Coexistence of Wolbachia with Buchnera aphidicola and a Secondary Symbiont in the Aphid Cinara cedri

JOURNAL OF BACTERIOLOGY, Oct. 2004, p. 6626 6633 Vol. 186, No. 19 0021-9193/04/$08.00 0 DOI: 10.1128/JB.186.19.6626 6633.2004 Copyright 2004, American Society for Microbiology. All Rights Reserved. Coexistence of Wolbachia with Buchnera aphidicola and a Secondary Symbiont in the Aphid Cinara cedri Laura Gómez-Valero, 1 Mario Soriano-Navarro, 1 Vicente Pérez-Brocal, 1 Abdelaziz Heddi, 2 Andrés Moya, 1 José Manuel García-Verdugo, 1 and Amparo Latorre 1 * Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de València, València, Spain, 1 and Laboratoire de Biologie Fonctionnelle Insectes et Interactions, UMR INRA/INSA de Lyon, Villeurbanne Cedex, France 2 Received 19 May 2004/Accepted 7 July 2004 Intracellular symbiosis is very common in the insect world. For the aphid Cinara cedri, we have identified by electron microscopy three symbiotic bacteria that can be characterized by their different sizes, morphologies, and electrodensities. PCR amplification and sequencing of the 16S ribosomal DNA (rdna) genes showed that, in addition to harboring Buchnera aphidicola, the primary endosymbiont of aphids, C. cedri harbors a secondary symbiont (S symbiont) that was previously found to be associated with aphids (PASS, or R type) and an -proteobacterium that belongs to the Wolbachia genus. Using in situ hybridization with specific bacterial probes designed for symbiont 16S rdna sequences, we have shown that Wolbachia was represented by only a few minute bacteria surrounding the S symbionts. Moreover, the observed B. aphidicola and the S symbionts had similar sizes and were housed in separate specific bacterial cells, the bacteriocytes. Interestingly, in contrast to the case for all aphids examined thus far, the S symbionts were shown to occupy a similarly sized or even larger bacteriocyte space than B. aphidicola. These findings, along with the facts that C. cedri harbors the B. aphidicola strain with the smallest bacterial genome and that the S symbionts infect all Cinara spp. analyzed so far, suggest the possibility of bacterial replacement in these species. * Corresponding author. Mailing address: Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de València, Apartat 2085, 46071 València, Spain. Phone: (34) 963543649. Fax: (34) 963543670. E-mail: amparo.latorre@uv.es. Insects are prone to intracellular symbiotic associations (endosymbiosis) with microorganisms. Such associations are particularly widespread among members of the orders Homoptera (aphids, whiteflies, mealybugs, psyllids, and cicadas), Blattaria (cockroaches), and Coleoptera (beetles) (4, 6, 24). In most cases, insects display specific bacterium-bearing host cells (the bacteriocytes), and endosymbionts fail to grow and divide outside of the bacteriocytes. Therefore, the field of intracellular symbiosis was essentially unraveled through approaches such as PCR, electron microscopy, in situ hybridization, DNA sequencing, and molecular phylogeny. For instance, genome sequencing of five insect primary endosymbionts confirmed the nutritional role of and host-symbiont syntrophy proposed for their respective symbioses (1, 19, 40, 45, 51). In aphids (Hemiptera: Aphididae), the primary endosymbiont Buchnera aphidicola, one of the -Proteobacteria, is maternally transmitted by entering the embryos of each generation. The insect life cycle is complex, involving alternations of sexual and asexual reproduction. During their reproductive phase, aphids contain within their body cavity a bilobed structure called the bacteriome that consists of 60 to 90 uninucleate, polyploid bacteriocytes. Inside the bacteriocytes, B. aphidicola is enclosed within host-derived vesicles (4). In nature, all but a few aphid populations harbor B. aphidicola (4, 32). Exceptions include certain aphid species of the tribe Ceratiphidini that possess extracellular yeast-like symbionts belonging to the subphylum Ascomycotina in the abdominal hemocoel instead of B. aphidicola. The latter association was interpreted as being a symbiont replacement, with an intracellular bacterium being replaced with an extracellular fungus (14). The possibility of natural endosymbiont replacement in aphids has also been postulated by other authors (9, 28). In addition to harboring B. aphidicola, some aphid populations harbor other intracellular bacteria, which are commonly referred to as secondary symbionts (S symbionts). Typically, S symbionts are present in various lineages of aphids. However, in most aphids examined thus far, S symbionts are only represented in small numbers and occupy a limited bacteriocyte area compared to B. aphidicola (4, 17). Moreover, the S symbionts are not always confined in specific bacteriocytes and can be found in gut tissues, glands, body fluids, and cells that are surrounding, or even invading, the primary bacteriocytes. At present, five different S symbionts have been found. They include three different lineages within the -proteobacteria, provisionally called PASS or R type (7, 39, 50), PABS or T type (12, 39), and PAUS or U type (39), one lineage within the -proteobacteria, referred to as the PAR (pea aphid rickettsia) symbiont (8, 27, 48), and a Spiroplasma symbiont (16). S symbionts seem to be the result of multiple independent infections (37), and although they are usually maternally inherited (4, 6), their transmission may also occur horizontally from one host to another. Wolbachia is an obligatory intracellular -proteobacterium that infects diverse groups of insects and most species of filarial nematodes (3, 46, 52, 54). In arthropods, it causes reproductive alterations to the host, such as cytoplasmic incompatibility (CI), parthenogenesis, genetic male feminization, male killing, and virulence in Drosophila melanogaster (5, 26, 34, 36, 52, 54). Although there have been established cases of horizontal 6626

VOL. 186, 2004 THREE INTRACELLULAR SYMBIONTS COEXIST IN C. CEDRI 6627 transfer, Wolbachia organisms are inherited mainly through the maternal lineage of their hosts by vertical transmission. A phylogenetic analysis of worldwide Wolbachia strains by the use of fast-evolving genes such as ftsz and wsp split invertebrate Wolbachia strains into two major clades, designated A and B, and further divided them into subgroups (55, 57). Genomic size determination has shown that Wolbachia genomes are much smaller than the genomes of free-living bacteria (ranging from 0.95 to 1.5 Mb) (43) and closer to the genome sizes of other intracellular bacteria (1, 19, 40, 45, 51). Contrary to the genomes of these endosymbionts, the recently sequenced genome of Wolbachia pipientis wmel revealed the existence of very large amounts of repetitive DNA and mobile genetic elements, suggesting that they may have played a key role in shaping the evolution of Wolbachia (56). Cinara cedri belongs to the subfamily Lachninae of aphids and lives in colonies disposed on branches on the gymnosperm Cedrus atlantica and Cedrus deodora. Gil et al. (18) estimated the genome size of the B. aphidicola strain associated with C. cedri to be 450 kb by pulsed-field gel electrophoresis, making it the smallest bacterial genome reported so far. In the present paper, we describe and characterize two other bacteria found in the aphid C. cedri in addition to B. aphidicola: an S symbiont (PASS, or R type) that was previously described for aphids and Wolbachia. Furthermore, based on the relative abundance of the primary and secondary endosymbionts, we provide additional evidence for endosymbiont competition and replacement during evolution. MATERIALS AND METHODS Aphid material and DNA isolation. C. cedri aphids were collected from cedar trees (Cedrus atlantica) inllíria (Valencia, Spain) during May and October 2003. Several grams of C. cedri was used for the present analysis. Bacteriocytes containing both B. aphidicola and S symbionts were isolated as previously described (18). The total aphid DNA was isolated as described previously (23). Genomic bacterial DNAs were isolated according to a cetyltrimethylammonium bromide- NaCl protocol (2). Electron microscopy. Adults, embryos, and eggs from aphids were dissected under a microscope in 0.9% NaCl, prefixed in 0.1 M phosphate buffer (PB) (ph 7.4) at 4 C for 24 h, and washed several times in 0.1 M PB. They were then postfixed in 2% osmium tetroxide in 0.1 M PB for 90 min in darkness, dehydrated in ethanol, and embedded in araldite (Durcupan; Fluka). Semithin sections (1.5 m) were cut with a diamond knife and stained with toluidine blue. Ultrathin (0.05 m) sections were cut with a diamond knife, stained with lead citrate, and examined under a transmission electron microscope (JEOL-JEM1010). 16S rdna amplification and sequencing of B. aphidicola, S symbiont, and Wolbachia from C. cedri. To amplify the 16S ribosomal DNAs (rdnas) of both B. aphidicola and the S symbiont from C. cedri, we used the universal -proteobacterial primers 16Sup1 (5 -AGAGTTTGATCATGGCTCAGATTG-3 ) and 16Slo1 (5 -TACCTTGTTACGACTTCACCCCAG-3 ). The PCR conditions were 94 C for 2 min, followed by 30 cycles of 94 C for 15 s, 60 C for 30 s, and 72 C for 1 min. To assess the presence of P (primary) and S symbionts, we digested the PCR products with the SalI restriction enzyme, which specifically recognizes B. aphidicola, and three diagnostic enzymes, SacI, XbaI, and ClaI, that discriminate among R-, T-, and U-type S symbionts, respectively (37, 39). To further confirm the restriction analysis results, we cloned the PCR products into a pgem T-vector (Promega) and sequenced them by using the universal primers T7 and SP6. To detect the presence of -proteobacteria, we designed a pair of specific primers from database alignments of 16S rdnas by using an alignment of sequences from the following organisms: the PAR symbiont, Rickettsia japonica, and Wolbachia from Sitophilus oryzae, Drosophila mauritiana, and Drosophila sechellia (Table 1). The designed primers were as follows: 16SWup (5 -GCCTA ACACATGCAAGTCGAA-3 ) and 16SWlo (5 -AGCTTCGAGTGAAACCAA TTCCC-3 ), corresponding to positions 24 to 45 and 1379 to 1357, respectively, of the 16S rdna partial sequence of the Wolbachia strain associated with D. sechellia (accession number U17059). PCR mixtures consisted of 1.5 U of Taq DNA polymerase (Promega), a 200 M concentration of each deoxynucleoside triphosphate, a 300 nm concentration of each primer, and 10 ng of DNA template in a final volume of 50 l. An additional positive control of Wolbachia from S. oryzae (20) was used. The PCR amplification conditions were 94 C for 2 min, followed by 35 cycles of 94 C for 20 s, 63 C for 30 s, and 72 C for 90 s. The 1.3-kb PCR product obtained was purified (Qiaquick PCR purification kit; Qiagen) and directly sequenced by using the PCR primers. Simultaneous sequencing of the positive control discarded the possibility of a contamination by Wolbachia from S. oryzae. Searches in databases and a subsequent BlastN search confirmed the homology with Wolbachia within the -proteobacterial 16S rdnas. To resolve the phylogenetic position of the Wolbachia strain associated with C. cedri in the different subgroups into which Wolbachia has been divided, we amplified and sequenced a fragment of the wsp gene by using the primers wsp 81F (5 -TGGT CCAATAAGTGATGAAGAAAC-3 ) and wsp 691R (5 -AAAAATTAAACGC TACTCCA-3 ) according to the method described by Zhou et al. (57). Phylogenetic analysis. Sequences were aligned with the Clustal X algorithm (47). In the case of the wsp gene, we used a previously published alignment (57) (accession number DS32273) and added the sequence from this study (see Table 1 for a list of species and their individual accession numbers). Maximum likelihood (ML), maximum parsimony (MP), and distance (neighbor-joining) algorithms were used to perform phylogenetic analyses. The ML and MP algorithms were performed with PAUP 4.0, using the evolution model given by Modeltest (35), and the distance algorithm was performed with Mega software (22), using the Kimura-2 parameter model and pairwise deletion. Bootstrap analyses were done with 1,000 replications, except for the analysis of wsp with the ML algorithm, which was done with 300 replications. In situ hybridization. All experimental procedures for in situ hybridization were conducted under RNase-free conditions. For digoxigenin-labeled probes, we followed a protocol similar to a previously described one (17). For rhodamine-labeled probes, we followed another previously described procedure (20). Four 5 -end-labeled digoxigenin probes were used. EUB338 was used previously (17), and the remainder were designed for the present work to specifically detect B. aphidicola (BuCc), the S symbiont (SCc), and Wolbachia (WCc) in C. cedri. Two 5 -end-labeled rhodamine probes (W1 and W2) were also used (20). They are available upon request. RESULTS Electron microscopy. Semithin serial sections of 1.5 m from C. cedri were used to localize the different types of bacteriocytes within the aphid, which are known to be mainly located along the digestive tract (Fig. 1a). Two types of bacteriocytes were easily identifiable by their different tonalities after toluidine blue staining (Fig. 1b). By electron microscopy and morphological criteria, we identified three kinds of symbionts, namely, B. aphidicola, the S symbiont (both also visible under an optical microscope), and Wolbachia (Fig. 1c). The first two organisms were found exclusively within their corresponding bacteriocytes, whereas Wolbachia was present in defined, small cells. The B. aphidicola bacteriocytes possessed a dense cytoplasm with numerous mitochondria and abundant cisternae of the rough endoplasmic reticulum (RER) (Fig. 1d). Dictiosomes and vacuoles were also visible. The nucleus had large dimensions, was very irregular, and contained chromatin, which formed lumps. The plasmatic membrane of the bacteriocytes could be observed surrounding each bacterium. The bacteria appeared to be granulose, and some ribosome-like structures could be identified. The S-symbiont bacteriocytes possessed an electrolucid cytoplasm with some short cisternae of the RER, in contrast with the abundance found in the primary bacteriocytes (Fig. 1e). The mitochondria were less numerous than those found in B. aphidicola bacteriocytes. The S symbionts were sphere-like and were surrounded by the plasmatic membrane of the bacteriocyte. Finally, the bacteriocytes containing

6628 GÓMEZ-VALERO ET AL. J. BACTERIOL. TABLE 1. Hosts and corresponding associated symbionts, codes, partial gene sequences, and accession numbers of species used in the present work Host Symbiont Code c Partial gene sequence Accession no. -Proteobacteria Acyrtosiphon pisum B. aphidicola BAp 16S rdna M27039 Cinara cedri B. aphidicola BCc 16S rdna AY620431 a Cinara tujafilina S symbiont SCtj (R) 16S rdna AY136146 Brachycaudus cardui S symbiont SBca (S) 16S rdna AY13643 Uroleucon pieloui S symbiont SUpi (U) 16S rdna AY136163 Cinara cedri S symbiont SCce (R) 16S rdna AY620432 a Escherichia coli b E. coli 16S rdna U00096 -Proteobacteria Alluropodus melanoleucos Rickettsia japonica R.jap 16S rdna L36213 Acyrtosiphon pisum PAR R par 16S rdna U42084 Sytophilus oryzae Wolbachia wory 16S rdna AF035160 Drosophila mauritiana Wolbachia wmau 16S rdna U17060 Drosophila sechellia Wolbachia wsec 16S rdna U17059 Cinara cedri Wolbachia wced 16S rdna AY620430 a Drosophila melanogaster (yw 67C23 ) Wolbachia wmel1 wsp AF020072 Drosophila melanogaster (Aubiry 253) Wolbachia wmel2 wsp AF020063 Drosophila melanogaster (Cairns) Wolbachia wmelcs1 wsp AF020064 Drosophila melanogaster (Canton-S) Wolbachia wmelcs2 wsp AF020065 Drosophila melanogaster (Harwich) Wolbachia wmelh wsp AF020066 Drosophila simulans (Coff Harbour) Wolbachia wcof wsp AF020067 Aedes albopictus Wolbachia wa1ba wsp AF020059 Glossina morsitans Wolbachia wmors wsp AF020079 Nasonia vitripennis Wolbachia wvita wsp AF020081 Glossina centralis Wolbachia wcen wsp AF020078 Drosophila simulans (Riverside) Wolbachia wri1 wsp AF020070 Drosophila auraria Wolbachia wri2 wsp AF020062 Muscidifurax uniraptor Wolbachia wuni wsp AF020071 Drosophila simulans (Hawaii) Wolbachia wha1 wsp AF020068 Drosophila sechellia Wolbachia wha2 wsp AF020073 Ephestia cautella (A) Wolbachia wcaua wsp AF020075 Phlebotomus papatasi Wolbachia wpap wsp AF020082 Glossina austeni Wolbachia waus wsp AF020077 Tribolium confusum Wolbachia wcon wsp AF020083 Laodelphax striatellus Wolbachia wstri wsp AF020080 Trichogramma deion Wolbachia wdei wsp AF020084 Culex pipiens Wolbachia wpip1 wsp AF020061 Culex quinquefasciatus Wolbachia wpip2 wsp AF020060 Drosophila simulans (Mauritiana) Wolbachia wma wsp AF020069 Drosophila simulans (Noumea) Wolbachia wno wsp AF020074 Aedes albopictus Wolbachia walbb wsp AF020059 Ephesia cautella Wolbachia wcaub wsp AF020076 Tagosodes orizicolus Wolbachia wori wsp AF020085 Cinara cedri Wolbachia wcce wsp AY620433 a a Sequences was obtained in the present work. b Escherichia coli is a free-living bacterium. c For the code of Wolbachia species, we followed the work of Zhou et al. (57). Wolbachia were small and were usually located surrounding the S-symbiont bacteriocytes (Fig. 1f). They were scarce and very difficult to localize by light microscopy. The cytoplasm was thin and generally dense and did not contain sizeable accumulations of RER. Occasionally, the bacteria seemed to invade neighboring S symbionts (not shown). PCR and sequencing of endosymbiont 16S rdna. Amplification of endosymbiont 16S rdnas with universal -proteobacterial primers yielded a band of approximately 1,500 bp. Digestion of the PCR product with diagnostic enzymes that discriminate among B. aphidicola and R-, T-, and U-type S symbionts (39) revealed the presence of two different 16S rdna fragments that corresponded to two symbionts, specifically B. aphidicola and an R-type (or PASS) S symbiont (data not shown). Cloning, sequencing, and phylogenetic analyses of the two 16S rdna bands further confirmed these results (see below). Amplification with the specific primers designed to amplify -proteobacteria yielded a band of 1,259 bp that was sequenced and compared with selected -proteobacterial 16S rdna sequences from databases by the use of BlastN. Phylogenetic positions of C. cedri endosymbionts. Phylogenetic trees obtained after applying three different phylogenetic procedures yielded similar topologies (Fig. 2). Figure 2a shows the topology of a tree obtained with 16S rdnas from B. aphidicola associated with Acyrthosiphon pisum (BAp) and from three S symbionts (R, T, and U types) associated with different aphids (37) and with the two 16S rdna -proteobac-

VOL. 186, 2004 THREE INTRACELLULAR SYMBIONTS COEXIST IN C. CEDRI 6629 FIG. 1. (a) Longitudinal section of a C. cedri adult. Arrows indicate different bacteriocytes. Bar, 500 m. (b) Semithin serial sections of 1.5 m from C. cedri. P, primary symbiont (B. aphidicola) bacteriocytes; S, S symbiont (PASS or R-type); n, nuclei. Bar, 10 m. (c to f) Electron microscopic images. (c) The three types of bacteria associated with C. cedri can be seen. 1, B. aphidicola; 2, S symbiont; 3, Wolbachia. Arrows show the division of an S symbiont. Bar, 2 m. (d) Cytoplasm of B. aphidicola. m, mitochondria; R, RER. Bar, 0.5 m. (e) Cytoplasm of S-symbiont bacteriocytes. Bar, 0.5 m. (f) Cytoplasmic expansion of a bacteriocyte containing specifically Wolbachia (arrows) between S-symbiont bacteriocytes. Bar, 2 m. terial sequences obtained from C. cedri. The sequence corresponding to B. aphidicola (BCc) clustered with the BAp sequence, while the second one clustered with the sequence corresponding to the S symbiont associated with Cinara tujafilina, which was previously assigned to the R type (or PASS) (37). This sequence was called SCce (R) (Table 1). The first phylogenetic analysis tree with the -proteobacterial sequences (Fig. 2b) clearly showed that the third bacterium found in C. cedri corresponds to the Wolbachia strain (wcce in Table 1). Due to the paucity of 16S rdna nucleotide substitution in Wolbachia, the tree was useful to differentiate between Rickettsia and Wolbachia but not between Wolbachia strains. To accomplish this task, we performed a phylogenetic analysis as described by Zhou et al. (57), including the new sequence. As seen in Fig. 2c, the Wolbachia strain from C. cedri (wcce) clustered with strains wcon and wstri, which are associated with Tribolium confusum and Laodelphax striallus, respectively (Table 1). In situ endosymbiont localization. To physically localize C. cedri-associated bacteria, we performed in situ hybridization with specific oligonucleotide probes for 16S rdnas. Since it was stated previously that insect tissues generally emit strong autofluorescence (17), we developed nonfluorescent in situ hybridization methods using digoxigenin-labeled probes. Using the eubacterial universal EUB338 16S rdna probe, we succeeded in identifying and localizing the bacteriocytes of C. cedri in both adults and embryos (Fig. 3a). Two other digoxigenin-specific probes, BuCc and SCc, identified two types of bacteriocytes, those filled with B. aphidicola (Fig. 3b) and those filled with S symbionts (Fig. 3c). This observation confirmed the results obtained by electron microscopy and demonstrated the bacterial specificity of the probes. Furthermore, an intriguing bacterial space distribution was detected in the three figures, which exhibit three serial sections. Whereas B. aphidicola bacteriocytes were found outside of the bacteriome (Fig. 3b), S-symbiont bacteriocytes were concentrated in the interior

6630 GÓMEZ-VALERO ET AL. J. BACTERIOL. FIG. 2. Phylogenetic trees obtained with the neighbor-joining algorithm by using Kimura-2p and pairwise deletion. (a) Tree based on sequences of 16S rdnas from selected -proteobacteria. BCc and SCceR correspond to B. aphidicola and the R-type S symbiont (PASS) associated with the aphid C. cedri, respectively. (b) Tree based on sequences of 16S rdnas from selected -proteobacteria. (c) Tree based on wsp sequences. The topologies obtained by using the MP and ML methods were similar. See Table 1 for the codes of all species analyzed. Bootstrap values in panels a and b correspond those obtained by the distance, ML, and MP algorithms, respectively. Values below 50% were not reported. In panels b and c, wced corresponds to Wolbachia associated with C. cedri. In panel c, bootstrap values correspond to those obtained by the distance algorithm. ML and MP values were reported only for the branches corresponding to the Wolbachia B group. For the A group, only bootstrap values obtained by the distance method were reported. For the names of species and subgroups, we have followed a previously described system (57). (Fig. 3c). Interestingly, Fig. 3b and c and other examined slides (data not shown) show that the S symbiont seems to cover at least 60% of the total bacteriome surface. Hybridization with the WCc probe designed for Wolbachia detection did not give clear results, probably due to the small amount and/or the minute size of these bacteria (Fig. 1c and f). To increase the signal, after checking that C. cedri does not emit any autofluorescence in red wavelengths, we performed fluorescence in situ hybridization by using a combination of two previously described rhodamine-labeled Wolbachia probes (20). As a result, several red spots were seen, with some of them disseminated throughout the whole body and some others concentrated around the S-symbiont bacteriocytes (Fig. 3d), as shown in Fig. 1f. These results also suggest that Wolbachia is always intracellular and absent from the hemolymph. DISCUSSION The presence of three distinct endosymbiotic bacteria has already been identified in insects such as the tsetse fly (11). For aphids, most studies referring to multisymbiosis report double infections with B. aphidicola and S symbionts (49). The association between aphids and B. aphidicola is very ancient, and the congruence of phylogenetic trees of aphids and B. aphidicola indicates a unique infection event about 84 to 164 million years ago, followed by the coevolution of both partners (30, 53). During the accommodation to symbiotic life, B. aphidicola has suffered a drastic genome reduction as well as many important molecular and biochemical changes (4, 10, 29, 31). In contrast to B. aphidicola, S symbionts seem to be the result of multiple independent infections, and in addition to their maternal transmission, they can also be horizontally transmitted from one host to another (37, 39), therefore not sharing a long evolutionary history with their hosts. Most studies on the presence of S-symbiont occurrences in aphids have been conducted on members of the tribe Macrosiphini from the subfamily Aphidinae. For the subfamily Lachninae, S symbionts have been visualized by histological techniques in Stomaphis yanonis, Nippolachnus piri, and Cinara pini (15, 17) and

VOL. 186, 2004 THREE INTRACELLULAR SYMBIONTS COEXIST IN C. CEDRI 6631 FIG. 3. In situ hybridization of tissue sections from C. cedri adults by using digoxigenin- and rhodamine-labeled probes (see Materials and Methods). (a) EUB338 digoxigenin probe; (b) B. aphidicola probe (BuCc); (c) S-symbiont probe (SCc); (d) fluorescence in situ hybridization of Wolbachia (red). amplified by PCR from three new species belonging to the Cinara genus (C. cupressi, C. maritimae, and C. tujafilina). In these three species, the S symbiont was characterized as PASS (or R type) (37), which is the same bacterium found associated with C. cedri in the present work. This new finding provides support for the previously stated hypothesis of a stable association between R-type S symbionts and the Cinara host clade (37). The different techniques used to visualize endosymbionts in aphids have shown that P and S symbionts exhibit different morphologies, and in all cases the sizes of B. aphidicola bacteriocytes overwhelmed those of S-symbiont bacteriocytes. In the present study, however, we found that the P and S symbionts associated with C. cedri have similar sizes (Fig. 1 and 3b and c), and the S symbiont seems to occupy a similar amount of or more space (up to 60%) than B. aphidicola. Therefore, since B. aphidicola associated with C. cedri possesses the smallest bacterial genome reported so far (450 kb), the possibility of a symbiont replacement in an advanced stage can be suggested. A comparison of the three B. aphidicola sequenced genomes (40, 45, 51) revealed a high degree of gene order conservation since the last common symbiotic ancestor (for a different perspective, see reference 38). However, wide variations in genome size have been found, since B. aphidicola strains from several aphid subfamilies showed differences of up to 200 kb (18), indicating that genome degradation is still an ongoing process that is probably related to variations in the host lifestyle. Therefore, the evolution of the B. aphidicola genome appears to be degenerative rather than adaptive (29). This degenerative process raises the question of whether these endosymbionts are approaching a minimal genome stage for symbiotic life or are being driven toward extinction. In the latter case, S-symbiont competition with such a reduced and degraded B. aphidicola genome may compensate for the loss of the ability of B. aphidicola to support host fitness and eventually may replace the P symbiont. Many aphid species lack S symbionts, which suggests that they may not be essential for host survival (4, 39). Nevertheless, different positive effects, such as rescue from heat damage (9, 27), host plant specialization and reproduction (41, 48), and resistance to parasitoid attack and other natural enemies (13, 33), have been described, indicating some putative roles for the S symbionts. The most direct evidence of S symbionts overtaking the role of B. aphidicola has been obtained by Koga et al. (21) through experiments investigating the biological effects of PASS on A. pisum strains. They have proven that infections with PASS enabled the survival and reproduction of B. aphidicola-free aphids. Interestingly, they showed that PASS invaded the bacteriocyte space in parallel with B. aphidicola elimination, establishing a novel endosymbiotic system. If this is so, then the distribution of P- and S-symbiont bacteriocytes in C. cedri could be interpreted as the P-symbiont being replaced with the S symbiont. In insects, the best evidence of an endosymbiont replacement was recently found in Sitophilus spp. of the family Dryophthoridae (24). The currently ongoing sequencing of B. aphidicola from C. cedri in our laboratory will help us to understand whether this reduced genome is still able to perform the necessary functions for symbiotic life or if some of these functions have been overtaken by the abundant S symbionts. In spite of the high prevalence of Wolbachia throughout arthropod species, no aphid species has been described to be associated with these bacteria before. Rickettsia (PAR) is the only -proteobacterium that was previously found in aphids, as it was found in the hemolymph of A. pisum with different levels of infection (7, 8, 27, 49). Here we report the presence of Wolbachia in the aphid C. cedri. If it is confirmed that C. cedri populations are naturally infected with Wolbachia, the phenotypic effects on the host should be further investigated. Nevertheless, according to its phylogenetic position (Fig. 2c), we hypothesize that CI is the most probable phenotypic effect (57). The other two host effects (i.e., parthenogenesis and male killing) cannot be excluded. Indeed, since noninfected C. cedri possesses sexual and asexual lineages, the presence of Wolbachia could increase the prevalence of asexual lineages, as previously described for the Hymenopteran group (42). Genes encoding homologs of the type IV secretion system, used by many pathogenic bacteria to secrete macromolecules, are also present and expressed in Wolbachia (25). It has been postulated that Wolbachia secretes certain molecules that may participate in the expression of CI through the type IV secretion system. It would be interesting to investigate whether an active vir operon containing these genes is also present in strain wcce and whether the secretion of macromolecules affects the two other symbionts that are already established in C. cedri. Variations in genome size in Wolbachia strains reach about 550 kb (43), which is even higher than the up to 200-kb variations found among B. aphidicola strains (18). This suggests that, similar to the case for B. aphidicola, adaptations to different host lifestyles are taking place in Wolbachia. The most

6632 GÓMEZ-VALERO ET AL. J. BACTERIOL. reduced genomes correspond to Wolbachia strains infecting nematodes (0.95 to 1.1 Mb), which are considered to be more like classical mutualists, and the largest genome was found in the parasitic A group of Wolbachia (43). However, contrary to the gene order conservation observed in B. aphidicola strains, frequent rearrangement events during Wolbachia evolution have been observed (56), which could have important consequences on the virulence of the strains. A comparison of the physical and genetic maps of the virulent Wolbachia strain wmelpop with those of the closely related benign strain wmel have shown that the two genomes are largely conserved, with the exception of a single inversion in the chromosome (44). The rearrangements in Wolbachia likely correspond to the introduction and massive expansion of repeat element families that are absent from other obligate intracellular species (56). Since Wolbachia has not been previously found in aphids, its presence in C. cedri may be the product of an infection by horizontal transfer from some other insect. Parasitoids have been proposed as a possible vector for transmitting Wolbachia (52). Thus, we hypothesized that the parasitoids of the subfamily Lachninae, the Aphidinae of the genus Pauesia, may be good candidates for horizontal transfer events. So far, no Wolbachia strain has been described as being present in any species of this genus. ACKNOWLEDGMENTS Financial support was provided by projects BFM2003-00305 from Ministerio de Ciencia y Tecnología and Grupos 03/204 from Generalitat Valenciana. We thank J. M. Michelena and P. González for aphid species identification and Rosario Gil and Juan José Canales for critical reading of the manuscript. We also acknowledge the Servicio de Secuenciación de ácidos nucléicos y proteínas at SCSIE (Universitat de València) for sequencing support. REFERENCES 1. Akman, L., A. Yamashita, H. 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