Buchnera has changed flatmate but the repeated replacement of co-obligate symbionts is not associated with the ecological expansions of their aphid hosts.

Symbiotic associations with bacteria have facilitated important evolutionary transitions in insects and resulted in long-term obligate interactions. Recent evidence suggests that these associations are not always evolutionarily stable and that symbiont replacement, and/or supplementation of an obligate symbiosis by an additional bacterium, has occurred during the history of many insect groups. Yet, the factors favouring one symbiont over another in this evolutionary dynamic are not well understood; progress has been hindered by our incomplete understanding of the distribution of symbionts across phylogenetic and ecological contexts. While many aphids are engaged into an obligate symbiosis with a single Gammaproteobacterium, Buchnera aphidicola, in species of the Lachninae subfamily, this relationship has evolved into a 'ménage à trois', in which Buchnera is complemented by a cosymbiont, usually Serratia symbiotica. Using deep sequencing of 16S rRNA bacterial genes from 128 species of Cinara (the most diverse Lachninae genus), we reveal a highly dynamic dual symbiotic system in this aphid lineage. Most species host both Serratia and Buchnera but, in several clades, endosymbionts related to Sodalis, Erwinia or an unnamed member of the Enterobacteriaceae have replaced Serratia. Endosymbiont genome sequences from four aphid species confirm that these coresident symbionts fulfil essential metabolic functions not ensured by Buchnera. We further demonstrate through comparative phylogenetic analyses that cosymbiont replacement is not associated with the adaptation of aphids to new ecological conditions. We propose that symbiont succession was driven by factors intrinsic to the phenomenon of endosymbiosis, such as rapid genome deterioration or competitive interactions between bacteria with similar metabolic capabilities.

Assessment of a 16S rRNA amplicon Illumina sequencing procedure for studying the microbiome of a symbiont-rich aphid genus.

The bacterial communities inhabiting arthropods are generally dominated by a few endosymbionts that play an important role in the ecology of their hosts. Rather than comparing bacterial species richness across samples, ecological studies on arthropod endosymbionts often seek to identify the main bacterial strains associated with each specimen studied. The filtering out of contaminants from the results and the accurate taxonomic assignment of sequences are therefore crucial in arthropod microbiome studies. We aimed here to validate an Illumina 16S rRNA gene sequencing protocol and analytical pipeline for investigating endosymbiotic bacteria associated with aphids. Using replicate DNA samples from 12 species (Aphididae: Lachninae, Cinara) and several controls, we removed individual sequences not meeting a minimum threshold number of reads in each sample and carried out taxonomic assignment for the remaining sequences. With this approach, we show that (i) contaminants accounted for a negligible proportion of the bacteria identified in our samples; (ii) the taxonomic composition of our samples and the relative abundance of reads assigned to a taxon were very similar across PCR and DNA replicates for each aphid sample; in particular, bacterial DNA concentration had no impact on the results. Furthermore, by analysing the distribution of unique sequences across samples rather than aggregating them into operational taxonomic units (OTUs), we gained insight into the specificity of endosymbionts for their hosts. Our results confirm that Serratia symbiotica is often present in Cinara species, in addition to the primary symbiont, Buchnera aphidicola. Furthermore, our findings reveal new symbiotic associations with Erwinia- and Sodalis-related bacteria. We conclude with suggestions for generating and analysing 16S rRNA gene sequences for arthropod-endosymbiont studies.

Regular or covert sex defines two lineages and worldwide superclones within the leaf-curl plum aphid (Brachycaudus helichrysi, Kaltenbach).

Asexual reproduction occurs widely in plants and animals, particularly in insects. Aphid species usually reproduce by cyclic parthenogenesis, but many species include obligate asexual lineages. We recently showed that the leaf-curl plum aphid, Brachycaudus helichrysi, actually encompasses two lineages, B. helichrysi H1 and H2. Ecological data suggest that these lineages have different life cycles. We conducted a large population genetics study, based on 14 microsatellite loci, to infer their respective life cycles and investigate their population structure and geographical distribution. Brachycaudus helichrysi H1 displayed the genetic signature of cyclical parthenogenesis, using plum trees as primary hosts for sexual reproduction, as classically described for B. helichrysi. This global survey showed that the Central Asian population of H1 was clearly differentiated from American-European populations. By contrast, B. helichrysi H2 displayed the typical signature of obligate asexual reproduction. H2 encompassed at least eight highly successful genotypes or superclones. This lack of ability to undergo sexual reproduction was confirmed for one of the superclones by sex induction experiments. We found only one B. helichrysi H2 population that underwent sexual reproduction, which was collected from peach trees, in Northern India. Our results confirm that H1 and H2 have different life cycles. Brachycaudus helichrysi H1 is clearly heteroecious using plum trees as primary hosts, while B. helichrysi H2 encompasses several anholocyclic lineages, and some heteroecious populations that until now have only been found associated with peach trees as primary hosts. We discuss implications of these findings for the pest status of B. helichrysi lineages.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 December 2009-31 January 2010.

This article documents the addition of 220 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Allanblackia floribunda, Amblyraja radiata, Bactrocera cucurbitae, Brachycaudus helichrysi, Calopogonium mucunoides, Dissodactylus primitivus, Elodea canadensis, Ephydatia fluviatilis, Galapaganus howdenae howdenae, Hoplostethus atlanticus, Ischnura elegans, Larimichthys polyactis, Opheodrys vernalis, Pelteobagrus fulvidraco, Phragmidium violaceum, Pistacia vera, and Thunnus thynnus. These loci were cross-tested on the following species: Allanblackia gabonensis, Allanblackia stanerana, Neoceratitis cyanescens, Dacus ciliatus, Dacus demmerezi, Bactrocera zonata, Ceratitis capitata, Ceratitis rosa, Ceratits catoirii, Dacus punctatifrons, Ephydatia mülleri, Spongilla lacustris, Geodia cydonium, Axinella sp., Ischnura graellsii, Ischnura ramburii, Ischnura pumilio, Pistacia integerrima and Pistacia terebinthus.