the secondary endosymbiotic bacterium of the pea aphid ... · ll05 lusignan, france 29 march 1995...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/00/$04.0010

July 2000, p. 2748–2758 Vol. 66, No. 7

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

The Secondary Endosymbiotic Bacterium of the Pea AphidAcyrthosiphon pisum (Insecta: Homoptera)

TAKEMA FUKATSU,1* NARUO NIKOH,1,2 RENA KAWAI,1 AND RYUICHI KOGA1

National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Tsukuba,305-8566,1 and Bio-Oriented Technology Research Advancement Institution, Omiya, 331-8537,2 Japan

Received 4 February 2000/Accepted 17 April 2000

The secondary intracellular symbiotic bacterium (S-symbiont) of the pea aphid Acyrthosiphon pisum wasinvestigated to determine its prevalence among strains, its phylogenetic position, its localization in the hostinsect, its ultrastructure, and the cytology of the endosymbiotic system. A total of 14 aphid strains wereexamined, and the S-symbiont was detected in 4 Japanese strains by diagnostic PCR. Two types of eubacterial16S ribosomal DNA sequences were identified in disymbiotic strains; one of these types was obtained from theprimary symbiont Buchnera sp., and the other was obtained from the S-symbiont. In situ hybridization andelectron microscopy revealed that the S-symbiont was localized not only in the sheath cells but also in a noveltype of cells, the secondary mycetocytes (S-mycetocytes), which have not been found previously in A. pisum. Thesize and shape of the S-symbiont cells were different when we compared the symbionts in the sheath cells andthe symbionts in the S-mycetocytes, indicating that the S-symbiont is pleomorphic under different endosym-biotic conditions. Light microscopy, electron microscopy, and diagnostic PCR revealed unequivocally that thehemocoel is also a normal location for the S-symbiont. Occasional disordered localization of S-symbionts wasalso observed in adult aphids, suggesting that there has been imperfect host-symbiont coadaptation over theshort history of coevolution of these organisms.

To date, about 4,400 species of aphids (Homoptera, Aphi-didae) have been described (5). Almost all of them have anintracellular symbiotic bacterium, Buchnera sp. (4, 7, 41, 44):the exceptions are some cerataphidine aphids in which thebacterial symbiont has been replaced by an ascomycetousyeastlike endosymbiotic fungus (6, 17, 21, 25, 38). In the aphidbody, Buchnera cells are harbored in the cytoplasm of myce-tocytes (or bacteriocytes), which are hypertrophied cells in theabdomen that are specialized for endosymbiosis. The aphidsand their Buchnera symbionts are considered intimately mutu-alistic; the symbionts cannot live when they are removed fromthe host cells (3), and the aphids become sterile or die when

they are deprived of their symbionts (35, 36, 46). Aphids aresupposed to provide their Buchnera symbionts with a stableniche and nutrients, and it has been demonstrated that Buch-nera cells synthesize essential amino acids and other nutrientsfor the host (4, 10, 11). Since Buchnera cells are passed fromone generation to the next by ovarial transmission and have nofree-living state (7), they are considered a maternally inheritedgenetic element. The evolutionary origin of Buchnera symbi-onts is believed to be quite ancient. Morphological, histologi-cal, biochemical, and molecular phylogenetic lines of evidencehave consistently suggested that the Buchnera symbionts ofvarious distantly related aphid species had a single origin; these

TABLE 1. Strains of A. pisum (Harris) used in this study

Strain Original locality Collection date Original host plant Lab host plant Provider P-symbiont S-symbiont

AIST99 Tsukuba, Ibaraki, Japan 7 April 1999 Vicia sativa Vicia faba T. Fukatsu 1a 2EF99 Suginami-ku, Tokyo, Japan 22 April 1999 Vicia sativa Vicia faba T. Fukatsu 1 2HG99 Bunkyo-ku, Tokyo, Japan 22 April 1999 Vicia sativa Vicia faba T. Fukatsu 1 1IS NDb ND ND Vicia faba H. Ishikawa 1 (AB033776)c 1 (AB033778)MR88 Morioka, Iwate, Japan 15 August 1988 Pisum sativum Vicia faba K. Honda 1 (AB033775) 1 (AB033779)SM ND ND ND Vicia faba Y. Narai 1 (AB033774) 2TKC93 Tokachi, Hokkaido, Japan 23 June 1993 Glycine max Vicia faba K. Honda 1 (AB033772) 1 (AB033777)LL01 Lusignan, France Before 1988 Medicago sativa Vicia faba Y. Rahbe 1 (AB033773) 2LL02 Lusignan, France Before 1988 Medicago sativa Vicia faba Y. Rahbe 1 2LL04 Lusignan, France 29 March 1995 Medicago sativa Vicia faba Y. Rahbe 1 2LL05 Lusignan, France 29 March 1995 Medicago sativa Vicia faba Y. Rahbe 1 2LL06 Cornell University, Ithaca, N.Y. 19 July 1996 Trifolium sp. Vicia faba Y. Rahbe 1 2LF08 Fleurieu-sur-Saone, France 21 July 1996 Trifolium pratense Vicia faba Y. Rahbe 1 2LC09 Cornell University, Ithaca, N.Y. 19 July 1996 Medicago sativa Medicago sativa Y. Rahbe 1 2

a 1, present, as determined in this study; 2, absent, as determined in this study.b ND, no data.c The numbers in parentheses are accession numbers of the 16S rDNA sequences.

* Corresponding author. Mailing address: National Institute of Bio-science and Human-Technology, Agency of Industrial Science andTechnology, Tsukuba, 305-8566, Japan. Phone: 81-298-61-6087. Fax:81-298-61-6080. E-mail: [email protected].

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bacteria descended from a bacterium that was acquired by thecommon ancestor of extant aphids (7, 20, 41, 43, 45). Becauseof their predominance and importance in aphids, Buchneraspp. and the mycetocytes harboring them are often referred toas the primary symbionts (P-symbionts) and the primary my-cetocytes (P-mycetocytes), respectively. Phylogenetically, theBuchnera P-symbiont belongs to the g subdivision of the divi-sion Proteobacteria (51). Buchnera represents one of the mostextensively investigated endosymbiotic microbes of insects.

In addition to Buchnera P-symbionts, a number of aphids areknown to contain a second type of intracellular symbiotic bac-teria (7, 20, 22, 26). These additional bacteria are harboredseparately in a different type of mycetocytes, which constitutea mycetome (or bacteriome) with the P-mycetocytes, and arevertically transmitted to the aphid offspring (7, 18). They arefound in many but not all lineages of aphids, and differ re-markably in morphology and localization in different lineages.It is thought that they have polyphyletic evolutionary origins (7,20, 22, 26). These symbionts are collectively called secondarysymbionts (S-symbionts). In contrast to the studies of P-sym-bionts, only a few modern studies of the S-symbionts of aphidshave been performed (8, 20, 21, 26, 51); some early exceptionswere histological studies in which conventional light micros-copy was used. The only previous molecular phylogenetic char-acterization of S-symbionts was a study performed with the peaaphid Acyrthosiphon pisum, whose P- and S-symbionts belongto distinct lineages in the g subdivision of the Proteobacteria(51).

The previous findings for the S-symbiont of A. pisum are,however, rather fragmentary and somewhat confusing. Grif-fiths and Beck (30) and McLean and Houk (40) first describedthe S-symbiont of A. pisum by using electron microscopy. Inthese studies, the S-symbionts were found in syncytial sheathcells that were located at the periphery of the mycetome andwere closely associated with the P-mycetocytes. In the cyto-plasm of the sheath cells, small rod-shaped bacteria occurredtogether with well-developed endoplasmic reticulum, the Golgiapparatus, and mitochondria. Using light microscopy, Douglasand Dixon (12) observed that rod-shaped S-symbionts werepresent in the sheath cells of young larvae but were onlyloosely associated with the mycetocytes in older insects. Incontrast, Fukatsu and Ishikawa (19, 20) did not detect anyintracellular bacteria other than the P-symbionts in immuno-histochemical studies. Grenier et al. (29) also found no rod-shaped intracellular bacteria but discovered that one strain ofA. pisum contained tubular extracellular microorganisms in its

hemocoel. Although Unterman et al. (51) identified the 16Sribosomal DNA (rDNA) sequence of the S-symbiont, they didnot confirm that the sequence was derived from the rod-shaped bacteria in the sheath cells. Using a specific PCR tech-

FIG. 1. RFLP analysis of bacterial 16S rDNA amplified and cloned from thetotal DNA of A. pisum IS. Lanes 1 through 10 contained cloned 16S rDNAfragments digested by RsaI (left) or HinfI (right) and resolved in a 2% agarosegel. Lanes 1 through 4, 7, 8, and 10, clones containing the P-symbiont sequence;lanes 5, 6, and 9, clones containing the S-symbiont sequence. Lane M containedDNA size markers (2,000, 1,500, 1,000, 700, 500, 400, 300, 200, and 100 bp, fromtop to bottom).

FIG. 2. Diagnostic PCR detection of endosymbionts in A. pisum strains. (A)Universal detection of eubacterial endosymbionts with primers 16SA1 and16SB1. (B) Specific detection of the P-symbiont with primers 16SA1 and ApisP.(C) Specific detection of the S-symbiont with primers 16SA1 and ApisS. (D)Specific detection of the S-symbiont with primers 16SA1 and PASScmp. (E)Specific detection of Rickettsia spp. with primers 16SA1 and Rick16SR. Lane 1,strain IS; lane 2, TKC93; lane 3, MR88; lane 4, HG99; lane 5, EF99; lane 6, SM;lane 7, AIST99; lane 8, LL01; lane 9, LL02; lane 10, LL04; lane 11, LL05; lane12, LC06; lane 13, LF08; lane 14, LC09; lane 15, bruchid beetle Kytorhinussharpianus containing Rickettsia sp. (24); lane 16, no-template control; lane M,DNA size markers (2,000, 1,500, 1,000, 700, 500, 400, 300, 200, and 100 bp, fromtop to bottom). Although the results for only one individual of each strain areshown, the reproducibility of the results was confirmed by examining more than12 individuals of each strain.

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nique, Chen and Purcell (8) detected the S-symbiont sequencein more than 80% of the California clones of A. pisum whichthey examined. When hemolymph of S-symbiont-positive in-sects was microinjected into S-symbiont-negative insects, theS-symbiont sequence was successfully transferred to the recip-ients and, notably, inherited by their offspring. Thus, it wassuggested that the carrier of the S-symbiont sequence occursfreely in the hemocoel, although bacterial cells were not foundin the hemolymph when microscopy was used. Based on theseresults, it is difficult to find consensus concerning the morphol-ogy, localization, and microbial nature of the S-symbiont inA. pisum. Furthermore, the presence of other types of faculta-tive bacterial associates makes the situation more complicated.For instance, Chen et al. (9) identified a rod-shaped, mater-nally inherited bacterium which was a member of the genusRickettsia in the hemocoel of many strains of A. pisum, andHarada and Ishikawa (31), Grenier et al. (29), and Haradaet al. (32, 33) have isolated many types of gut bacteria fromA. pisum.

In the present study, we investigated the prevalence, phylo-genetic position, localization, cytology, and ultrastructure ofthe S-symbiont of A. pisum by using diagnostic PCR, molecu-lar phylogeny, histology, in situ hybridization, and electronmicroscopy. Notably, we discovered a novel type of cells har-boring the S-symbionts in addition to the sheath cells andcharacterized the facultative and pleomorphic nature of theS-symbiont in A. pisum; based on our findings previous reportscould be reinterpreted and synthesized.

MATERIALS AND METHODS

Materials. The strains of A. pisum used in this study are listed in Table 1. Allof these strains are laboratory-maintained strains. Because they originated froma single parthenogenetic female or a few parthenogenetic females and weremaintained through numerous parthenogenetic generations, the lines are clonalisofemale lines. Japanese strains were reared on seedlings of the broad bean,Vicia faba, at 20°C by using a long-day regimen (16 h of light and 8 h of darkness).Newly molted unwinged adults were preserved in acetone until molecular andhistological analyses were conducted (16). Unwinged adults of European andAmerican strains maintained at INRA-INSA, Lyon, France, were shipped to usby Y. Rahbe in acetone.

PCR, cloning, and sequencing of 16S rDNA. The DNA of an individual insectkept in acetone was extracted by using a QIAamp tissue kit (Qiagen). Using thewhole-insect DNA, we amplified almost all of the bacterial 16S rDNA (length,about 1.5 kb) by PCR by using primers 16SA1 (59-AGAGTTTGATCMTGGCTCAG-39) and 16SB1 (59-TACGGYTACCTTGTTACGACTT-39) and the fol-lowing temperature profile: 94°C for 2 min, followed by 30 cycles consisting of94°C for 1 min, 50°C for 1 min, and 70°C for 2 min. Cloning of the PCR products,typing of the clones by restriction fragment length polymorphism (RFLP) anal-ysis, and DNA sequencing were conducted as previously described (23).

Diagnostic PCR. Using specific reverse primers PASScmp (59-GCAATGTCTTATTAACACAT-39) and ApisS (59-GCCATCAGGCAGTTTC-39) for the S-symbiont, primer ApisP (59-TCTTTTGGGTAGATCC-39) for the P-symbiont,and primer Rick16SR (59-CATCCATCAGCGATAAATCTTTC-39) for Rickett-sia spp. in combination with universal forward primer 16SA1, we performed adiagnostic PCR detection analysis of the 16S rDNA of the endosymbiotic bac-teria by using the following temperature profile: 94°C for 2 min, followed by 30cycles consisting of 94°C for 1 min, 55°C for 1 min, and 70°C for 2 min.

Molecular phylogenetic analysis. A multiple alignment of 16S rDNA se-quences was prepared by using the methods of Feng and Doolittle (15) andGotoh (28). The final alignment was inspected and corrected manually. Ambig-uously aligned regions were excluded from the phylogenetic analysis. Nucleotidesites that included an alignment gap(s) were also omitted from the aligned dataset. Neighbor-joining trees (47) were constructed with Kimura’s two-parameterdistance (37) by using the CLUSTAL W program package (50). Maximum-like-

lihood trees (13) were constructed by using the MORPHY 2.3 program package(1). Maximum-parsimony trees were constructed by using the PAUP 4.0b2 pro-gram package (49). A bootstrap test (14) was conducted with 1,000 resamplings.

Histology. Histological preparation, in situ hybridization, and enzymatic probedetection were performed essentially as previously described (26). Insects pre-served in acetone were transferred to alcoholic formalin (ethanol-formalin, 3:1),and their heads and thoraxes were removed with forceps to facilitate infiltrationof reagents. After the insects were kept in the fixative overnight, they weredehydrated and cleared with an ethanol-xylene series and embedded in paraffin.Serial tissue sections (thickness, 5 mm) were cut with a rotary microtome andwere mounted on silane-coated glass slides. The sections were dewaxed with axylene-ethanol series and air dried prior to in situ hybridization.

In situ hybridization. BIO-PASScmp (59-,biotin.GCAATGTCTTATTAACACAT-39) targeting the S-symbiont was complementary to primer PASS-59 (8).BIO-ApisS (59-,biotin.GCCATCAGGCAGTTTC-39) and DIG-ApisP (59-,digoxigenin.TCTTTTGGGTAGATCC-39) were designed to specifically de-tect the S- and P-symbionts, respectively. BIO-EUB338 and DIG-EUB338,which generally recognize eubacterial 16S rRNA (2, 26), were used to visualizeboth the S- and P-symbionts. About 200 ml of hybridization buffer (20 mM Tris-HCl [pH 8.0], 0.9 M NaCl, 0.01% sodium dodecyl sulfate, 30% formamide)containing 50 pmol of probe per ml was applied to a tissue section, and thepreparation was covered with a coverslip and incubated in a humidified chamberat room temperature overnight. To eliminate nonspecific binding of the probe,the tissue section was rinsed in washing buffer (20 mM Tris-HCl [pH 8.0], 0.9 MNaCl, 0.01% sodium dodecyl sulfate, 30% formamide) for 10 min at 37°C. Afterthe tissue section was washed with 13 SSC (13 SSC is 0.15 M NaCl plus 0.015M sodium citrate), bound probe was detected as previously described (26).Biotin-labeled probes were visualized by using a Vectastain Elite ABC kit(Vector). Digoxigenin-labeled probes were detected by using a DIG nucleic aciddetection kit (Boehringer Mannheim). To confirm the specificity of the hybrid-ization procedure, the following control experiments were conducted: no-probecontrol experiment, RNase digestion control experiment, and competitive sup-pression control experiment performed with excess unlabeled probe (26).

Examination of hemolymph. The abdominal dorsa of adult aphids, the sur-faces of which had been sterilized and washed with 70% ethanol and sterilewater, were fixed onto glass slides with Scotch tape carefully so that the insectswere not damaged. The legs were removed with forceps, and the hemolymphcoming from the injury was collected with a glass capillary. About 1 ml ofhemolymph was subjected to either DNA extraction with a QIAamp tissue kit ordirect microscopic observation. For the latter procedure, the hemolymph wasapplied to microscopic immersion oil on a glass slide, covered with a coverslip,and observed with a light microscope.

Electron microscopy. The embryos of unwinged adult aphids were removedunder a dissecting microscope in the presence of 2.5% glutaraldehyde in 0.1 Mphosphate buffer (pH 7.4), prefixed in the fixative at 4°C overnight, postfixed in2% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) at 4°C for 60 min, andsubjected to block staining with 0.5% uranyl acetate for 60 min. The embryoswere dehydrated with an ethanol series and embedded in Epon 812. Ultra-thin sections were cut with an ultramicrotome (Ultracut-N; Leichert-Nissei),mounted on collodion-coated copper mesh, stained with uranyl acetate and leadcitrate, and observed with a transmission electron microscope (model H-7000;Hitachi) at 75 kV.

Nucleotide sequence accession numbers. The 16S rDNA sequences of theP- and S-symbionts of the A. pisum strains described in this paper have beendeposited in the DDBJ, EMBL, and GenBank nucleotide sequence databasesunder accession numbers AB033772 through AB033779 (Table 1).

RESULTS

Analysis of the 16S rDNA of the P- and S-symbionts ofstrain IS. Almost the entire length of eubacterial 16S rDNA ina strain IS adult was amplified by PCR, and the products weresubjected to cloning. When the clones obtained were examinedto determine their RFLP patterns, two major types of cloneswere identified (Fig. 1). Three clones of the first type and fiveclones of the second type were sequenced. The three se-quences of the first type were identical and exhibited a veryhigh level of similarity to the sequence of the P-symbiont ofA. pisum in the database. Only one substitution and two indels

FIG. 3. In situ hybridization of the P- and S-symbionts of A. pisum. (A) Mycetome of a strain IS embryo probed with BIO-EUB338. Tubular S-symbionts in aS-mycetocyte and globular P-symbionts in many P-mycetocytes are present. (B) Mycetome of a strain IS embryo probed with BIO-PASScmp. Tubular S-symbionts ina S-mycetocyte and small S-symbionts in sheath cells are specifically visualized in the mycetome. (C) Mycetome of a strain IS embryo probed with BIO-EUB338. Inthis S-mycetocyte, the S-symbionts are short rods. (D) Mycetome of a strain IS embryo probed with BIO-EUB338. Sheath cells containing small S-symbionts areassociated with the P-mycetocytes. (E) Mycetome of a strain MR88 embryo probed with BIO-EUB338. A S-mycetocyte harboring tubular S-symbionts is locatedbetween P-mycetocytes. (F) Mycetome of a strain TKC93 embryo probed with BIO-EUB338. The same disymbiotic organization is observed. Bar 5 10 mm. The arrowsindicate the locations of sheath cells. Abbreviations: P, P-symbiont: S, S-symbiont.

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were found among 1,473 aligned nucleotide sites. The fivesequences of the second type were identical except for twonucleotide sites. At sites 137 and 232 the sequences of threeclones contained G, while the sequences of the other twoclones contained A. These sequences exhibited very high levelsof similarity to the sequence of the S-symbiont in the database.Out of 1,462 aligned nucleotide sites not including the twopolymorphic sites, only two substitutions were found.

Diagnostic PCR detection of the S-symbiont in variousstrains of A. pisum. To examine the presence of the S-symbiontin various strains of A. pisum, we performed diagnostic PCRexperiments with specific reverse primers in combination withuniversal forward primer 16SA1 (Fig. 2). When specific primerApisP was used, the P-symbiont was detected in all 14 strainsexamined (Fig. 2B). When specific primer ApisS was used, onthe other hand, the S-symbiont was detected only in fourstrains, strains IS, MR88, TKC93, and HG99 (Fig. 2C); thesefindings were supported by the results of PCR performed withspecific primer PASScmp (Fig. 2D). Although Chen et al. (9)identified a Rickettsia species in many strains of A. pisum, PCRperformed with specific primer Rick16SR demonstrated thatall 14 strains were Rickettsia negative (Fig. 2E).

16S rDNA sequences of the P- and S-symbionts of variousstrains. In addition to the 16S rDNA sequences of strain ISsymbionts, we determined the 16S rDNA sequences of theP- and S-symbionts of four strains. Both the P- and S-symbiontsequences were found in disymbiotic strains MR88 and TKC93,whereas only the P-symbiont sequence was found in monosym-biotic strains AIST99 and LL01. The sequences of the P-sym-bionts of different strains were almost identical, as were thesequences of the S-symbionts. Molecular phylogenetic analysisshowed that, as previously reported, the P-symbionts clusteredwith Buchnera strains obtained from other species belonging tothe Aphidinae, whereas the S-symbionts were related to en-teric bacteria, such as Serratia, Enterobacter, Erwinia, and Esch-erichia strains (data not shown).

In situ hybridization of the S-symbiont. In order to deter-mine the morphology and localization of the S-symbiont invivo, tissue sections of the insects were subjected to in situhybridization with oligonucleotide probes that target bacterial16S rRNA (Fig. 3). In disymbiotic strain IS, two types of in-tracellular bacteria were detected with probe BIO-EUB338,which recognizes eubacteria universally (Fig. 3A, C, and D).One type of bacteria was globular and predominant and washarbored in round uninucleate mycetocytes that constituted ahuge mycetome in the abdomen of each embryo. On the basisof these histological and morphological features this bacteriumwas identified as the P-symbiont, Buchnera sp.; this identifica-tion was confirmed by in situ hybridization with probe DIG-ApisP, which recognizes the P-symbiont specifically (data notshown). In addition to the P-symbionts, intracellular bacteriathat had a different shape and localization were also found.When preparations were probed with BIO-EUB338, small por-tions of cytoplasm containing tiny short rods were observed(Fig. 3D). The rod-shaped cells were closely associated withthe P-mycetocytes on the periphery of each embryonic myce-tome. These histological features corresponded to the featuresof sheath cells previously reported to harbor S-symbionts. Inaddition, the S-symbionts were found in special cells that havenot been found previously in A. pisum (Fig. 3A through C).These cells, designated secondary mycetocytes (S-myceto-cytes), were similar in size to the P-mycetocytes. In embryos,the mycetomes were usually composed of tens of P-myceto-cytes and one S-mycetocyte or a few S-mycetocytes. The S-mycetocytes were normally full of tubes or long rods (Fig. 3A),although the bacteria appeared to be shorter rods in some

cases (Fig. 3C). The bacteria in the S-mycetocytes were largerand longer than the bacteria in the sheath cells. However,when tissue sections were probed with BIO-PASScmp, whichspecifically recognizes the S-symbiont, the bacteria were visu-alized in both the S-mycetocytes and the sheath cells (Fig. 3B).Hybridization with probe BIO-ApisS gave the same results(data not shown). Therefore, we concluded that in A. pisumthe S-symbionts are harbored in two types of cells, theS-mycetocytes and the sheath cells. In other disymbioticstrains, including strains MR88 and TKC93, S-mycetocytescontaining tubular S-symbionts were also present (Fig. 3E andF). The S-symbiont-specific probes certainly detected the bac-teria in the S-mycetocytes and sheath cells, as shown in Fig.3B (data not shown). In the monosymbiotic strains, in con-trast, the S-symbionts were not detected by in situ hybridiza-tion (data not shown).

Disordered localization of the S-symbiont. In adults of strainIS, we occasionally observed abnormal localization of the S-symbionts (Fig. 4). In these insects, S-symbionts were detectednot only in the S-mycetocytes but also in other cells and tissues.In embryos, normally the P- and S-symbionts are specificallyharbored in different types of cells. However, Fig. 4A showsan embryonic mycetome in which some of the P-mycetocyteswere also infected by S-symbionts. Although some mycetocyteslooked normal, some were infected with a mixture of cells, andothers were predominantly occupied by S-symbionts. In addi-tion, other embryonic tissues around the mycetome were alsosporadically infected. In maternal tissues, normally huge P-mycetocytes are the only cells that contain P-symbionts, and nospecial cells harboring S-symbionts are found. However, Fig.4B shows a maternal mycetocyte filled with S-symbionts inplace of P-symbionts; around this mycetocyte fat body cells andhemocoel also contained S-symbionts. We have infrequentlyencountered insects exhibiting such symptoms; none or a fewof the tens of individuals sampled and processed at the sametime for histological studies have shown them. So far, however,such insects have been found in at least three samples thatwere independently collected.

Examination of hemolymph. We examined the hemolymphof Japanese strains of A. pisum to determine whether symbi-onts were present by using diagnostic PCR and light micros-copy. As shown in Fig. 2, in the disymbiotic strains both P- andS-symbionts were detected by PCR while in the monosymbioticstrains only P-symbionts were detected (data not shown); thesefindings indicated that at least a small number of symbiontswere present in the hemolymph of A. pisum. When the hemo-lymph was directly observed by light microscopy, we foundmany tubular or rod-shaped particles which were morpholog-ically reminiscent of the S-symbionts observed by in situ hy-bridization only in disymbiotic strains (data not shown).

Electron microscopy of the S-symbiont. The fine structureand localization of the S-symbiont in strain IS embryos wereinvestigated by transmission electron microscopy (Fig. 5).Sheath cells harboring many rod-shaped S-symbionts werefound throughout the mycetome and were closely associatedwith the P-mycetocytes (Fig. 5A). In addition to the S-symbi-onts, the cytoplasm of the sheath cells contained mitochondria,endoplasmic reticulum, and ribosomes (Fig. 5C), as describedpreviously. Notably, in the monosymbiotic strains the sheathcells had similar ultrastructural features, although they did notcontain S-symbionts (Fig. 5D). The large S-mycetocytes har-bored a number of S-symbionts, which were generally largerthan the S-symbionts in the sheath cells, although the overallshape of the bacteria was difficult to determine with ultrathinsections. The cytoplasm of the S-mycetocytes was highly vacu-olated and in this respect differed from that of the sheath cells



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(Fig. 5B). We found that even in embryos a number of S-symbionts were present in extracellular locations, althoughthey were still closely associated with the sheath cells andS-mycetocytes (Fig. 5B, E, and F).

DISCUSSION

Since the first electron microscopic observations of endo-symbiosis in pea aphids (30, 40), it has been repeatedly claimedand widely accepted that the S-symbionts in A. pisum areharbored by the sheath cells (4, 10, 11, 34, 42). In the presentstudy, however, we demonstrated that in Japanese strains ofA. pisum, the S-symbionts are also found in large special cells,the S-mycetocytes in the mycetomes (Fig. 3 and 5). Although ithas been found for many aphids that secondary intracellularbacteria are harbored in the cytoplasm of large mycetocytesthat are cytologically distinct from the P-mycetocytes (7, 18, 20,22, 26), this is the first description of this type of mycetocyte inA. pisum. The morphology and ultrastructure of the S-myce-tocytes were clearly distinguishable from the morphology andultrastructure of the sheath cells. Thus, the S-symbionts areharbored in two different types of host cells specialized forendosymbiosis.

Our results indicated that in A. pisum there are three typesof cells involved in endosymbiosis: the P-mycetocytes, S-myce-tocytes, and sheath cells. The developmental and evolutionaryorigins of these cells are very interesting but poorly under-stood. The size, shape, and location of the S-mycetocytes werereminiscent of the size, shape, and location of the P-myceto-cytes (Fig. 3), and we observed that the S-symbionts occasion-ally invaded the P-mycetocytes (Fig. 4). These facts suggestthat the S-mycetocytes are developmentally homologous to theP-mycetocytes, although the ultrastructure of the S-myceto-cytes was not similar to the ultrastructure of the P-mycetocytes(Fig. 5). Cytologically, it appeared that the sheath cells werequite different from the P- and S-mycetocytes. The mecha-nisms which enable S-symbionts to target different types ofcells are also intriguing.

Are S-mycetocytes found in disymbiotic strains in general, orare they restricted to Japanese strains? To answer this ques-

tion, a more extensive histological study to determine the pres-ence of S-symbionts in A. pisum strains is needed. However, itis quite likely that S-mycetocytes are present at least in anAmerican disymbiotic strain. When McLean and Houk (40)observed a smear preparation of mycetocytes of a CaliforniaA. pisum strain by light microscopy, they found a large cellcontaining aggregations of bacilliform bacteria (40). Judgingfrom its size and cytological traits, the cell was probably a S-mycetocyte rather than a sheath cell, although McClean andHouk did not examine the cell by electron microscopy. Themycetome of an A. pisum embryo is normally composed of tensof P-mycetocytes and sheath cells and only one S-mycetocyteor a few S-mycetocytes. It is conceivable that in previous elec-tron microscopic studies the researchers failed to obtain im-ages of S-mycetocytes because of the scarcity of these cells inthe material. In fact, we had to examine a large number ofultrathin sections to observe an S-mycetocyte, while sheathcells were found in most ultrathin sections containing myce-tomes.

Intracellular bacteria in the sheath cells and intracellularbacteria in the S-mycetocytes differed in size and morphology(Fig. 3 and 5). In the sheath cells the bacteria were smallrods, whereas in the S-mycetocytes the bacteria were largerand longer. However, in situ hybridization experiments inwhich specific oligonucleotide probes were used unequivocallyshowed that the bacteria in the two types of cells are geneti-cally identical (Fig. 3B). The differences in morphology wereattributed to the pleomorphism of S-symbiont cells inducedunder different environmental conditions, a trait commonlyfound in microorganisms (48).

In addition to the sheath cells and the S-mycetocytes, thehemocoel was identified as a third location of S-symbiont cells.A considerable population of S-symbionts was present in thehemocoel. Electron microscopic examination clearly showedthat even in embryos extracellular S-symbionts were closelyassociated with the sheath cells and the S-mycetocytes (Fig. 5).These results suggest that the hemocoel is a normal location ofS-symbionts. Although the presence of S-symbionts in the he-molymph has been suggested previously by the results of PCR

FIG. 4. Disordered localization of the S-symbiont found in several unwinged adults of A. pisum IS. (A) Embryonic P-mycetocytes infected by S-symbionts.Abnormally, the P- and S-symbionts coexist in several cells. (B) Maternal P-mycetocyte filled with S-symbionts. The P-symbionts are almost completely replaced byS-symbionts. The symbionts were visualized by in situ hybridization with probe BIO-EUB338. Bar 5 10 mm.



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FIG. 5. Electron microscopy of the endosymbiosis in A. pisum embryos. (A through C, E, and F) Strain IS containing both P- and S-symbionts. (D) Strain SM lackingthe S-symbiont. (A) Strain IS sheath cell located between large P-mycetocytes. Small rod-shaped S-symbionts are located intracellularly. (B) Strain IS S-mycetocyteharboring many tubular S-symbionts. The S-symbionts are apparently larger than the S-symbionts in sheath cells. The cytoplasm of the S-mycetocyte is highlyvacuolated. (C) Magnified image of a strain IS sheath cell containing S-symbionts, mitochondria, and endoplasmic reticulum. (D) Magnified image of a sheath cell ofa strain SM aphid without the S-symbiont. The sheath cell contains developed mitochondria and endoplasmic reticulum but no S-symbionts. (E) ExtracellularS-symbionts associated with strain IS sheath cells on the periphery of a mycetome. (F) Magnified image of S-symbionts on the periphery of a strain IS sheath cell. Bars 52 mm. Abbreviations: ER, endoplasmic reticulum; Mt, mitochondrion; N, nucleus; P-Myc, primary mycetocyte; ShC, sheath cell; S-Myc, secondary mycetocyte.



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FIG. 5—Continued.



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and injection experiments (8), this is the first study in which themorphology and localization of extracellular S-symbionts havebeen determined microscopically. Insects have humoral andcellular immune systems that effectively attack and kill bacte-rial and fungal intruders in the body fluid (27, 39). Thus, theextracellular location of S-symbionts suggests that these cellsare able to avoid the host immune system in some way. Inaddition, S-symbionts are able to target the sheath cells and theS-mycetocytes specifically. To investigate endosymbiotic mech-anisms at a molecular level, the S-symbiont of A. pisum mightprovide a good experimental system because manipulation andartificial transmission of the S-symbiont are possible (8).

16S rDNA sequence analyses strongly suggested that theS-symbionts of Japanese A. pisum strains identified in this studyand the S-symbionts of American strains described in previousworks (8, 51) are the same bacterium. The S-symbiont of A. pi-sum was closely related to enteric bacteria, such as Serratia,Enterobacter, Erwinia, and Escherichia strains, which may suggestthat its evolutionary origin was a gut bacterium. In fact, variousgut bacteria have been isolated from A. pisum; one such predom-inant bacterium was identified as an Erwinia species (31–33).

In strain IS adults examined histologically, we occasionallyobserved surprising localization of the S-symbiont (Fig. 4). Inthese adults, the S-symbionts, which infected various tissuesand cells, appeared to be out of control. Unfortunately, wedid not establish whether these insects exhibited pathologicalsymptoms when they were alive. Since we examined only asmall number of cases, it is not clear whether such disorderedbehavior is exceptional and observed only sometimes or is in-duced by particular physiological and environmental conditions.Our observations may reflect imperfect coadaptation in the en-dosymbiosis involving the S-symbiont and A. pisum, presumablydue to the short history of coevolution of these organisms.

With the disymbiotic strains, PCR experiments showed thatall of the individuals contained the S-symbiont after severalyears of maintenance in the laboratory (Fig. 2). As previouslyreported (8), the offspring of disymbiotic mothers all inheritedthe S-symbiont through parthenogenesis. Therefore, the S-symbiont is passed to the next generation by vertical transmis-sion with high fidelity, at least under laboratory conditions.Although the transmission process has not been studied indetail, it must occur at an early embryonic stage in the body ofthe mother, because S-symbionts were found in the S-myceto-cytes and sheath cells of young embryos (Fig. 3 and 5). Con-sidering that injection of hemolymph can establish a heritableinfection of the S-symbiont (8; T. Fukatsu, unpublished data),it is conceivable that the S-symbiont is transmitted to embryosvia the hemolymph.

On the other hand, both Chen and Purcell (8) and we havedemonstrated that the S-symbiont does not infect all individ-uals in populations of A. pisum, suggesting that vertical trans-mission of the S-symbiont may not be perfect under naturalconditions. If the transmission rate is not 100%, the rate ofS-symbiont infection must decline, and eventually the S-sym-biont must disappear from populations as host generationsproceed. However, S-symbiont infection is certainly main-tained in natural populations, which suggests that some pro-cesses may counter imperfect vertical transmission. One pos-sibility is that the S-symbiont makes the host slightly more fit,while another possibility is horizontal transmission of the S-symbiont.

To date, there has been no evidence of horizontal transmis-sion of the S-symbiont from disymbiotic A. pisum to monosym-biotic A. pisum under laboratory mixed-rearing conditions (8;Fukatsu, unpublished data). Interestingly, however, it has beenreported that a 16S rDNA sequence identical to that of the

S-symbiont was found in an aphid belonging to different genus,Macrosiphum rosae (8), suggesting that interspecific horizontaltransmission of the S-symbiont may have occurred under nat-ural conditions. The S-symbiont of A. pisum was stably main-tained and vertically transmitted when it was injected into adifferent aphid species, Acyrthosiphon kondoi (8), suggestingthat the S-symbiont is not strictly host specific and thus may betransmitted horizontally. Although speculative, several hori-zontal transmission routes are conceivable. Parasitoid waspsmight be vectors for the S-symbiont and perform microinjec-tion in the wild. Considering its phylogenetic affinity to gutbacteria, the S-symbiont might sometimes be excreted withhoneydew. If oral ingestion can occasionally establish an infec-tion, honeydew, squashed aphids, and phloem sap of plantsheavily populated by aphids could be sources of infection.

At this stage, the biological effects of the S-symbiont on thehost aphid are not known; however, it is known that the S-symbiont is not essential for the host because there are mono-symbiotic strains and populations. Nutritional, physiological,and population dynamics studies on A. pisum strains with andwithout the S-symbiont should be performed in order to de-termine whether the S-symbiont is almost neutral, parasitic, orslightly advantageous for the host under various environmentalconditions. Considering the spatial proximity and integrity ofthe P- and S-symbionts in the endosymbiotic system, it is con-ceivable that the S-symbiont may interact with and modify theestablished mutualism between the aphid and Buchnera sp. insome way.

The S-symbiont of A. pisum is very interesting because it canbe considered an intermediate between facultative endosym-biotic bacteria, such as Wolbachia spp., and highly specializedmutualistic intracellular symbionts, such as Buchnera spp. (41).Like Wolbachia spp., the S-symbiont is not essential for thehost and sometimes is horizontally transmitted across lineagesand species. Like Buchnera spp., the S-symbiont is harbored byspecialized host cells for endosymbiosis. In future studies onthe S-symbiont, we may be able to gain insight into the evolu-tionary transition from facultative guest microbe to obligatelymutualistic symbiont.

We identified two intracellular symbiotic bacteria, the P- andS-symbionts, in the A. pisum strains examined in this study.Frequent occurrence of Rickettsia sp. has been reported forAmerican strains (9), although this organism was not detectedin this study. As far as we know, these three microbes arethe major endosymbiotic bacteria of the pea aphid that havebeen described so far. However, it is not certain that this is thecomplete picture of the endosymbiotic microbiota. Furtherinvestigations may reveal other types of interesting endosym-biotic associates in A. pisum.

ACKNOWLEDGMENTS

We thank K. Honda, H. Ishikawa, Y. Narai, and Y. Rahbe for aphidsamples, A. Sugimura, S. Kumagai, and K. Sato for technical andsecretarial assistance, and T. Wilkinson and Y. Rahbe for reading themanuscript.

This research was supported by the Industrial Science and Technol-ogy Frontier Program “Technological Development of Biological Re-sources in Bioconsortia” of the Ministry of International Trade andIndustry of Japan and by the Program for Promotion of Basic Re-search Activities for Innovation Biosciences (ProBRAIN) of the Bio-Oriented Technology Research Advancement Institution.

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