Jean Peccoud

Host range expansion of an introduced insect pest through multiple colonizations of specialized clones

Asexuality confers demographic advantages to invasive taxa, but generally limits adaptive potential for colonizing of new habitats. Therefore, pre‐existing adaptations and habitat tolerance are essential in the success of asexual invaders. We investigated these key factors of invasiveness by assessing reproductive modes and host‐plant adaptations in the pea aphid, Acyrthosiphon pisum, a pest recently introduced into Chile. The pea aphid encompasses lineages differing in their reproductive mode, ranging from obligatory cyclical parthenogenesis to fully asexual reproduction. This species also shows variation in host use, with distinct biotypes specialized on different species of legumes as well as more polyphagous populations. In central Chile, microsatellite genotyping of pea aphids sampled on five crops and wild legumes revealed three main clonal genotypes, which showed striking associations with particular host plants rather than sampling locations. Phenotypic analyses confirmed their strong host specialization and demonstrated parthenogenesis as their sole reproductive mode. The genetic relatedness of these clonal genotypes with corresponding host‐specialized populations from the Old World indicated that each clone descended from a particular Eurasian biotype, which involved at least three successful introduction events followed by spread on different crops. This study illustrates that multiple introductions of highly specialized clones, rather than local evolution in resource use and/or selection of generalist genotypes, can explain the demographic success of a strictly asexual invader.

Inheritance patterns of secondary symbionts during sexual reproduction of pea aphid biotypes

Herbivorous insects frequently harbor bacterial symbionts that affect their ecology and evolution. Aphids host the obligatory endosymbiont Buchnera, which is required for reproduction, together with facultative symbionts whose frequencies vary across aphid populations. These maternally transmitted secondary symbionts have been particularly studied in the pea aphid, Acyrthosiphon pisum, which harbors at least 8 distinct bacterial species (not counting Buchnera) having environmentally dependent effects on host fitness. In particular, these symbiont species are associated with pea aphid populations feeding on specific plants. Although they are maternally inherited, these bacteria are occasionally transferred across insect lineages. One mechanism of such nonmaternal transfer is paternal transmission to the progeny during sexual reproduction. To date, transmission of secondary symbionts during sexual reproduction of aphids has been investigated in only a handful of aphid lineages and 3 symbiont species. To better characterize this process, we investigated inheritance patterns of 7 symbiont species during sexual reproduction of pea aphids through a crossing experiment involving 49 clones belonging to 9 host‐specialized biotypes, and 117 crosses. Symbiont species in the progeny were detected with diagnostic qualitative PCR at the fundatrix stage hatching from eggs and in later parthenogenetic generations. We found no confirmed case of paternal transmission of symbionts to the progeny, and we observed that maternal transmission of a particular symbiont species (Serratia symbiotica) was quite inefficient. We discuss these observations in respect to the ecology of the pea aphid.

Massive horizontal transfer of transposable elements in insects

Significance Eukaryotes normally receive their genetic material from their parents but may occasionally, like prokaryotes do, acquire DNA from unrelated organisms through horizontal transfer (HT). In animals and plants, HT mostly concerns transposable elements (TEs), probably because these pieces of DNA can move within genomes. Assessing the impact of HTs on eukaryote evolution and the factors shaping the dynamics of these HTs requires large-scale systematic studies. We have analyzed the genomes from 195 insect species and found that no fewer than 2,248 events of HT of TEs occurred during the last 10 My, particularly between insects that were closely related and geographically close. These results suggest that HT of TEs plays a major role in insect genome evolution. Horizontal transfer (HT) of genetic material is central to the architecture and evolution of prokaryote genomes. Within eukaryotes, the majority of HTs reported so far are transfers of transposable elements (TEs). These reports essentially come from studies focusing on specific lineages or types of TEs. Because of the lack of large-scale survey, the amount and impact of HT of TEs (HTT) in eukaryote evolution, as well as the trends and factors shaping these transfers, are poorly known. Here, we report a comprehensive analysis of HTT in 195 insect genomes, representing 123 genera and 13 of the 28 insect orders. We found that these insects were involved in at least 2,248 HTT events that essentially occurred during the last 10 My. We show that DNA transposons transfer horizontally more often than retrotransposons, and unveil phylogenetic relatedness and geographical proximity as major factors facilitating HTT in insects. Even though our study is restricted to a small fraction of insect biodiversity and to a recent evolutionary timeframe, the TEs we found to be horizontally transferred generated up to 24% (2.08% on average) of all nucleotides of insect genomes. Together, our results establish HTT as a major force shaping insect genome evolution.

Continuous Influx of Genetic Material from Host to Virus Populations

Many genes of large double-stranded DNA viruses have a cellular origin, suggesting that host-to-virus horizontal transfer (HT) of DNA is recurrent. Yet, the frequency of these transfers has never been assessed in viral populations. Here we used ultra-deep DNA sequencing of 21 baculovirus populations extracted from two moth species to show that a large diversity of moth DNA sequences (n = 86) can integrate into viral genomes during the course of a viral infection. The majority of the 86 different moth DNA sequences are transposable elements (TEs, n = 69) belonging to 10 superfamilies of DNA transposons and three superfamilies of retrotransposons. The remaining 17 sequences are moth sequences of unknown nature. In addition to bona fide DNA transposition, we uncover microhomology-mediated recombination as a mechanism explaining integration of moth sequences into viral genomes. Many sequences integrated multiple times at multiple positions along the viral genome. We detected a total of 27,504 insertions of moth sequences in the 21 viral populations and we calculate that on average, 4.8% of viruses harbor at least one moth sequence in these populations. Despite this substantial proportion, no insertion of moth DNA was maintained in any viral population after 10 successive infection cycles. Hence, there is a constant turnover of host DNA inserted into viral genomes each time the virus infects a moth. Finally, we found that at least 21 of the moth TEs integrated into viral genomes underwent repeated horizontal transfers between various insect species, including some lepidopterans susceptible to baculoviruses. Our results identify host DNA influx as a potent source of genetic diversity in viral populations. They also support a role for baculoviruses as vectors of DNA HT between insects, and call for an evaluation of possible gene or TE spread when using viruses as biopesticides or gene delivery vectors.

Genetic characterisation of new host-specialised biotypes and novel associations with bacterial symbionts in the pea aphid complex

The pea aphid, Acyrthosiphon pisum Harris, constitutes a complex of sympatric populations that are specialised to distinct species of Fabaceae. So far, 12 such populations have been characterised genetically as genetic clusters associated with one or few legumes species. These clusters form a continuum of genetic divergence linking host‐associated races, which show moderate hybridization, to nascent species. They are also known to be associated with different species of endosymbiotic bacteria, which have environmentally dependent effects on aphid fitness. Here, we report on the genetic characterisation of new host‐associated populations in this species complex. We sampled pea aphids in eastern France on Genista tinctoria, G. sagittalis, Onobrychis viciifolia, and Hippocrepis comosa. Bayesian clustering methods based on genotypes obtained at 25 microsatellite loci showed that each sampled plant species hosted a specific pea aphid population. Comparison with previously characterised biotypes showed that the population associated with Hippocrepis comosa was no different from that on Securigera varia. Migrant aphids from other host plants and hybrids were found at various frequencies on the newly sampled plant species. They were particularly frequent on Onobrychis viciifolia, occasional between the two Genista‐associated populations, and rare on Hippocrepis comosa. PCR‐based screening of bacterial species revealed new associations between aphid biotypes and facultative endosymbionts, chiefly the combination of Serratia symbiotica and Hamiltonella defensa within most individuals collected on Genista. The newly identified biotypes, which add up to a total of 15 within the pea aphid complex, offer new material to study the mechanisms and genetic bases of host‐specialisation and ecological speciation in this model aphid.

Widespread host-dependent hybrid unfitness in the pea aphid species complex.

Linking adaptive divergence to hybrid unfitness is necessary to understand the ecological factors contributing to reproductive isolation and speciation. To date, this link has been demonstrated in few model systems, most of which encompass ecotypes that occupy relatively early stages in the speciation process. Here we extend these studies by assessing how host‐plant adaptation conditions hybrid fitness in the pea aphid, Acyrthosiphon pisum. We made crosses between and within five pea aphid biotypes adapted to different host plants and representing various stages of divergence within the complex. Performance of F1 hybrids and nonhybrids was assessed on a “universal” host that is favorable to all pea aphid biotypes in laboratory conditions. Although hybrids performed equally well as nonhybrids on the universal host, their performance was much lower than nonhybrids on the natural hosts of their parental populations. Hence, hybrids, rather than being intrinsically deficient, are maladapted to their parents’ hosts. Interestingly, the impact of this maladaptation was stronger in certain hybrids from crosses involving the most divergent biotype, suggesting that host‐dependent postzygotic isolation has continued to evolve late in divergence. Even though host‐independent deficiencies are not excluded, hybrid maladaptation to parental hosts supports the hypothesis of ecological speciation in this complex.

Genomic regions repeatedly involved in divergence among plant‐specialized pea aphid biotypes

Understanding the genetic bases of biological diversification is a long‐standing goal in evolutionary biology. Here, we investigate whether replicated cases of adaptive divergence involve the same genomic regions in the pea aphid, Acyrthosiphon pisum, a large complex of genetically differentiated biotypes, each specialized on different species of legumes. A previous study identified genomic regions putatively involved in host‐plant adaptation and/or reproductive isolation by performing a hierarchical genome scan in three biotypes. This led to the identification of 11 FST outliers among 390 polymorphic microsatellite markers. In this study, the outlier status of these 11 loci was assessed in eight biotypes specialized on other host plants. Four of the 11 previously identified outliers showed greater genetic differentiation among these additional biotypes than expected under the null hypothesis of neutral evolution (α < 0.01). Whether these hotspots of genomic divergence result from adaptive events, intrinsic barriers or reduced recombination is discussed.

Molecular Test to Assign Individuals within the Cacopsylla pruni Complex

Crop protection requires the accurate identification of disease vectors, a task that can be made difficult when these vectors encompass cryptic species. Here we developed a rapid molecular diagnostic test to identify individuals of Cacopsylla pruni (Scopoli, 1763) (Hemiptera: Psyllidae), the main vector of the European stone fruit yellows phytoplasma. This psyllid encompasses two highly divergent genetic groups that are morphologically similar and that are characterized by genotyping several microsatellite markers, a costly and time-consuming protocol. With the aim of developing species-specific PCR primers, we sequenced the Internal Transcribed Spacer 2 (ITS2) on a collection of C . pruni samples from France and other European countries. ITS2 sequences showed that the two genetic groups represent two highly divergent clades. This enabled us to develop specific primers for the assignment of individuals to either genetic group in a single PCR, based on ITS2 amplicon size. All previously assigned individuals yielded bands of expected sizes, and the PCR proved efficient on a larger sample of 799 individuals. Because none appeared heterozygous at the ITS2 locus (i.e., none produced two bands), we inferred that the genetic groups of C . pruni , whose distribution is partly sympatric, constitute biological species that have not exchanged genes for an extended period of time. Other psyllid species (Cacopsylla, Psylla, Triozidae and Aphalaridae) failed to yield any amplicon. These primers are therefore unlikely to produce false positives and allow rapid assignment of C . pruni individuals to either cryptic species.

Disentangling the Causes for Faster-X Evolution in Aphids

Abstract The faster evolution of X chromosomes has been documented in several species, and results from the increased efficiency of selection on recessive alleles in hemizygous males and/or from increased drift due to the smaller effective population size of X chromosomes. Aphids are excellent models for evaluating the importance of selection in faster-X evolution because their peculiar life cycle and unusual inheritance of sex chromosomes should generally lead to equivalent effective population sizes for X and autosomes. Because we lack a high-density genetic map for the pea aphid, whose complete genome has been sequenced, we first assigned its entire genome to the X or autosomes based on ratios of sequencing depth in males (X0) to females (XX). Then, we computed nonsynonymous to synonymous substitutions ratios (dN/dS) for the pea aphid gene set and found faster evolution of X-linked genes. Our analyses of substitution rates, together with polymorphism and expression data, showed that relaxed selection is likely to be the greatest contributor to faster-X because a large fraction of X-linked genes are expressed at low rates and thus escape selection. Yet, a minor role for positive selection is also suggested by the difference between substitution rates for X and autosomes for male-biased genes (but not for asexual female-biased genes) and by lower Tajima’s D for X-linked compared with autosomal genes with highly male-biased expression patterns. This study highlights the relevance of organisms displaying alternative chromosomal inheritance to the understanding of forces shaping genome evolution.

Fast Evolution and Lineage-Specific Gene Family Expansions of Aphid Salivary Effectors Driven by Interactions with Host-Plants

Effector proteins play crucial roles in plant-parasite interactions by suppressing plant defenses and hijacking plant physiological responses to facilitate parasite invasion and propagation. Although effector proteins have been characterized in many microbial plant pathogens, their nature and role in adaptation to host plants are largely unknown in insect herbivores. Aphids rely on salivary effector proteins injected into the host plants to promote phloem sap uptake. Therefore, gaining insight into the repertoire and evolution of aphid effectors is key to unveiling the mechanisms responsible for aphid virulence and host plant specialization. With this aim in mind, we assembled catalogues of putative effectors in the legume specialist aphid, Acyrthosiphon pisum, using transcriptomics and proteomics approaches. We identified 3,603 candidate effector genes predicted to be expressed in A. pisum salivary glands (SGs), and 740 of which displayed up-regulated expression in SGs in comparison to the alimentary tract. A search for orthologs in 17 arthropod genomes revealed that SG-up-regulated effector candidates of A. pisum are enriched in aphid-specific genes and tend to evolve faster compared with the whole gene set. We also found that a large fraction of proteins detected in the A. pisum saliva belonged to three gene families, of which certain members show evidence consistent with positive selection. Overall, this comprehensive analysis suggests that the large repertoire of effector candidates in A. pisum constitutes a source of novelties promoting plant adaptation to legumes.