AEM
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Other Versions of this Article:
AEM.00022-08v1
74/13/4236    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Google Scholar
Right arrow Articles by Lamelas, A.
Right arrow Articles by Latorre, A.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lamelas, A.
Right arrow Articles by Latorre, A.
Agricola
Right arrow Articles by Lamelas, A.
Right arrow Articles by Latorre, A.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, July 2008, p. 4236-4240, Vol. 74, No. 13
0099-2240/08/$08.00+0     doi:10.1128/AEM.00022-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Evolution of the Secondary Symbiont "Candidatus Serratia symbiotica" in Aphid Species of the Subfamily Lachninae {triangledown},{dagger}

Araceli Lamelas,1 Vicente Pérez-Brocal,1,2 Laura Gómez-Valero,1,3 María José Gosalbes,1 Andrés Moya,1 and Amparo Latorre1*

Institut Cavanilles de Biodiversitat i Biologia Evolutiva and Departament de Genètica, Universitat de València, Apartado de Correos 22085, 46071 Valencia, Spain,1 London School of Hygiene and Tropical Medicine, Department of Infectious and Tropical Diseases, London WC1E 7HT, United Kingdom,2 Unité de Génomique des Microorganismes Pathogènes, Institut Pasteur, 28 Rue du Dr Roux, 75724 Paris, France3

Received 3 January 2008/ Accepted 12 May 2008


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 Nucleotide sequence accession...
 REFERENCES
 
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.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
Nucleotide sequence accession...
 REFERENCES
 
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).


View this table:
[in this window]
[in a new window]

  		TABLE 1. Taxonomic status, S-symbionts, locations, and accession numbers of the aphid species included in the present study

 
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).


Figure 1
View larger version (25K):
[in this window]
[in a new window]

  		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.

 
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).


Figure 2
View larger version (103K):
[in this window]
[in a new window]

  		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).

 
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.
Top
 ABSTRACT
 INTRODUCTION
 Nucleotide sequence accession...
REFERENCES
 
The DNA sequences determined in this study were deposited in the EMBL/GenBank nucleotide sequence databases with the accession numbers shown in Table 1.


   ACKNOWLEDGMENTS
 
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.


   FOOTNOTES
 
* Corresponding author. Mailing address: Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de València, Apartado de Correos 22085, 46071 Valencia, Spain. Phone: (34)-96-3543649. Fax: (34)-96-3543670. E-mail: amparo.latorre{at}uv.es Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back

{triangledown} Published ahead of print on 23 May 2008. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 Nucleotide sequence accession...
 REFERENCES
 

  1. Baumann, P. 2005. Biology bacteriocyte-associated endosymbionts of plant sap-sucking insects. Annu. Rev. Microbiol. 59:155-189.[CrossRef][Medline]
  2. Buades, C., J. M. Michelena, A. Latorre, and A. Moya. 1999. Accelerated evolution in bacterial endosymbionts of aphids. Int. Microbiol. 2:11-14.[Medline]
  3. Buchner, P. 1965. Endosymbiosis of animals with plant microorganisms. Interscience Publishers, New York, NY.
  4. Fukatsu, T., and H. Ishikawa. 1998. Differential immunohistochemical visualization of the primary and secondary intracellular symbiotic bacteria of aphids. Appl. Entomol. Zoolog. 33:321-326.
  5. Fukatsu, T., N. Nikoh, R. Kawai, and R. Koga. 2000. The secondary endosymbiotic bacterium of the pea aphid Acyrthosiphon pisum (Insecta: Homoptera). Appl. Environ. Microbiol. 66:2748-2758.[Abstract/Free Full Text]
  6. Fukatsu, T., K. Watanabe, and Y. Sekiguchi. 1998. Specific detection of intracellular symbiotic bacteria of aphids by oligonucleotide-probed in situ hybridization. Appl. Entomol. Zool. 33:461-472.
  7. Gil, R., B. Sabater-Munoz, A. Latorre, F. J. Silva, and A. Moya. 2002. Extreme genome reduction in Buchnera spp.: toward the minimal genome needed for symbiotic life. Proc. Natl. Acad. Sci. USA 99:4454-4458.[Abstract/Free Full Text]
  8. Gómez-Valero, L., M. Soriano-Navarro, V. Pérez-Brocal, A. Heddi, A. Moya, J. M. Garcia-Verdugo, and A. Latorre. 2004. Coexistence of Wolbachia with Buchnera aphidicola and a secondary symbiont in the aphid Cinara cedri. J. Bacteriol. 186:6626-6633.[Abstract/Free Full Text]
  9. Heie, O. E. 1987. Paleontology and phylogeny, p. 367-391. In A. K. Minks and P. Harrewijn (ed.), World crop pest, vol. 2A. Aphids: their biology, natural enemies and control. Elsevier, Amsterdam, The Netherlands.
  10. Latorre, A., A. Moya, and F. J. Ayala. 1986. Evolution of mitochondrial DNA in Drosophila subobscura. Proc. Natl. Acad. Sci. USA 83:8649-8653.[Abstract/Free Full Text]
  11. Leonardo, T. E., and E. B. Mondor. 2006. Symbiont modifies host life-history traits that affect gene flow. Proc. Biol. Sci. 273:1079-1084.[CrossRef][Medline]
  12. Martínez-Torres, D., C. Buades, A. Latorre, and A. Moya. 2001. Molecular systematics of aphids and their primary endosymbionts. Mol. Phylogenet. Evol. 20:437-449.[CrossRef][Medline]
  13. McCutcheon, J. P., and N. A. Moran. 2007. Parallel genomic evolution and metabolic interdependence in an ancient symbiosis. Proc. Natl. Acad. Sci. USA 104:19392-19397.[Abstract/Free Full Text]
  14. Moran, N. A., J. A. Russell, R. Koga, and T. Fukatsu. 2005. Evolutionary relationships of three new species of Enterobacteriaceae living as symbionts of aphids and other insects. Appl. Environ. Microbiol. 71:3302-3310.[Abstract/Free Full Text]
  15. Moran, N. A., P. Tran, and N. M. Gerardo. 2005. Symbiosis and insect diversification: an ancient symbiont of sap-feeding insects from the bacterial phylum Bacteroidetes. Appl. Environ. Microbiol. 71:8802-8810.[Abstract/Free Full Text]
  16. Moya, A., J. Peretó, R. Gil, and A. Latorre. 2008. Learning how to live together: genomic insights into prokaryote-animal symbioses. Nat. Rev. Genet. 9:218-229.[CrossRef][Medline]
  17. Oliver, K. M., N. A. Moran, and M. S. Hunter. 2005. Variation in resistance to parasitism in aphids is due to symbionts not host genotype. Proc. Natl. Acad. Sci. USA 102:12795-12800.[Abstract/Free Full Text]
  18. Ortiz-Rivas, B., A. Moya, and D. Martínez-Torres. 2004. Molecular systematics of aphids (Homoptera: Aphididae): new insights from the long-wavelength opsin gene. Mol. Phylogenet. Evol. 30:24-37.[CrossRef][Medline]
  19. Pérez-Brocal, V., R. Gil, S. Ramos, A. Lamelas, M. Postigo, J. M. Michelena, F. J. Silva, A. Moya, and A. Latorre. 2006. A small microbial genome: the end of a long symbiotic relationship? Science 314:312-313.[Abstract/Free Full Text]
  20. Remaudière, G., and M. Remaudière. 1997. Catalogue des Aphididae du monde. Institut National de la Recherche Agronomique, Paris, France.
  21. Russell, J. A., A. Latorre, B. Sabater-Munoz, A. Moya, and N. A. Moran. 2003. Side-stepping secondary symbionts: widespread horizontal transfer across and beyond the Aphidoidea. Mol. Ecol. 12:1061-1075.[CrossRef][Medline]
  22. Russell, J. A., and N. A. Moran. 2006. Costs and benefits of symbiont infection in aphids: variation among symbionts and across temperatures. Proc. Biol. Sci. 273:603-610.[CrossRef][Medline]
  23. Sandstrom, J., A. Telang, and N. A. Moran. 2000. Nutritional enhancement of host plants by aphids—a comparison of three aphid species on grasses. J. Insect Physiol. 46:33-40.[Medline]
  24. Scarborough, C. L., J. Ferrari, and H. C. Godfray. 2005. Aphid protected from pathogen by endosymbiont. Science 310:1781.[Abstract/Free Full Text]
  25. Shigenobu, S., H. Watanabe, M. Hattori, Y. Sakaki, and H. Ishikawa. 2000. Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Nature 407:81-86.
  26. Staden, R., K. F. Beal, and J. K. Bonfield. 2000. The Staden package, 1998. Methods Mol. Biol. 132:115-130.[Medline]
  27. Takiya, D. M., P. L. Tran, C. H. Dietrich, and N. A. Moran. 2006. Co-cladogenesis spanning three phyla: leafhoppers (Insecta: Hemiptera: Cicadellidae) and their dual bacterial symbionts. Mol. Ecol. 15:4175-4191.[CrossRef][Medline]
  28. Tamas, I., L. Klasson, B. Canback, A. K. Naslund, A. S. Eriksson, J. J. Wernegreen, J. P. Sandstrom, N. A. Moran, and S. G. Andersson. 2002. 50 million years of genomic stasis in endosymbiotic bacteria. Science 296:2376-2379.[Abstract/Free Full Text]
  29. Tsuchida, T., R. Koga, and T. Fukatsu. 2004. Host plant specialization governed by facultative symbiont. Science 303:1989.[Free Full Text]
  30. van Ham, R. C., J. Kamerbeek, C. Palacios, C. Rausell, F. Abascal, U. Bastolla, J. M. Fernandez, L. Jimenez, M. Postigo, F. J. Silva, J. Tamames, E. Viguera, A. Latorre, A. Valencia, F. Moran, and A. Moya. 2003. Reductive genome evolution in Buchnera aphidicola. Proc. Natl. Acad. Sci. USA 100:581-586.[Abstract/Free Full Text]

Applied and Environmental Microbiology, July 2008, p. 4236-4240, Vol. 74, No. 13
0099-2240/08/$08.00+0     doi:10.1128/AEM.00022-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Other Versions of this Article:
AEM.00022-08v1
74/13/4236    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Google Scholar
Right arrow Articles by Lamelas, A.
Right arrow Articles by Latorre, A.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lamelas, A.
Right arrow Articles by Latorre, A.
Agricola
Right arrow Articles by Lamelas, A.
Right arrow Articles by Latorre, A.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. Microbiol. Mol. Biol. Rev. Eukaryot. Cell All ASM Journals