resultsuggests that a threshold density of Rickettsia bacteria in eggsis required to trigger development of female embryos. Al-though there have not been specific studies of Rickettsia, therole of bacterial density in the manipulation of reproductionhas been shown in a number of symbiotic associations betweeninsects and cytoplasmic incompatibility-inducing Wolbachia(19, 38, 47, 65). Male killing (36) and induction of partheno-genesis (33, 94) also appear to be positively correlated with theWolbachia titer.

The pattern of distribution of Rickettsia in the reproductivetissues of P. soemius was studied by using FISH analysis. In theovary, Rickettsia bacteria are present in the germarium, in thegerm line cells, in the nurse cells, and in the developing oo-cytes. This pattern has also been observed for Wolbachia inseveral insect hosts (22, 62), as well as for a distantly relatedlineage of bacteria that manipulate reproduction, Cardinium(44, 95, 97). The high concentration of Rickettsia in the ovaryand eggs of P. soemius suggests that the efficiency of transmis-sion of bacteria is high and that there is strong expression ofthe PI phenotype, as confirmed by infection of all thelytokousfemales and the production of only female progeny during 20generations of lab rearing. The mechanism used by Rickettsiasymbionts for transovarial transmission in their hosts is notknown, but active microtubule-mediated transport and passivetransport during cytoplasmic dumping into the oocyte from thenurse cells through the ring canals have been suggested forWolbachia transmission (72). Although our observations do

not elucidate the transport mechanism of the P. soemius sym-biont, they suggest that the nurse cells have a role as a reservoirof bacteria that are delivered into the developing oocytes. Thistype of transport has been observed for symbiotic Rickettsia ofLiposcelis bostrycophila (54). Finally, Rickettsia was found to beconcentrated at the posterior pole of the oocyte during mid tolate and late oogenesis, as well as at the posterior pole of theegg during early embryogenesis. The same pattern has beenobserved for Wolbachia in many insect host species (13, 22, 63,70, 91, 98). As the posterior pole is the site where the germcells form during embryogenesis, concentration of symbionts atthis site has been thought to be a mechanism for increasing theprobability that bacteria are integrated into the germ cells andthen transmitted to host progeny (31, 63, 77).

It has been found that some Rickettsia strains have positiveeffects on host fitness (92), and in particular, these symbiontshave been shown to play an essential role in oogenesis in thebooklouse L. bostrycophila (54) and possibly also in the barkbeetle Coccotrypes dactyliperda (93). In some cases, infectionsby reproductive parasites can have beneficial effects on theirarthropod hosts. For example, Cardinium infection is associ-ated with an increase in fecundity in a mite (84), while Wol-bachia is necessary for oogenesis (22, 67) or for normal egghatching (21, 66) in some insect hosts. The production of asignificantly lower number of viable adult offspring by antibi-otic-treated P. soemius females could indicate that the pres-ence of Rickettsia increases fitness, or alternatively, it could bedue to a toxic effect resulting from treatment with antibiotics.The finding that rifampin treatment is not detrimental to the

FIG. 3. Distribution of Rickettsia (bright red) in the eggs of thelytokous P. soemius. (A and B) Freshly laid egg heavily infected with Rickettsia.(C and D) Early embryogenesis. (C) Embryo surface with bacteria clustered at opposite poles of the mitotic spindles in dividing nuclei. (D) Insideof the same egg, showing high concentrations of bacteria around the pole cells. DAPI-stained nuclei are blue. (A, C, and D) Fluorescence channel.(B) Overlay of fluorescence and bright-field channels. Bars, 50 �m.



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longevity of treated adult females suggests that antibiotic tox-icity probably does not result in a lower number of viable adultoffspring. On the other hand, our results did not show at whatlevel (oogenesis, embryogenesis, larval development) Rickett-sia could induce a fitness benefit. In fact, although there was atrend toward a reduction in fitness in treated females, the datafor fecundity (total number of eggs oviposited), the percentageof unhatched eggs, and the level of mortality of the progenyduring development from larvae to pupae were not signifi-cantly different for treated and untreated females. However,the results presented here, although not sufficient to supportthe hypothesis that there is a clear benefit to the fitness of thehost, seem to indicate that infection does not have a detrimen-tal effect on the fecundity, fertility, and longevity of infected P.soemius females.

Analysis of 16S rRNA and gltA gene sequences revealed that

the PI Rickettsia of P. soemius showed the highest level ofsimilarity with the tick symbiont R. bellii (99% and 97% nu-cleotide similarity for 16S rRNA and gltA gene sequences,respectively). Phylogenetic analyses based on 16S rRNA andgltA gene sequences placed the P. soemius symbiont in a well-supported basal clade that includes R. bellii and the symbiontsof non-blood-feeding arthropods. On the basis of 16S rRNAgene data, the symbiont of P. soemius appeared to be distantlyrelated to the PI Rickettsia of N. formosa (no data for the gltAgene of this strain were available from public databases). Eventhough these two PI bacteria both occur in eulophid wasps,they did not form a monophyletic group. The PI Rickettsia ofN. formosa was related to pathogenic species in the typhus andspotted fever groups, as shown by previous phylogenetic anal-yses (53, 54). Using the most recent and well-resolved phylog-eny of Rickettsia (86), the PI Rickettsia of P. soemius can be

FIG. 4. FISH analysis of thelytokous P. soemius eggs (posterior half). Rickettsiae are bright spots and contrast with the autofluorescencebackground of the ooplasm. (A to C) Comparison of eggs produced by (A) naturally infected, (B) antibiotic-treated, and (C) uninfected biparental(negative control) females. (D to F) Egg (produced by a naturally infected female) simultaneously stained with (D) the Rickettsia-specific probe(red) and (E) the universal bacterial probe EUB338 (green). (F) Merged image of panels D and E showing bacteria stained with both probes(yellow). Bars, 50 �m.



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placed in the ancestral bellii group, while the PI Rickettsia of N.formosa is a member of the transitional group, a lineage at thetop of the tree that shares immediate ancestry with the spottedfever group and whose members have genotypic and pheno-typic characteristics intermediate between those of the typhusand spotted fever groups (25, 26). Recently, three other rick-ettsiae whose biology is unknown, each of which infects adifferent species of eulophid wasps, have been found to clusterin the transitional group (86), leaving open the possibility thatthey and the N. formosa symbiont might form a monophyleticgroup of transitional PI Rickettsia strains.

One hypothesis to account for the absence of monophyly forthe PI rickettsiae of P. soemius and N. formosa is that theability to manipulate the reproduction (induction of partheno-genesis) of closely related hosts (eulophid wasps) may havebeen acquired independently by unrelated rickettsiae duringevolution. An alternative hypothesis is that parthenogenesis-inducing genes were laterally transferred from PI Rickettsiaharbored by a thelytokous wasp species to non-PI Rickettsiaharbored by a bisexual wasp species. Recently, it has becomeevident that lateral gene transfer may play an important role inthe evolution of rickettsial genomes (10, 25). In particular,lateral gene transfer appears to have occurred at a high fre-quency between rickettsiae that harbor plasmids, which wasthe case for the ancestral species R. bellii and the transitionalspecies R. felis (26). An example of recombination betweenrickettsiae in the bellii and transitional groups has recentlybeen discovered in the ladybird beetle Coccidula rufa (86). As

the PI Rickettsia of P. soemius is closely related to R. bellii andthe PI Rickettsia of N. formosa is closely related to R. felis, it ispossible that these symbionts exchange genes, including genesinvolved in PI. However, lateral gene transfer could have oc-curred only if horizontal transmission of Rickettsia betweendifferent parasitoid species took place. Considering the biologyof many eulophid parasitoids, horizontal transmission of sym-bionts might be possible. P. soemius and N. formosa, for exam-ple, can parasitize the same leafminer host species (8), and it islikely that they can develop as facultative hyperparasitoids byfeeding on a different parasitoid species (2, 11, 52, 78). Thenfemale larvae of a bisexual species (e.g., P. soemius) harboringnon-PI Rickettsia could acquire PI Rickettsia during develop-ment (hyperparasitization) on larval stages of an infected the-lytokous species (e.g., N. formosa). Although there is no ex-perimental evidence of horizontal transmission of Rickettsiabetween eulophid species so far, interspecies horizontal trans-mission from host to parasitoid larva has been demonstrated tooccur in nature for the Rickettsia symbiont of the whiteflyBemisia tabaci, a member of the bellii group (17). In this case,however, Rickettsia ingested during parasitoid larval develop-ment persists in the adult wasp and localizes in the reproduc-tive tissues, but it is not vertically transmitted to the parasitoidprogeny. Furthermore, natural interspecies horizontal trans-mission during hyperparasitization has been shown to occur forWolbachia, and the recipient parasitoid species lost the symbi-onts after a few generations (34). If horizontal transmission ofRickettsia occurred between infected eulophid species (e.g.,transmission from N. formosa to P. soemius), resident symbi-onts could have acquired parthenogenesis-inducing genes bylateral transfer from PI Rickettsia. Eventually, even though PIRickettsia did not become established in the recipient host (ahyperparasitoid [e.g., P. soemius]), its PI genes did becomeestablished, giving rise to thelytokous reproduction. Recently,it has been discovered that lateral transfer of novel genes fromother intracellular bacteria, like Cardinium, could have con-tributed to the evolution of Rickettsia genomes (24). Based onthis finding, the possibility that a parthenogenesis-inducinggene of PI Rickettsia was acquired by lateral transfer fromother manipulators of reproduction that inhabited the samehost cannot be excluded. In thelytokous P. soemius, such anevent would have been followed by loss of the donor bacte-rium.

To date, complete sequencing of the Rickettsia genome andcomparative analyses have focused on species that are symbi-onts of blood-feeding arthropods and species that are generallypathogenic to vertebrates (26). Determining the compositionof the genomes of nonpathogenic insect-associated rickettsiae,like PI symbionts, and determining whether these organismscan be horizontally transmitted between different host speciesare important for understanding the evolutionary history ofthese lineages. It is also important to understand the contri-butions of mechanisms of lateral gene transfer, such as plas-mids and bacteriophages, which may transfer the ability tomanipulate reproduction within and among Rickettsia lineages,as well as between Rickettsia and other intracellular bacteria.

ACKNOWLEDGMENTS

We thank Paolo Navone, who first collected and provided the the-lytokous parthenogenetic wasp P. soemius, Salvatore Cozzolino for

FIG. 5. Karyotype of thelytokous P. soemius. (A) Female diploidcomplement (2n � 12). (B) Male haploid complement (n � 6). Bars,5 �m.



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providing sequencing facilities, and Kerry Oliver and Stephan Schmitz-Esser for their useful comments on the manuscript. This work wasperformed at Istituto per la Protezione delle Piante, CNR, Portici(NA), Italy.

This work was supported by CNR grant AG.P01.013.

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Rickettsia Symbionts Cause Parthenogenetic Reproduction in ...negatively affect some aspects of host ﬁtness, causing reduc-tions in body weight, fecundity, and longevity in the pea