microbiology ecology vol1 issue 4

8/12/2019 microbiology ecology vol1 issue 4

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Characterization of a Facultative Endosymbiotic Bacterium of theea Aphid Acyrthosiphon pisum

Tsuchida1,2, R. Koga1, X.Y. Meng1, T. Matsumoto3 and T. Fukatsu1

Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566, JapanDepartment of Biology, University of York, York YO10 5YW, UKDepartment of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo 153-8902, Japan

ceived: 1 October 2003 / Accepted: 9 February 2004 / Online publication: 24 January 2005

bstract

he pea aphid U-type symbiont (PAUS) was investigatedcharacterize its microbiological properties. Fluores-

nce in situ hybridization (FISH) and electron micros-py revealed that PAUS was a rod-shaped bacteriumund in three different locations in the body of the peahid Acyrthosiphon pisum: sheath cells, secondary my-tocytes, and hemolymph. Artificial transfer experi-ents revealed that PAUS could establish stable infectiond vertical transmission when introduced into unin-

cted pea aphids. When 28 aphid species collected in

pan were subjected to a diagnostic PCR assay, fourecies of the subfamily Aphidinae (Aphis citricola,Aphisrii,Macrosiphum avenae, andUroleucon giganteus) and

species of the subfamily Pemphiginae (Colopha kansu-i) were identified to be PAUS-positive. The sporadiccidences of PAUS infection without reflecting the aphid

hylogeny can be best explained by occasional horizontalansfers of the symbiont across aphid lineages.

troduction

most all aphids harbor a maternally inherited c-pro-obacterium called Buchnera in the cytoplasm of my-tocytes (or bacteriocytes), large cells specialized fordosymbiosis [3, 9]. Since Buchnera provides the hostth essential amino acids and other nutrients [10],

uchnera-free aphids suffer retarded growth, sterility, orath [20, 26]. Over the long history of the endosymbi-ic association [25], the genome of Buchnera has lostany genes needed for independent life, resulting inastic genome reduction and the inability to survive

utside host cells [30]. Because of its prevalence and

importance for the host,Buchnerais often referred to as aprimary symbiont of aphids.

In addition to the primary symbiont Buchnera, anumber of aphids harbor different types of verticallytransmitted endosymbiotic bacteria [3, 1214, 16, 17].These bacteria, most of which are not essential for thehost aphids, have been collectively referred to as sec-ondary symbionts (or accessory symbionts). To date, fivefacultative secondary symbionts have been identifiedfrom the pea aphidAcyrthosiphon pisum. A rod- or tube-shaped bacterium, which is closely related to Serratiaspp., was identified in secondary mycetocytes, sheath

cells, and hemolymph and referred to as PASS (after peaaphid secondary symbiont) [5, 6, 2123, 31], R-typesymbiont [28, 29], or S-symbiont [16, 34]. A rod-shapedbacterium, which is closely related to a symbiont ofwhitefly, Bemisia tabaci, was identified in mycetocytesand hemolymph and referred to as PABS (after pea aphidBemisia-type symbiont) [7, 8, 22, 31] or T-type symbiont[28, 29]. A rod-shaped bacterium, which belongs to thegenusRickettsia, was detected in hemolymph and referredto as Rickettsia symbiont [32] or PAR (after pea aphidRickettsia) [4, 6, 22, 24, 31]. A symbiotic bacterium of the

genusSpiroplasma, detected in hemolymph, was referredto as Spiroplasma symbiont [18]. Finally, a symbioticbacterium belonging to the c-Proteobacteria was referredto as PAUS (after pea aphid U-type symbiont) [22, 31,32] or U-type symbiont [28, 29].

Our knowledge of phenotypic effects on host aphidsassociated with infection by secondary symbionts islimited. There have been several reports only on sec-ondary symbionts of A. pisum. PASS, Rickettsia, andSpiroplasma have been reported to confer slightly nega-tive effects on the host fitness under certain environ-

mental conditions [6, 18]. PASS was shown to benefit thehost at high temperatures [24]. It was reported that PASSand PABS confer resistance of the host against parasitoidrrespondence to: T. Fukatsu; E-mail: [email protected]

6 DOI: 10.1007/s00248-004-0216-2 d Volume 49, 126133 (2005) d Springer Science+Business Media, Inc. 2005


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wasps [26]. Infection with PASS was shown to restore thesurvival and reproduction of Buchnera-free aphids thatshould otherwise be sterile [21].

Notably, several studies have suggested that PAUSinfection is also relevant to the biology of A. pisum.Frequencies of PAUS infection exhibited a geographicalcline across the mainland Japan [32]. Statistical analyses

suggested that the distribution of PAUS could be corre-lated with environmental factors such as host plant spe-cies, temperature, and precipitation of the regions [32].In California, PAUS infection was associated with hostplant specialization of the aphid [22]. In France, PAUSinfection was restricted to a particular host race of theaphid [31]. Recently, it was experimentally shown thatPAUS infection broadens host plant range of the aphid[33]. In addition to A. pisum, diverse aphid species havebeen shown to harbor endosymbionts allied to PAUS [28,29]. Hence, more attention should be paid to the bio-

logical aspects of this facultative symbiont. Thus far,however, PAUS has been identified solely by PCR andsequencing of its 16S rDNA. Microbiological propertiesof PAUS are totally unknown.

In this study, morphology, tissue and cellular local-ization,fine structure, inheritance and transmission, andhost aphids of PAUS were investigated in detail by usinglight and electron microscopy, in situ hybridization,diagnostic PCR, DNA sequencing, and an artificialtransfer technique.

Materials and Methods

Materials. Insect materials used in this study are listedin Table 1. Laboratory strains of A. pisum were main-tained on seedlings of the broad bean,Vicia faba, at 20Cin a long day regime (L:D = 16 h: 8 h). Field-collectedaphid samples were preserved in acetone for DNAextraction and molecular analysis [15].

Histology. Histological procedure was as de-scribed [14]. Ovarioles dissected from adult aphids werefixed in Carnoys solution (ethanol:chloroform:aceticacid = 6:3:1) overnight.

The fixed embryos were dehydrated and clearedthrough an ethanolxylene series, and embedded inparaffin. Serial tissue sections 5 lm thick were cut with arotary microtome and mounted on silane-coated glassslides. The sections were dewaxed through a xyleneethanol series, air-dried, and subjected to in situhybridization.

Examination of Hemolymph. Hemolymph collec-tion was performed as described [16]. The abdominaldorsa of adult aphids, the surfaces of which had beensterilized and washed with 70% ethanol and sterile water,

were carefullyfixed onto glass slides using adhesive tape.The legs of the insects were removed with forceps, andthe hemolymph coming from the injury was collectedwith a glass capillary. Bacterial preparation was madeessentially as described [1]. Hemolymph collected from50 aphids was fixed with 4% paraformaldehyde in PBSfor 2 h at 4C and was centrifuged to remove thefixative.

The pelletted cells were resuspended in distilled water,and 20-lL drops of the suspension were spotted ontosilane-coated glass slides and air-dried. The slides weredehydrated through ethanol series, air-dried, and sub-

jected to in situ hybridization.

Fluorescence in Situ Hybridization. The followingprobes were used for fluorescence in situ hybridization(FISH) targeted to 16S rRNA: Buchnera-specific probeApisP2a-FITC [5-FITC-CCTCTTTTGGGTAGATCC-3]and PAUS-specific probe TAM-U16 [5-TAM-

GTAGCAAGCTACTCCCCGAT-3 ]. Around 150 lL ofhybridization buffer (20 mM Tris-HCl, pH 8.0; 0.9 MNaCl; 0.01% sodium dodecyl sulfate; 30% formamide)containing 10 pmol/mL each of the probes and 200 ng/mL of 4,6-diamino-2-phenylindole (DAPI) was appliedto a slide. The slides were incubated in a humidifiedchamber at 46C overnight, rinsed in 1 SSC (0.15 MNaCl, 15 mM sodium citrate) for 10 min, mounted withan antifading solution (PBS containing 90% glycerol and1% 1,4-diazobicyclo[2,2,2]octane), and observed underan epifluorescent microscope. Specificity of the hybrid-

ization was confirmed based on the following controlexperiments: no probe control experiment, RNasedigestion control experiment, and competitive suppres-sion control experiment performed with excess unlabeledprobe [14].

Electron Microscopy. Adult aphids were dissectedin 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH7.4). The dissected embryos were prefixed in thefixativeat 4C overnight, postfixed in 2% osmium tetroxide inthe phosphate buffer at 4C for 3 h, and subjected to

block staining with 1% uranyl acetate for 1 h. The em-bryos were dehydrated through an ethanol series andembedded in Spurr resin. Ultrathin sections were madewith an ultramicrotome (Ultracut-N; Leichert-Nissei),mounted on collodion-coated copper mesh, stained withuranyl acetate and lead citrate, and observed with atransmission electron microscope (model H-7000; Hit-achi).

Artificial Transfer of PAUS. Hemolymph transferwas performed by a microinjection technique as de-

scribed [18]. Hemolymph from adults of the PAUS-in-fected strain TUt was injected into 3-day-old (second orthird instar) nymphs of the PAUS-free strain AIST. As acontrol treatment, hemolymph from the strain AIST was

T. TSUCHIDA ET AL.: CHARACTERIZATION OF AN APHID FACULTATIVESYMBIONT 127


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ble 1.Aphids examined in this study

bfamilya species (strain) Locality Host plant (collector b) Nc PAUSd

) Laboratory strainsidinae

Acyrthosiphon pisum(AIST)e Tsukuba, Ibaraki Vicia saliva f

Acyrthosiphon pisum(TUt)e Tsuchiura, Ibaraki Trifolium repens(TT) f +

) Field-collected sampleshidinaeAcyrthosiphon kondoi Aizu, Fukushima Trifolium repens(K.H) 1

Morioka, Iwate Trifolium repens(KH) 1Shonai, Yamagata Trifolium repens(KH) 1

Acyrthosiphon magnoliae Han-nou, Saitama Sambucus sieboldiana 10Tsuchiura, Ibaraki Sambucus sieboldiana 10

Aphis citricola Suginami, Tokyo Choenomeles speciosa 10 +Heiwajima, Tokyo Hydrangea macrophylla 10Suginami, Tokyo Citrus natsudaidai 10

Aphis craccivora Tsukuba, Ibaraki Vicia sativa 10Ohta, Tokyo Robinia pseudoacacia(IM) 10Okayama, Okayama Vicia sativa 10Sakai, Osaka Vicia sativa 10

Toyoda, Aichi Vicia sativa 10Numazu, Shizuoka Vicia sativa 10Tsuchiura, Ibaraki Trifolium repens 10Yachiyo, Ibaraki Vicia sativa 10

Aphis gossypii Tsukuba, Ibaraki Hibiscus syriacus 10Aphis nerii Taito, Tokyo Nerium oleander 10 +Aphis rumicis Tsuchiura, Ibaraki Rumex japonicus 10Brevicoryne brassicae Kisarazu, Chiba Brassica rapa 10Cavariella araliae Han-nou, Saitama Aralia elata 10Hyalopterus pruni Tsukuba, Ibaraki Phragmites australis 10

xoptera odinae Suginami, Tokyo Rhusjavanica 20Macrosiphoniella kuwayamai Funehiki, Fukushima Artemisia princeps 10Macrosiphoniella yomogifoliae Han-nou, Saitama Artemisia princeps 10

Koriyama, Fukushima Artemisia princeps 10Macrosiphum avenae Ichihara, Chiba Akebia quinata 10 +Macrosiphum euphorbiae Tsukuba, Ibaraki Tulipa sp. 10Megoura crassicauda Fukiage, Kagoshima Vicia sativa(HS) 20

Sakai, Osaka Vicia sativa 12Tsukuba, Ibaraki Vicia sativa 10Chikugo, Fukuoka Vicia sativa(AK) 5Wakayama, Wakayama Vicia sativa 3Toyonaka, Osaka Vicia sativa 10Hamamatsu, Shizuoka Vicia sativa 10Numazu, Shizuoka Vicia sativa 10Yachiyo, Ibaraki Vicia sativa 10

Megoura lespedezae Tsukuba, Ibaraki Prunus yedoensis 10

itobion ibarae Tsukuba, Ibaraki Rosa sp. 10Tsuchiura, Ibaraki Rosa sp. 10Tsuchiura, Ibaraki Rosa sp. 10Onomichi, Hiroshima Rosa sp. 10

Tuberocephalus sakurae Tsukuba, Ibaraki Prunus yedoensis 10Tuberocephalus sasakii Tsukuba, Ibaraki Prunus yedoensis 20

Tsukuba, Ibaraki Prunus yedoensis 10Uroleucon giganteus Han-nou, Saitama Circum sp. 20 +Uroleucon nigrotuberculatum Ohta, Tokyo Solidago altissima 10

MPHIGINAEColopha kansugei Fukuoka, Fukuoka Carex morrowii(SA) 3 +

Kashiwa, Chiba Carex morrowii(SA) 3 +Paracolopha morrisoni Han-nou, Saitama Zelkova serrata 20

(continues)

8 T. TSUCHIDA ET AL.: CHARACTERIZATION OF AN APHIDFACULTATIVESYMBIONT


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injected into nymphs of the strain AIST to confirm thatinjection itself does not damage or affect the recipient

insects. Progeny of the injected insects were examined forstable and heritable PAUS infection by specific PCRdetection.

DNA Extraction, Diagnostic PCR, Cloning, and

Sequencing. Insect samples were subjected to DNAextraction using a conventional proteinase K digestionand phenolchloroform extraction method. The purifiedDNA was dissolved in an adequate volume of TE buffer(10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA). Specific PCRdetection of PAUS was conducted using AmpliTaq Gold

DNA polymerase (Roche, Basel) and its supplementedbuffer system as described [32]. A 16S rDNA segment ofPAUS was amplified using the primers U99F [5-AT-CGGGGAGTAGCTTGCTAC-3] [29] and 16SB4 [5-CTAGAGATCGTCGCCTAGGTA-3] [32] under a tem-perature profile of 95C for 10 min followed by 35 cyclesof 95C for 30 s 55C for 30 s 72 C for 1 min. Toconfirm successful DNA extraction, 16S rDNA ofBuch-nera and/or mitochondrial cytochrome oxidase I (COI)gene of host aphid were subjected to PCR. Buchnera 16SrDNA was amplified using the primers Buch l6S1F [5-

GAGCTTGCTCTCTTTGTCGGCAA-3] and Buch16S1R [5-CTTCTGCGGGTAACGTCACGAA-3] [32]under the temperature profile described above. Mito-chondrial COI gene was detected using the primersLCO1490 [5- GGTCAACAAATCATAAAGATATTGG-3] and HCO2198 [5-TAAACTTCAGGGTGACCAAAAAATCA-3] [11] under essentially the same temperatureprofile but annealing at 50C. Amplified segments of 16SrDNA putatively from PAUS were cloned and sequencedas described [16].

Nucleotide Sequence Accession Numbers. The16S rDNA sequences of PAUS-allied bacteria from theaphidsA. pisum, Aphis citricola, Aphis nerii, Macrosiphumavenae, Uroleucon giganteus, and Colopha kansugei were

deposited in the DDBJ/EMBL/GenBank nucleotide se-quence database under accession numbers AB112788

AB112793.

Results

FISH Detection of PAUS in Tissue Sections. Tissuesections of naturally PAUS-infected aphids were sub-

jected to FISH targeting 16S rRNA of PAUS. In the in-fected strain TUt, PAUS was detected in the sheath cellslocated on the periphery of embryonic mycetomes(Fig. 1A). In addition, PAUS was found in larger cells,secondary mycetocytes (Fig. 1B), although only a fraction

of the embryos (around 10% of more than 200 embryosfrom 20 adults examined) exhibited this type of locali-zation. In the PAUS-free aphid strain AIST, no signals ofPAUS were detected by FISH (data not shown).

FISH Detection of PAUS in Hemolymph. Hemol-ymph from the PAUS-infected aphids, which was care-fully collected without damaging the insects, was smearedon glass slides. When the hemolymph preparations werestained with DAPI, rod-shaped bacteria (25 lmlength) were predominantly observed (Fig. 2A), whereas

few coccal bacteria such as Buchnera were found. Therod-shaped bacteria hybridized to the PAUS-specificprobe (Fig. 2B), but not to the Buchnera-specific probe(data not shown). In the hemolymph from the PAUS-freeaphids, no PAUS cells were detected by FISH (data notshown).

Electron Microscopy of PAUS. Fine structure andlocalization of PAUS were investigated by transmissionelectron microscopy. Sheath cells were found on theperiphery of the mycetome in close association with the

primary mycetocytes harboring Buchnera. Many rod-shaped bacterial cells were present in the sheath cells(Fig. 3A). These bacteria clearly exhibited cell wallstructure, which was not seen on Buchneracells (Fig. 3B).

Table 1.continued

Subfamilya species (strain) Locality Host plant (collector b) Nc PAUSd

LACHNINAELachnus roboris Tsukuba, Ibaraki Quercus sp. 10

Ohta, Tokyo Castanopsis sp. 10Lachnus tropicalis Tsukuba, Ibaraki Castenea crenata 10

Tsukuba, Ibaraki Castenea crenata 10

Tsukuba, Ibaraki Castenea crenata 10Nippolachnus piri Tsukuba, Ibaraki Rhaphiolepis umbellata(NK) 10Stomaphissp. Akiruno, Tokyo Acer pictum 20

aHigher taxa according to Blackman and Eastop [2].bIM: I. Mori; HS: H. Shibao; AK: A. Kume; KH: K. Honda; SA: S. Akimoto; NK: N. Kohmoto; no indication: T. Fukatsu.cNumber of pooled individuals subjected to DNA extraction and subsequent diagnostic PCR.d+: PAUS-positive using diagnostic PCR.eFree of any of the other secondary symbionts, PASS, PABS, Rickettsia, andSpiroplasma.fNot applicable.



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number of bacterial rods were also found extracellu-rly (Fig. 3C). These locations of the bacterial rods werensistent with the location of PAUS demonstrated bySH (see Figs. 1 and 2). The bacterial rods were notserved in the primary mycetocytes, and not found in

e PAUS-free aphid strain (data not shown).

Artificial Transfer of PAUS by Hemolymph Injec-

n. Hemolymph from adult insects of the PAUS-fected strain TUt was injected into 3-day-old nymphs

the PAUS-free strain AIST, whereby three injected

nes were generated. The injected nymphs became adult5 days after the injection and began to deposit nymphsrthenogenetically. The newborn nymphs were exam-ed for PAUS infection by diagnostic PCR. Nymphs

born 58 days after the injection were free of PAUS.Infected nymphs appeared 910 days after the injection.

At later stages, all nymphs inherited PAUS. When 10first-instar nymphs per generation were examined overtwo successive generations, PAUS was inherited to all theoffspring tested. To date, one of the lines has beenmaintained for 6 months through 24 generations and stillexhibits 100% infection with PAUS (Table 2).

Host Range of PAUS. In an attempt to estimatethe incidence of PAUS infection across aphid populationsand species in Japan, field-collected aphid samples, rep-resenting 579 individuals, 57 populations, 28 species, and

three subfamilies, were subjected to diagnostic PCR usingPAUS-specific primers for 16S rDNA (Table 1). SpecificPCR product was detected from five species: Aphis citri-cola, Aphis nerii, Macrosiphum avenae, Uroleucon gigan-

Figure 2. Fluorescence in situ hybridization of hemolymph

preparations from A. pisum adults of the PAUS-infected strain

TUt. (A) DAPI staining. (B) FISH with specific probe for 16S

rRNA of PAUS. Bars: 10 lm.

gure 1. Fluorescence in situ hybridization of PAUS (red) and

chnera(green) on tissue sections ofA. pisum embryos of the

AUS-infected strain TUt. (A) PAUS in sheath cells, which were in

se association with primary mycetocytes harboring Buchnera.

) PAUS in a secondary mycetocyte (arrow) and sheath cells.

rs: 50 lm.



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teus, and Colopha kansugei. The nucleotide sequences ofthe 16S rDNA segment, 171 bps in length, were similar toeach other [93.0% (159/171) 100% (171/171)] and tothe sequences of PAUS from A. pisum and other aphidspreviously reported [28, 29].

Discussion

To date, five facultative secondary symbionts, PASS,PABS, PAUS, Rickettsia, and Spiroplasma, have beenidentified from A. pisum in addition to the essentialsymbiont Buchnera [47, 16, 18, 22, 28, 29, 31, 32, 34].Among them, microbiological properties of PAUS havebeen poorly characterized, in contrast to the other foursecondary symbionts whose microbiological propertieshave been examined to some extent. This study providesthe first histological and experimental investigations of

PAUS.FISH analyses revealed that PAUS was found in three

different locations in the same host body: sheath cells,secondary mycetocytes, and hemolymph. Sheath cells andhemolymph of infected aphid embryos always containedPAUS, whereas PAUS-harboring secondary mycetocytewas found only in a fraction of the embryos (Figs. 1 and2). The PAUS-harboring sheath cells were in close asso-ciation with the Buchnera-harboring primary myceto-cytes on the periphery of each embryonic mycetome(Fig. 3 AC). The spatial proximity suggests that there

might be various biological interactions between theessential and facultative endosymbiotic bacteria, as hasbeen demonstrated for the PASSBuchnera association[21].

In the course of endosymbiotic evolution, the gen-ome ofBuchnera has lost many genes needed for inde-pendent life, which resulted in drastic genome reductionand inability to survive outside the host cells [30]. Forexample, cell wall ofBuchnera is reduced [19], and thegenome of Buchnera lacks some genes for the biosyn-thetic pathway of cell wall [30]. Electron microscopic

observation identified PAUS as bacterial rods with cellwalls (Fig. 3B). The presence of a cell wall may reflect arecent origin of the endosymbiotic association betweenPAUS and the aphid.

The in vivo localization of PAUS, sheath cells, sec-ondary mycetocytes, and hemolymph, was quite similarto those of other secondary symbionts, PASS and PABS[16, 21, 29]. The similar localization patterns of PAUS,PASS, and PABS suggest that the common molecular andcellular mechanisms might underlie the infection andmaintenance of these secondary symbionts. However,

localization of PAUS in secondary mycetocytes was de-tected in only a fraction of the embryos examined(Fig. 1B) whereas localization of PASS in secondarymycetocytes was constantly observed [16, 21], suggesting

Figure 3.Electron microscopy ofA. pisumembryos of the PAUS-

infected strain TUt. (A) Rod-shaped PAUS cells in the cytoplasm

of a sheath cell, located between a fat body cell (upper right) and a

primary mycetocyte harboring Buchnera (lower left). (B) An en-

larged image of PAUS cells, in which cell wall structures are seen.(C) Extracellular PAUS cells associated with a sheath cell on the

periphery of mycetome. Arrows and asterisks indicate PAUS and

Buchnera, respectively. Bars: 2 lm. Abbreviations, ER: endoplasmic

reticulum; FBC: fat body cell; Mt: mitochondrion; N: nucleus; P-

Myc: primary mycetocyte; ShC: sheath cell.



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at their cellular tropism may be different to some ex-nt.

In this study, we investigated the infectivity andability of PAUS by artificial transfer into uninfectedost insects. Injection of hemolymph from infected in-

cts into uninfected ones established a stable PAUSfection in the recipients. Furthermore, the introduced

AUS was vertically transmitted to the offspring of thecipient stably over 24 generations (Table 2). These re-lts indicated that PAUS infection is stably maintainedrough host generations, at least under laboratory con-tions. It was also suggested that PAUS is potentiallypable of not only vertical but also horizontal trans-ission in populations ofA. pisum.

Transfer experiments of PAUS identified a consid-able time lag between the injection and the establish-

ent of vertical transmission. Adult aphids of 58 dayster injection did not transmit PAUS to their offspring;rtial transmission was detected 912 days after injec-

on, and complete transmission followed (Table 2). Twoypotheses can explain this pattern. One hypothesis isat there is a critical developmental stage at which the

mbryos are susceptible to PAUS infection. Anotherypothesis is that there is a threshold density of PAUSat is needed for successful transmission to the embryosthe ovarioles. To estimate which of these hypotheses is

ore appropriate, quantitative and histological studies

n PAUS infection during the development ofA. pisume required. Notably, similar infection time lags haveen identified from other secondary symbionts, PASS] and Spiroplasma [18].

PAUS (also referred to as U-type symbionts) haveen identified not only from A. pisum but also fromher diverse aphid species. Sandstrom et al. [29] re-

orted that, of 17 aphid species examined, PAUS wasesent in six species of the Aphidinae (Macrosiphonielladovicianae, Uroleucon astronomus, U. solidagensis, U.neum, U. helianthicola, and A. pisum). Russell et al.

8] found that, of 76 aphid species examined, PAUS wasrbored by four species of the Aphidinae (Brachycaudusrdui, Macrosiphum euphorbiae, M. rosae,and Uroleucondbeckiae), one species of the Chaitophorinae (Chaito-

phorus populati), and one species of the Pemphiginae(Pemphigus betae). In this study, of 29 Japanese aphidspecies examined, PAUS was detected from five species ofthe Aphidinae (Aphis citricola, A. nerii, Macrosiphumavenae, Uroleucon giganteus, and A. pisum) and one

species of the Pemphiginae (Colopha kansugei) (Table 1).Considering that the numbers of aphid individuals,populations, and species examined in these studies arequite limited, further survey will no doubt increase thelist of aphid species infected with this type of symbiont.To gain insights into potential host range of PAUS,artificial transfer experiments across aphid lineages willbe of great interest.

The phylogenetic distribution of PAUS, sporadicallyfound across at least three aphid subfamilies, poorly re-flected the systematics of the host aphids (Table 1) [28].

The pattern can be best explained by the idea that PAUShave experienced occasional horizontal transfers acrossaphid lineages, while the symbiont has normally beensubjected to stable vertical transmission (Table 2). Al-though the route of horizontal transfer is unknown,involvement of parasitoids or plant phloem fluid hasbeen suggested [8, 18, 28].

As for effects of PAUS infection on A. pisum, severalstudies have provided some meaningful data. Frequenciesof PAUS infection exhibited a geographical cline acrossmainland Japan [32]. On the basis of statistical analyses,

the distribution of PAUS was positively correlated withwhite clover as host plant, lower temperature, and smallerprecipitation [32]. In California, PAUS infection wasassociated with host plant specialization of the aphid forwhite clover [22]. In France, PAUS infection was re-stricted to a particular host race of the aphid specializedfor red clover [31]. Recently, it was experimentally shownthat PAUS infection broadened the host plant range ofthe aphid [33]. These observations suggest that PAUSinfection might affect the adaptation of the host aphid tothese environmental factors. Alternatively, PAUS might

be simply associated with the aphid strains and raceswithout substantial effects on the adaptation [23]. Theseideas may be not mutually exclusive, considering thatdifferent aphid populations must have experienced dif-

ble 2.Artificial transfer of the PAUS from strain TUt to strain AIST by injection of hemolymph

ected lines

Injected generation Successive generations

56 (89)a 78 (1011) 910 (1213) 1112 (1415) 1314 (1617) 15 (18) 1c 2d 24e

ST-Tut 0/4b 0/6 2/6 4/6 6/6 8/8 10/10 10/10 20/20ST-TUt 2 0/4 0/6 3/6 4/6 6/6 8/8 10/10 10/10 20/20ST-TUt 3 0/4 0/6 2/6 5/6 6/6 8/8 10/10 10/10 20/20

ays after injection (days after birth).umber of infected offspring/number of offspring examined.tablished from a nymph deposited 1213 days after injection of the injected generation.tablished from a newborn nymph of the generation 1.

bout 6 months after injection.



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ferent environmental conditions and evolutionary histo-ries. To gain insights into what interactions exist amongthe host aphid, Buchnera, PAUS, and environmentalfactors, further microbiological investigations will beneeded such as spatiotemporal population dynamics ofPAUS under different environmental conditions in rela-tion to fitness effects on the host aphid.

Acknowledgments

We thank A. Sugimura, H. Ouchi, S. Kumagai, S. Tat-suno, and K. Sato for technical and secretarial assistance;A. Kume, H. Shibao, I. Mori, K. Honda, N. Kohmoto,and S. Akimoto for aphid samples; and T. Wilkinson forcritically reading the manuscript. This research wassupported by the Program for Promotion of Basic Re-search Activities for Innovation Biosciences (ProBRAIN)of the Bio-Oriented Technology Research Advancement

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