Transcriptomic basis of sex loss in the pea aphid.

BACKGROUND: Transitions from sexual to asexual reproduction are common in eukaryotes, but the underlying mechanisms remain poorly known. The pea aphid-Acyrthosiphon pisum-exhibits reproductive polymorphism, with cyclical parthenogenetic and obligate parthenogenetic lineages, offering an opportunity to decipher the genetic basis of sex loss. Previous work on this species identified a single 840 kb region controlling reproductive polymorphism and carrying 32 genes. With the aim of identifying the gene(s) responsible for sex loss and the resulting consequences on the genetic programs controlling sexual or asexual embryogenesis, we compared the transcriptomic response to photoperiod shortening-the main sex-inducing cue-of a sexual and an obligate asexual lineage of the pea aphid, focusing on heads (where the photoperiodic cue is detected) and embryos (the final target of the cue). RESULTS: Our analyses revealed that four genes (one expressed in the head, and three in the embryos) of the region responded differently to photoperiod in the two lineages. We also found that the downstream genetic programs expressed during embryonic development of a future sexual female encompass ∼1600 genes, among which miRNAs, piRNAs and histone modification pathways are overrepresented. These genes mainly co-localize in two genomic regions enriched in transposable elements (TEs). CONCLUSIONS: Our results suggest that the causal polymorphism(s) in the 840 kb region somehow impair downstream epigenetic and post-transcriptional regulations in obligate asexual lineages, thereby sustaining asexual reproduction.

An integrated protocol for targeted mutagenesis with CRISPR-Cas9 system in the pea aphid.

CRISPR-Cas9 technology is a very efficient functional analysis tool and has been developed in several insects to edit their genome through injection of eggs with guide RNAs targeting coding sequences of genes of interest. However, its implementation in aphids is more challenging. Aphids are major pests of crops worldwide that alternate during their life cycle between clonality and sexual reproduction. The production of eggs after mating of sexual individuals is a single yearly event and is necessarily triggered by a photoperiod decrease. Fertilized eggs then experience an obligate 3-month diapause period before hatching as new clonal colonies. Taking into consideration these particularities, we developed in the pea aphid Acyrthosiphon pisum a step-by-step protocol of targeted mutagenesis based on the microinjection within fertilized eggs of CRISPR-Cas9 components designed for the editing of a cuticular protein gene (stylin-01). This protocol includes the following steps: i) the photoperiod-triggered induction of sexual morphs (2 months), ii) the mating and egg collection step (2 weeks), iii) egg microinjection and melanization, iv) the 3-month obligate diapause, v) the hatching of new lineages from injected eggs (2 weeks) and vi) the maintenance of stable lineages (2 weeks). Overall, this 7-month long procedure was applied to three different crosses in order to estimate the impact of the choice of the genetic combination on egg production dynamics by females as well as hatching rates after diapause. Mutation rates within eggs before diapause were estimated at 70-80%. The hatching rate of injected eggs following diapause ranged from 1 to 11% depending on the cross and finally a total of 17 stable lineages were obtained and maintained clonally. Out of these, 6 lineages were mutated at the defined sgRNAs target sites within stylin-01 coding sequence, either at the two alleles (2 lineages) or at one allele (4 lineages). The final germline transmission rate of the mutations was thus around 35%. Our protocol of an efficient targeted mutagenesis opens the avenue for functional studies through genome editing in aphids.

Hydroxylated human homotrimeric collagen I in Agrobacterium tumefaciens-mediated transient expression and in transgenic tobacco plant.

Potential contamination of animal-derived collagen with pathogens has led to the demand for safe recombinant sources of this complex molecule. In continuation of our previous work [Ruggiero et al. (2000) FEBS Lett. 469, 132-136], here we show that it is possible to produce recombinant hydroxylated homotrimeric collagen in tobacco plants that are co-transformed with a human type I collagen and a chimeric proline-4-hydroxylase (P4H). This is to our knowledge the first time that transient expression in tobacco was used to improve the quality of a recombinant protein produced in plants through co-expression with an animal cell-derived modifying enzyme. We demonstrated the functionality of the new chimeric P4H and thus improved the thermal stability of recombinant collagen I from plants to 37 degrees C.