EMS mutagenesis in the pea aphid Acyrthosiphon pisum.

In aphids, clonal individuals can show distinct morphologic traits in response to environmental cues. Such phenotypic plasticity cannot be studied with classical genetic model organisms such as Caenorhabditis elegans or Drosophila melanogaster. The genetic basis of this biological process remain unknown, as mutations affecting this process are not available in aphids. Here, we describe a protocol to treat third-stage larvae with an alkylating mutagen, ethyl methanesulfonate (EMS), to generate random mutations within the Acyrthosiphon pisum genome. We found that even low concentrations of EMS were toxic for two genotypes of A. pisum. Mutagenesis efficiency was nevertheless assessed by estimating the occurrence of mutational events on the X chromosome. Indeed, any lethal mutation on the X-chromosome would kill males that are haploid on the X so that we used the proportion of males as an estimation of mutagenesis efficacy. We could assess a putative mutation rate of 0.4 per X-chromosome at 10 mM of EMS. We then applied this protocol to perform a small-scale mutagenesis on parthenogenetic individuals, which were screened for defects in their ability to produce sexual individuals in response to photoperiod shortening. We found one mutant line showing a reproducible altered photoperiodic response with a reduced production of males and the appearance of aberrant winged males (wing atrophy, alteration of legs morphology). This mutation appeared to be stable because it could be transmitted over several generations of parthenogenetic individuals. To our knowledge, this study represents the first example of an EMS-generated aphid mutant.

Evolutionary study of duplications of the miRNA machinery in aphids associated with striking rate acceleration and changes in expression profiles.

BACKGROUND: The sequencing of the genome of the pea aphid Acyrthosiphon pisum revealed an unusual expansion of the miRNA machinery, with two argonaute-1, two dicer-1 and four pasha gene copies. In this report, we have undertaken a deeper evolutionary analysis of the phylogenetic timing of these gene duplications and of the associated selective pressures by sequencing the two copies of ago-1 and dcr-1 in different aphid species of the subfamily Aphidinae. We have also carried out an analysis of the expression of both copies of ago-1 and dcr-1 by semi-quantitative PCR in different morphs of the pea aphid life cycle. RESULTS: The analysis has shown that the duplication of ago-1 occurred in an ancestor of the subfamily Aphidinae while the duplication of dcr-1 appears to be more recent. Besides, it has confirmed a pattern of one conserved copy and one accelerated copy for both genes, and has revealed the action of positive selection on several regions of the fast-evolving ago-1b. On the other hand, the semi-quantitative PCR experiments have revealed a differential expression of these genes between the morphs of the parthenogenetic and the sexual phases of Acyrthosiphon pisum. CONCLUSIONS: The discovery of these gene duplications in the miRNA machinery of aphids opens new perspectives of research about the regulation of gene expression in these insects. Accelerated evolution, positive selection and differential expression affecting some of the copies of these genes suggests the possibility of a neofunctionalization of these duplicates, which might play a role in the display of the striking phenotypic plasticity of aphids.

Expansion of genes encoding piRNA-associated argonaute proteins in the pea aphid: diversification of expression profiles in different plastic morphs.

Piwi-interacting RNAs (piRNAs) are known to regulate transposon activity in germ cells of several animal models that propagate sexually. However, the role of piRNAs during asexual reproduction remains almost unknown. Aphids that can alternate sexual and asexual reproduction cycles in response to seasonal changes of photoperiod provide a unique opportunity to study piRNAs and the piRNA pathway in both reproductive modes. Taking advantage of the recently sequenced genome of the pea aphid Acyrthosiphon pisum, we found an unusually large lineage-specific expansion of genes encoding the Piwi sub-clade of Argonaute proteins. In situ hybridisation showed differential expressions between the duplicated piwi copies: while Api-piwi2 and Api-piwi6 are "specialised" in germ cells their most closely related copy, respectively Api-piwi5 and Api-piwi3, are expressed in the somatic cells. The differential expression was also identified in duplicated ago3: Api-ago3a in germ cells and Api-ago3b in somatic cells. Moreover, analyses of expression profiles of the expanded piwi and ago3 genes by semi-quantitative RT-PCR showed that expressions varied according to the reproductive types. These specific expression patterns suggest that expanded aphid piwi and ago3 genes have distinct roles in asexual and sexual reproduction.

Expansion of the miRNA pathway in the hemipteran insect Acyrthosiphon pisum.

The pathways that allow short noncoding RNAs such as the microRNAs (miRNAs) to mediate gene regulation and control critical cellular and developmental processes involve a limited number of key protein components. These proteins are the Dicer-like RNases, double-stranded RNA (dsRNA)-binding proteins, and the Argonaute (AGO) proteins that process stem-loop hairpin transcripts of endogenous genes to generate miRNAs or long dsRNA precursors (either exogenous or endogenous). Comparative genomics studies of metazoans have shown the pathways to be highly conserved overall; the major difference observed is that the vertebrate pathways overlap in sharing a single Dicer (DCR) and AGO proteins, whereas those of insects appear to be parallel, with distinct Dicers and AGOs required for each pathway. The genome of the pea aphid is the first available for a hemipteran insect and discloses an unexpected expansion of the miRNA pathway. It has two copies of the miRNA-specific dicr-1 and ago1 genes and four copies of pasha a cofactor of drosha involved in miRNA biosynthesis. For three of these expansions, we showed that one copy of the genes diverged rapidly and in one case (ago1b) shows signs of positive selection. These expansions occurred concomitantly within a brief evolutionary period. The pea aphid, which reproduces by viviparous parthenogenesis, is able to produce several adapted phenotypes from one single genotype. We show by reverse transcriptase-polymerase chain reaction that all the duplicated copies of the miRNA machinery genes are expressed in the different morphs. Investigating the function of these novel genes offers an exciting new challenge in aphid biology.

Transcriptomic analysis of intestinal genes following acquisition of pea enation mosaic virus by the pea aphid Acyrthosiphon pisum.

Viruses in the family Luteoviridae are strictly transmitted by aphids in a non-propagative, circulative and persistent mode. Virions ingested by aphids successively cross the gut and the accessory salivary gland epithelia before being released, together with saliva, into the plant vasculature. Virion transport through aphid cells occurs by a transcytosis mechanism. This study conducted a transcriptomic analysis of intestinal genes of the pea aphid Acyrthosiphon pisum following uptake of pea enation mosaic virus. Among the 7166 transcripts analysed, 128 were significantly regulated (105 genes downregulated and 23 upregulated). Of these genes, 5 % were involved in intracellular trafficking, endocytosis and signal transduction, three important steps in the internalization and transport of virions. The limited levels of downregulation (maximum of 3.45-fold) and upregulation (maximum of 1.37-fold) suggest that the virus hijacks a constitutive endocytosis-exocytosis mechanism without heavily perturbing cell metabolism. Although limited to about 20 % of the pea aphid genes, this work represents the first large-scale analysis of aphid gene regulation following virus acquisition. A better knowledge of this virus-vector interaction will be possible only when tools representing the complete genomic capacity of the aphid become available.