Research Article
1 July 2008

Evolution of the Secondary Symbiont “Candidatus Serratia symbiotica” in Aphid Species of the Subfamily Lachninae


Buchnera aphidicola BCc, the primary endosymbiont of the aphid Cinara cedri (subfamily Lachninae), is losing its symbiotic capacity and might be replaced by the coresident “Candidatus Serratia symbiotica.” Phylogenetic and morphological analyses within the subfamily Lachninae indicate two different “Ca. Serratia symbiotica” lineages and support the longtime coevolution of both symbionts in C. cedri.
Aphids are plant sap-feeding insects that harbor the gamma-proteobacterium Buchnera aphidicola as their primary endosymbiont (3). B. aphidicola genome sequencing confirmed its role in supplying the nutrients lacking in the aphid diet (25). Several aphids harbor additional bacteria known as secondary symbionts (S-symbionts). S-symbionts are considered facultative, as despite conferring positive effects to their hosts (11, 17, 22, 24, 29), they are not essential to host survival and reproduction (1, 16). Although normally transmitted vertically through host generations, their distribution patterns suggest that sporadic horizontal transmission can occur (21).
B. aphidicola BCc, the primary endosymbiont of the cedar aphid Cinara cedri, possesses the smallest known B. aphidicola genome, with only 422 kb (7). Unlike the other sequenced B. aphidicola strains (25, 28, 30), this strain cannot synthesize tryptophan and riboflavin, which must come from another source (19). One particular feature of C. cedri is the abundant and nonfacultative presence of the S-symbiont “Candidatus Serratia symbiotica” (8). Moreover, “Ca. Serratia symbiotica” and B. aphidicola BCc are similar in size and shape and housed in separate, specific bacteriocytes. These observations and the rapid evolution of most of the retained genes led us to postulate that B. aphidicola BCc is losing its symbiotic capacity and might be replaced by “Ca. Serratia symbiotica” (19). Its complete genome sequence (in progress) will tell us whether this bacterium can perform all the metabolic functions necessary for host fitness or, alternatively, if some pathways have been lost. Then, the metabolic complementation of both bacteria would be the expected evolutionary outcome. In the sharpshooter Homalodisca coagulata, the endosymbionts “Candidatus Baumannia cicadellinicola” and “Candidatus Sulcia muelleri” have complementary biosynthetic abilities, needed to provide their host with nutrients lacking in the xylem sap (13). Molecular phylogenetic studies showed that both symbionts represent ancient associations (15), being “coprimary” symbionts (27).
Most studies on S-symbiont presence in aphids have been conducted on members of the subfamily Aphidinae, mainly on Acyrthosiphon pisum. Regarding the subfamily Lachninae, to which C. cedri belongs, the few studies carried out report the presence of gammaproteobacterial S-symbionts (4, 6, 21).
The aim of the present work was to determine whether “Ca. Serratia symbiotica” is consistently present in the subfamily Lachninae, thus indicating a long-term association with B. aphidicola, or whether its presence in the aphid C. cedri represents a singular case.
We have obtained sequences from 14 species of the subfamily Lachninae, either by sequencing the genes from aphids collected from natural populations between 2004 and 2006 or by retrieving them from databases (Table 1). Total DNA was extracted from aphids as described previously (10), and 16S rRNA genes were amplified (for primers and PCR conditions, see the supplemental material). The resulting PCR fragments were cloned into a pGEM-T vector (Promega) and diagnostic digestions made to assess whether the 16S rRNA gene belonged to B. aphidicola or a S-symbiont (23). Sequencing was performed with an ABI Prism BigDye Terminator v3.0 kit (Applied Biosystems), and the resulting sequences were analyzed with the Staden software package (26). BlastN searches confirmed the nature of the sequences. In addition to B. aphidicola, all aphid species harbored one S-symbiont, except Stomaphis cupressi. In most cases, the S-symbiont was “Ca. Serratia symbiotica” (Table 1).
We carried out 16S rRNA gene phylogenetic analyses with the sequences from B. aphidicola (Fig. 1A) and “Ca. Serratia symbiotica,” obtained in this work, and from different aphid subfamilies (in addition to Lachninae) of the family Aphididae, previously studied (21) (Fig. 1B) (see methods in the supplemental material). As expected, B. aphidicola from members of the subfamily Lachninae formed a well-solved cluster with respect to the two members of the family Aphididae used as outgroups (Fig. 1A). However, at the tribe level, although the two Eulachinini clades Cinara (Cinara) and Cinara (Cupressobium) are monophyletic groups, they did not cluster in a monophyletic tribe separated from the Lachnini, thus confirming uncertainties about the taxonomic status of these two tribes (12, 18).
Regarding the “Ca. Serratia symbiotica” phylogeny, the most interesting result is the existence of two major clusters (Fig. 1B). Cluster A encompasses representatives of the aphid subfamilies Aphidinae, Chaitophorinae, Eriosomatinae, and some Eulachnini members of the subfamily Lachninae from the clade Cinara (Cupressobium). Cluster B comprises solely members of the subfamily Lachninae, belonging to the clade Cinara (Cinara) from Eulachnini, plus species Lachnus roboris and Tuberolachnus salignus from tribe Lachnini. These clusters do not match those based on either the B. aphidicola 16S rRNA gene phylogeny (Fig. 1A) or the aphid phylogeny obtained with morphological traits (9).
To give support to the hypothesis of two “Ca. Serratia symbiotica” clusters, additional phylogenetic analyses were made with the protein-coding gene atpD in selected species from the subfamily Lachninae (for additional information, see the supplemental material). This gene was chosen because the ATPase operon has been lost in B. aphidicola BCc (19), and previous attempts to amplify a gene fragment in B. aphidicola from members of the subfamily Lachninae did not give positive results (2), indicating the possible loss of all the genes of this operon in B. aphidicola before the Lachninae split. The topology obtained (Fig. 1C) is similar to that with the ribosomal gene. Differences in branch length between the two clusters are more evident in this case, with longer branches in cluster B than in A. This feature indicates that this gene is evolving faster in “Ca. Serratia symbiotica” species in cluster B. Similar results for topology and branch length were obtained when 16S rRNA gene and atpD sequences were concatenated (data not shown).
In summary, we report the existence of at least two “Ca. Serratia symbiotica” clades in the subfamily Lachninae, which is compatible with the two aphid subgenera Cinara (Cupresobium) and Cinara (Cinara), proposed by entomologists according to morphological and other biological features (20). Moreover, while clade A encompasses “Ca. Serratia symbiotica” from aphids belonging to different subfamilies of the family Aphididae, clade B comprises only species from the subfamily Lachninae.
To further ascertain the presence of two different “Ca. Serratia symbiotica” clades, we performed electron microscopy studies of primary (B. aphidicola) and secondary (“Ca. Serratia symbiotica”) symbionts in two selected aphids as representatives of each clade: C. cedri (Eulachnini from clade B) and Cinara tujafilina (Eulachnini from clade A) (for details, see methods in the supplemental material). The most remarkable result concerns the differences in morphology of “Ca. Serratia symbiotica” (Fig. 2). C. cedri exhibited an unusually large cell size and spherical shape, which is characteristic of primary symbionts (1), and were detected only in their specific bacteriocytes. In C. tujafilina, by contrast, “Ca. Serratia symbiotica” displayed more typical cell size and shape (bacilliform) and was located in the sheath cells, in secondary bacteriocytes, and extracellularly, as previously reported for A. pisum (5, 14).
All these results suggest a long-term relationship between “Ca. Serratia symbiotica” and aphids of the subgenus Cinara (Cinara) of the subfamily Lachninae (and probably also of the tribe Lachnini). The hypothetical evolutionary scenario could be that infection by an ancestor of “Ca. Serratia symbiotica” took place before the tribes Lachnini and Eulachnini split. Afterwards, clades Cinara (Cinara) and Cinara (Cupressobium) diverged and evolved. Horizontal transfer events between members of Cinara (Cupressobium) and members of the other aphid subfamilies would explain why they cluster together with a very low level of divergence. However, in members of clade Cinara (Cinara), to which C. cedri belongs, “Ca. Serratia symbiotica” would have established a deep association due to the loss of some essential functions of B. aphidicola, which were taken over by “Ca. Serratia symbiotica” as previously proposed (19). The differences in branch length obtained indicate that in this lineage, the species has undergone accelerated evolution. More difficult to explain is the clustering of “Ca. Serratia symbiotica” of two members of the tribe Lachnini with members of clade B, and thus, further studies are needed with more representatives of the tribe Lachnini.
In conclusion, we postulate the presence of two types of “Ca. Serratia symbiotica” in aphids, one an S-symbiont but the other a primary-like endosymbiont.

Nucleotide sequence accession numbers.

The DNA sequences determined in this study were deposited in the EMBL/GenBank nucleotide sequence databases with the accession numbers shown in Table 1.
FIG. 1.
FIG. 1. Maximum likelihood phylogeny. (A) B. aphidicola 16S rRNA gene. The outgroups are B. aphidicola from Acyrthosiphon pisum and from Schizaphis graminum (GenBank accession numbers M27039 and NC_004061, respectively). (B) “Ca. Serratia symbiotica” 16S rRNA gene. Free-living bacteria are Serratia plymuthica (AY394724), Serratia marcescens (AF124038), and the outgroups Escherichia coli (AB045731) and Serratia boydii (AY696681). (C) Gene atpD from some selected “Ca. Serratia symbiotica” spp. Free-living bacteria are S. marcescens (ABI21950) and the outgroups Erwinia carotovora (IP_052595) and Yersinia pseudotuberculosis (BX936398). Numbers in nodes indicate support values in the form of proportions of bootstrap pseudoreplicates and Bayesian a posteriori probabilities for the corresponding inner branch. Branches with support values higher than 55% are collapsed. See Table 1 for species information.
FIG. 2.
FIG. 2. Electron microscopy of C. cedri (A) and C. tujafilina (B). Primary and secondary bacteriocytes harbor B. aphidicola (a) and “Ca. Serratia symbiotica” (b).
TABLE 1. Taxonomic status, S-symbionts, locations, and accession numbers of the aphid species included in the present study
Subfamily and tribeHost speciesS-symbiontLocationAccession no.  
    16S rRNA gene from S-symbiont16S rRNA gene from B. aphidicolaatp D from “Ca. Serratia symbiotica”
    EulachniniCinara (Cinara) schimitschekiCa. Serratia symbiotica”SpainEU348318EU334766 
 Cinara (Cinara) pineaCa. Serratia symbiotica”SpainEU348316EU334770EU348331
 Cinara (Cinara) gudarisCa. Serratia symbiotica”SpainEU348317EU334771 
 Cinara (Cinara) maghrebicaCa. Serratia symbiotica”SpainEU348319EU334772EU348330
 Cinara (Cinara) cedribCa. Serratia symbiotica”SpainAY620432 aAY620431 aEU360774
 Cinara (Cinara) cedribCa. Serratia symbiotica”SpainEU348324EU334777EU348327
 Cinara (Cinara) pinimaritimaeCandidatus Hamiltonella defensa”SpainEU348313EU334774 
 Cinara (Cinara) pilicornisCa. Serratia symbiotica”SpainEU348320EU334776EU348332
 Cinara (Cupressobium) tujafilinaCa. Serratia symbiotica”SpainEU348323EU334773EU348333
 Cinara (Cupressobium) juniperiunidentifiedSpainEU348311EU334768 
 Cinara (Cupressobium) cupressibCa. Serratia symbiotica”SpainEU348321EU334775EU348328
 Cinara (Cupressobium) cupressibCa. Serratia symbiotica”SpainEU348322EU334769EU348329
    LachniniStomaphis cupressiCandidatus Arsenophonus triatominarum”SpainEU348325EU334767 
 Maculolachnus submaculaunidentifiedSpainEU348312AJ296755 a 
 Lachnus roborisCa. Serratia symbiotica”SpainEU348314AJ296756 aEU348334
 Tuberolachnus salignusCa. Serratia symbiotica”SpainEU348315AJ296754 aEU348335
 Pterochloroides persicaeCa. Serratia symbiotica”SpainAY136155 a  
    MacrosiphiniMacrosiphoniella helichrysiCa. Serratia symbiotica”SpainAY136151 a  
 Acyrthosiphon pisumCa. Serratia symbiotica”JapanAB033777 a  
 Acyrthosiphon pisumCa. Serratia symbiotica”JapanAB033778 a  
 Acyrthosiphon pisumCa. Serratia symbiotica”JapanAB033779 a  
 Acyrthosiphon pisumCa. Serratia symbiotica”USAAF293617 a  
 Acyrthosiphon pisumCa. Serratia symbiotica”USAAY136139 a  
 Acyrthosiphon pisumCa. Serratia symbiotica”USAAY136140 a  
 Acyrthosiphon pisumCa. Serratia symbiotica”USAAY296732 a  
 Uroleucon caligatumCa. Serratia symbiotica”USAAF293624 a  
    AphidiniAphis craccivoraCa. Serratia symbiotica”SpainAY136137 a  
    ChaitophoriniPeriphyllus bulgaricusCa. Serratia symbiotica”SpainAY136157 a  
    FordiniSmynthurodes betaeCa. Serratia symbiotica”IsraelAY136159 a  
From the databases.
Different localities.


This work was supported by project BFU2007/06003 of the Ministerio de Educación y Ciencia (MEC). A. Lamelas is funded by a predoctoral fellowship from the Generalitat Valenciana (Spain).
We acknowledge J. M. Michelena for aphid identification and the Servicio de Secuenciación de Ácidos Nucléicos y Proteínas at SCSIE (Universitat de València) for sequencing support.

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Published In

cover image Applied and Environmental Microbiology
Applied and Environmental Microbiology
Volume 74Number 131 July 2008
Pages: 4236 - 4240
PubMed: 18502932


Received: 3 January 2008
Accepted: 12 May 2008
Published online: 1 July 2008


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Araceli Lamelas
Institut Cavanilles de Biodiversitat i Biologia Evolutiva and Departament de Genètica, Universitat de València, Apartado de Correos 22085, 46071 Valencia, Spain
Vicente Pérez-Brocal
Institut Cavanilles de Biodiversitat i Biologia Evolutiva and Departament de Genètica, Universitat de València, Apartado de Correos 22085, 46071 Valencia, Spain
London School of Hygiene and Tropical Medicine, Department of Infectious and Tropical Diseases, London WC1E 7HT, United Kingdom
Laura Gómez-Valero
Institut Cavanilles de Biodiversitat i Biologia Evolutiva and Departament de Genètica, Universitat de València, Apartado de Correos 22085, 46071 Valencia, Spain
Unité de Génomique des Microorganismes Pathogènes, Institut Pasteur, 28 Rue du Dr Roux, 75724 Paris, France
María José Gosalbes
Institut Cavanilles de Biodiversitat i Biologia Evolutiva and Departament de Genètica, Universitat de València, Apartado de Correos 22085, 46071 Valencia, Spain
Andrés Moya
Institut Cavanilles de Biodiversitat i Biologia Evolutiva and Departament de Genètica, Universitat de València, Apartado de Correos 22085, 46071 Valencia, Spain
Amparo Latorre [email protected]
Institut Cavanilles de Biodiversitat i Biologia Evolutiva and Departament de Genètica, Universitat de València, Apartado de Correos 22085, 46071 Valencia, Spain


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