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.

Juvenile hormone titre and related gene expression during the change of reproductive modes in the pea aphid.

Most aphids show reproductive polyphenism, i.e. they alternate their reproductive modes from parthenogenesis to sexual reproduction in response to short photoperiods. Although juvenile hormone (JH) has been considered a likely candidate for regulating the transition from asexual to sexual reproduction after photoperiod sensing, there are few studies investigating the direct relationship between JH titres and the reproductive-mode change. In addition, the sequencing of the pea aphid genome has allowed identification of the genes involved in the JH pathway, which in turn allows us to examine their expression levels in relation to the reproductive-mode change. Using liquid chromatography-mass spectrometry in the pea aphid, JHIII titre was shown to be lower in aphids producing sexual morphs under short-day conditions than in aphids producing parthenogenetic morphs under long-day conditions. The expression levels of genes upstream and downstream of JH action were quantified by real-time quantitative reverse-transcription-PCR across the reproductive-mode change. The expression level of JH esterase, which is responsible for JH degradation, was significantly higher in aphids reared under short-day conditions. This suggests that the upregulation of the JH degradation pathway may be responsible for the lower JHIII titre in aphids exposed to short-days, leading to the production of sexual morphs.

Neuropeptide and neurohormone precursors in the pea aphid, Acyrthosiphon pisum.

Aphids respond to environmental changes by developing alternative phenotypes with differing reproductive modes. Parthenogenetic reproduction occurs in spring and summer, whereas decreasing day lengths in autumn provoke the production of sexual forms. Changing environmental signals are relayed by brain neuroendocrine signals to the ovarioles. We combined bioinformatic analyses with brain peptidomics and cDNA analyses to establish a catalogue of pea aphid neuropeptides and neurohormones. 42 genes encoding neuropeptides and neurohormones were identified, of which several were supported by expressed sequence tags and/or peptide mass analyses. Interesting features of the pea aphid peptidome are the absence of genes coding for corazonin, vasopressin and sulfakinin and the presence of 10 different genes coding insulin related peptides, one of which appears to be very abundantly expressed.

A functional DNA methylation system in the pea aphid, Acyrthosiphon pisum.

Methylation of cytosine is one of the main epigenetic mechanisms involved in controlling gene expression. Here we show that the pea aphid (Acyrthosiphon pisum) genome possesses homologues to all the DNA methyltransferases found in vertebrates, and that 0.69% (+/-0.25%) of all cytosines are methylated. Identified methylation sites are predominantly restricted to the coding sequence of genes at CpG sites. We identify twelve methylated genes, including genes that interact with juvenile hormone, a key endocrine signal in insects. Bioinformatic prediction using CpG ratios for all predicted genes suggest that a large proportion of genes are methylated within the pea aphid.

AphidBase: a centralized bioinformatic resource for annotation of the pea aphid genome.

AphidBase is a centralized bioinformatic resource that was developed to facilitate community annotation of the pea aphid genome by the International Aphid Genomics Consortium (IAGC). The AphidBase Information System designed to organize and distribute genomic data and annotations for a large international community was constructed using open source software tools from the Generic Model Organism Database (GMOD). The system includes Apollo and GBrowse utilities as well as a wiki, blast search capabilities and a full text search engine. AphidBase strongly supported community cooperation and coordination in the curation of gene models during community annotation of the pea aphid genome. AphidBase can be accessed at http://www.aphidbase.com.

Transcriptomic and proteomic analyses of seasonal photoperiodism in the pea aphid.

BACKGROUND: Aphid adaptation to harsh winter conditions is illustrated by an alternation of their reproductive mode. Aphids detect photoperiod shortening by sensing the length of the night and switch from viviparous parthenogenesis in spring and summer, to oviparous sexual reproduction in autumn. The photoperiodic signal is transduced from the head to the reproductive tract to change the fate of the future oocytes from mitotic diploid embryogenesis to haploid formation of gametes. This process takes place in three consecutive generations due to viviparous parthenogenesis. To understand the molecular basis of the switch in the reproductive mode, transcriptomic and proteomic approaches were used to detect significantly regulated transcripts and polypeptides in the heads of the pea aphid Acyrthosiphon pisum. RESULTS: The transcriptomic profiles of the heads of the first generation were slightly affected by photoperiod shortening. This suggests that trans-generation signalling between the grand-mothers and the viviparous embryos they contain is not essential. By analogy, many of the genes and some of the proteins regulated in the heads of the second generation are implicated in visual functions, photoreception and cuticle structure. The modification of the cuticle could be accompanied by a down-regulation of the N-beta-alanyldopamine pathway and desclerotization. In Drosophila, modification of the insulin pathway could cause a decrease of juvenile hormones in short-day reared aphids. CONCLUSION: This work led to the construction of hypotheses for photoperiodic regulation of the switch of the reproductive mode in aphids.

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.

Sex versus parthenogenesis: a transcriptomic approach of photoperiod response in the model aphid Acyrthosiphon pisum (Hemiptera: Aphididae).

Most aphids develop a cyclic parthenogenesis life-cycle. After several generations of viviparous parthenogenetic females, it follows a single annual generation of sexual individuals, usually in autumn, that mate and lay the sexual eggs. Shortening of photoperiod at the end of the summer is a key factor inducing the sexual response. With the survey here reported we aimed at identifying a collection of candidate genes to participate at some point in the cascade of events that lead to the sexual phenotypes. Following a suppression subtractive hybridization methodology (SSH) on the model aphid Acyrthosiphon pisum, we built and characterised two reciprocal cDNA libraries (SDU and SDD) enriched respectively in genes up-regulated or down-regulated by short photoperiod conditions that lead to the sexual response in this aphid species. A total of 557 ESTs were obtained altogether representing 223 non-overlapping contigs. 29% of these were new sequences not present in previous aphid EST libraries. BLAST searches allowed putative identification of about 54% of the contigs present in both libraries. Relative quantification of expression through real-time quantitative PCR demonstrated the differential expression in relation with the photoperiod of 6 genes (3 up-regulated and 3 down-regulated by shortening the day length). Among these, expression of a tubulin gene, two cuticular proteins and a yet unidentified sequence along the day-night cycle was further investigated. Implications for current studies on gene regulation of the dichotomy sex vs. parthenogenesis in aphids are discussed.

Seasonal photoperiodism regulates the expression of cuticular and signalling protein genes in the pea aphid.

Seasonal photoperiodism in aphids is responsible for the spectacular switch from asexual to sexual reproduction. However, little is known on the molecular and physiological mechanisms involved in reproductive mode shift through the action of day length. Earlier works showed that aphid head, but not eyes, directly perceives the photoperiodic signal through the cuticle. In order to identify genes regulating the photoperiodic response, a 3321 cDNA microarray developed for the pea aphid, Acyrthosiphon pisum was used to compare RNA populations extracted from heads of short- and long-day reared aphids. Microarray analyses revealed that 59 different transcripts were significantly regulated, among which a majority encoded cuticular proteins and several encoded proteins involved in cellular signalling or signal transduction. These results were confirmed by quantitative RT-PCR experiments on two cuticular and three signalling protein genes. Complementary experiments eliminated moulting and circadian rhythms as putative confounding effects. Quantitative RT-PCR performed at additional developmental stages demonstrated the regulation of expression of cuticular and signalling protein genes during the whole process of photoperiod shortening. This suggests that photoperiodic changes could affect cuticle structure and cell to cell communication in the head of aphids in relation with the switch of reproductive modes.

T-DNA transfer from Agrobacterium tumefaciens to the ectomycorrhizal fungus Pisolithus microcarpus.

The model ectomycorrhizal fungus Pisolithus microcarpus isolate 441 was transformed by using Agrobacterium tumefaciens LBA1100 and AGL-1. The selection marker was the Shble gene of Streptoallotecius hidustanus, conferring resistance to phleomycin, under the control of the gpd gene promoter and terminator of Schizophyllum commune. Transformation resulted in phleomycin resistant clones which were confirmed by PCR to contain the resistance cassette. A. tumefaciens-mediated gene transfer would allow the development of RNA interference technology in P. microcarpus.

Expressed sequence tags of randomly selected cDNA clones from Eucalyptus globulus-Pisolithus tinctorius ectomycorrhiza.

Random sequencing of cDNA clones from Eucalyptus globulus-Pisolithus tinctorius ectomycorrhizal tissues was carried out to generate expressed sequence tags (ESTs). Database comparisons revealed that 42% of the cDNAs corresponded to previously sequenced genes. These ESTs represent efficient molecular markers to analyze changes in gene expression during the formation of the ectomycorrhizal symbiosis.

A novel class of ectomycorrhiza-regulated cell wall polypeptides in Pisolithus tinctorius.

Development of the ectomycorrhizal symbiosis leads to the aggregation of fungal hyphae to form the mantle. To identify cell surface proteins involved in this developmental step, changes in the biosynthesis of fungal cell wall proteins were examined in Eucalyptus globulus-Pisolithus tinctorius ectomycorrhizas by two-dimensional polyacrylamide gel electrophoresis. Enhanced synthesis of several immunologically related fungal 31- and 32-kDa polypeptides, so-called symbiosis-regulated acidic polypeptides (SRAPs), was observed. Peptide sequences of SRAP32d were obtained after trypsin digestion. These peptides were found in the predicted sequence of six closely related fungal cDNAs coding for ectomycorrhiza up-regulated transcripts. The PtSRAP32 cDNAs represented about 10% of the differentially expressed cDNAs in ectomycorrhiza and are predicted to encode alanine-rich proteins of 28.2 kDa. There are no sequence homologies between SRAPs and previously identified proteins, but they contain the Arg-Gly-Asp (RGD) motif found in cell-adhesion proteins. SRAPs were observed on the hyphal surface by immunoelectron microscopy. They were also found in the host cell wall when P. tinctorius attached to the root surface. RNA blot analysis showed that the steady-state level of PtSRAP32 transcripts exhibited a drastic up-regulation when fungal hyphae form the mantle. These results suggest that SRAPs may form part of a cell-cell adhesion system needed for aggregation of hyphae in ectomycorrhizas.

Cloning and and characterization of hydrophobins-encoding cDNAs from the ectomycorrhizal basdiomycete Pisolithus tinctorius.

Major alterations of fungal gene expression are induced by the development of ectomycorrhiza, a symbiosis between tree roots and filamentous fungi. Several cDNAs corresponding to highly expressed transcripts of the Basidomycete Pisolithus tinctorius (Pt) were isolated from symbiotic tissues. Two of these abundant transcripts (hydPt-1 and hydPt-2) encoded polypeptides belonging to the hydrophobin (Hyd) family, a group of small cysteine-rich fungal proteins involved im morphogenesis and plant-fungus interactions. As shown for other Hyd, the hydPt-1 and hydPt-2 mRNAs were barely detectable in mycelium grown in liquid culture and highly accumulated in aerial hyphae. In addition, these transcripts were also abundant in eucalyptus globulus-Pt ectomycorrhiza in early stages of differentiation, during the colonisation of roots.

The phosphoenolpyruvate carboxylase gene family of Sorghum: promoter structures, amino acid sequences and expression of genes.

Two different members of the phosphoenolpyruvate carboxylase(PEPC)-encoding multigene family (clones lambda CP21 and lambda CP46) have been isolated from a Sorghum vulgare lambda EMBL4 genomic library. The use of the 3'-noncoding regions to probe Northern blots of RNA from roots, etiolated leaves and green leaves indicated that lambda CP21 and lambda CP46 encode the C3- and C4-type leaf PEPC isoforms, respectively. The lambda CP21 clone is expressed in the three tissues and is not light-regulated, whereas lambda CP46 is only expressed in greening leaves. The nucleotide sequence of the 5'-flanking DNA (520 bp) has been determined for both genes. For lambda CP46, several direct repeats were located in this region with similarities to sequences found in other light-regulated genes, but not in lambda CP21. The deduced amino acid sequences of the two S. vulgare PEPC proteins are 75% identical.

Cloning symbiosis-related cDNAs from eucalypt ectomycorrhiza by PCR-assisted differential screening.

As part of a project to identify symbiosis-related genes, we report here a simple differential screening procedure for isolating up- and down-regulated fungal transcripts from a cDNA library of the developing Eucalyptus globulus-Pisolithus tinctorius mycorrhiza. cDNA inserts of randomly selected λZAP plaques were amplified by PCR and separated by agarose gel electrophoresis. The PCR-amplified cDNA samples were then screened by Southern blotting, using radiolabelled-cDNA probes of high specific activity. We have applied this method to fungal transcripts that are differentially expressed in ectomycorrhizas during the early stages of development. We estimate that about 50 % of the fungal mRNA population is regulated by development of the symbiosis; several up- and down-regulated cDNAs have been isolated for further analysis.

Immunolocalization of hydrophobin HYDPt-1 from the ectomycorrhizal basidiomycete Pisolithus tinctorius during colonization of Eucalyptus globulus roots.

• The immunolocalization of one of the hydrophobins of Pisolithustinctorius (HYDPt-1) is reported. Hydrophobin proteins play key roles in adhesion and aggregation of fungal hyphae, and it is already known that formation of ectomycorrhizas on eucalypt roots enhances the accumulation of hydrophobin mRNAs in the mycelium of Pisolithus tinctorius. • Purification of SDS-insoluble proteins from the mycelium of P. tinctorius showed the presence of a 13 kDa polypeptide with properties of class I hydrophobin. • Polyconal antibodies were raised against a recombinant HYDPt-1 polypeptide, and these were used for immunofluorescence-coupled transmission electron microscopy. • HYDPt-1 is a cell wall protein located at the surface of the hyphae with no preferential accumulation in the fungal cells of the different tissues of the ectomycorrhiza (i.e. extraradical hyphae, mantle or Hartig net).

Annotated expressed sequence tags for studies of the regulation of reproductive modes in aphids.

The damaging effect of aphids to crops is largely determined by the spectacular rate of increase of populational expansion due to their parthenogenetic generations. Despite this, the molecular processes triggering the transition between the parthenogenetic and sexual phases between their annual life cycle have received little attention. Here, we describe a collection of genes from the cereal aphid Rhopalosiphum padi expressed during the switch from parthenogenetic to sexual reproduction. After cDNA cloning and sequencing, 726 expressed sequence tags (EST) were annotated. The R. padi EST collection contained a substantial number (139) of bacterial endosymbiont sequences. The majority of R. padi cDNAs encoded either unknown proteins (56%) or housekeeping polypeptides (38%). The large proportion of sequences without similarities in the databases is related to both their small size and their high GC content, corresponding probably to the presence of 5'-unstranslated regions. Fifteen genes involved in developmental and differentiation events were identified by similarity to known genes. Some of these may be useful candidates for markers of the early steps of sexual differentiation.

Hydrophobins in ectomycorrhizas: heterologous transcription of the Pisolithus HydPt-1 gene in yeast and Hebeloma cylindrosporum.

Hydrophobins are fungal cell wall proteins involved in aggregation of hyphae. Upon the development of the ectomycorrhizal symbiosis between tree roots and fungal hyphae, the transcripts of hydrophobin genes markedly accumulated. As the precise role of these proteins in symbiosis is not yet known, we develop heterologous expression system of the Pisolithus hydrophobin HYDPt-1. This gene has been introduced in Saccharomyces cerevisiae and in the ectomycorrhizal basidiomycete Hebeloma cylindrosporum. Introns were required for hydPt-1 transcript accumulation in the basidiomycete H. cylindrosporum. Heterologous transcript accumulation did not alter the phenotype of either species. The lack of altered phenotype resulted from the absence of HYDPt-1 polypeptide accumulation in transformed strains.

Comparison of gene repertoires and patterns of evolutionary rates in eight aphid species that differ by reproductive mode.

In theory, the loss of sexual reproduction is expected to result in the accumulation of deleterious mutations. In aphids, two main types of life cycle, cyclic and obligate parthenogenesis, represent respectively "sexual" and "asexual" reproductive modes. We used the complete pea aphid genome and previously published expressed sequence tags (ESTs) from two other aphid species. In addition, we obtained 100,000 new ESTs from five more species. The final set comprised four sexual and four asexual aphid species and served to test the influence of the reproductive mode on the evolutionary rates of genes. We reconstructed coding sequences from ESTs and annotated these genes, discovering a novel peptide gene family that appears to be among the most highly expressed transcripts from several aphid species. From 203 genes found to be 1:1 orthologs among the eight species considered, we established a species tree that partly conflicted with taxonomy (for Myzus ascalonicus). We then used this topology to evaluate the dynamics of evolutionary rates and mutation accumulation in the four sexual and four asexual taxa. No significant increase of the nonsynonymous to synonymous ratio or of nonsynonymous mutation numbers was found in any of the four branches for asexual taxa. We however found a significant increase of the synonymous rate in the branch leading to the asexual species Rhopalosiphum maidis, which could be due to a change in the mutation rate or to an increased number of generations implied by its change of life cycle.