Genome sequence of the pea aphid Acyrthosiphon pisum

Abstract

Aphids are important agricultural pests and also biological models for studies of insect-plant interactions, symbiosis, virus vectoring, and the developmental causes of extreme phenotypic plasticity. Here we present the 464 Mb draft genome assembly of the pea aphid Acyrthosiphon pisum. This first published whole genome sequence of a basal hemimetabolous insect provides an outgroup to the multiple published genomes of holometabolous insects. Pea aphids are host-plant specialists, they can reproduce both sexually and asexually, and they have coevolved with an obligate bacterial symbiont. Here we highlight findings from whole genome analysis that may be related to these unusual biological features. These findings include discovery of extensive gene duplication in more than 2000 gene families as well as loss of evolutionarily conserved genes. Gene family expansions relative to other published genomes include genes involved in chromatin modification, miRNA synthesis, and sugar transport. Gene losses include genes central to the IMD immune pathway, selenoprotein utilization, purine salvage, and the entire urea cycle. The pea aphid genome reveals that only a limited number of genes have been acquired from bacteria; thus the reduced gene count of Buchnera does not reflect gene transfer to the host genome. The inventory of metabolic genes in the pea aphid genome suggests that there is extensive metabolite exchange between the aphid and Buchnera, including sharing of amino acid biosynthesis between the aphid and Buchnera. The pea aphid genome provides a foundation for post-genomic studies of fundamental biological questions and applied agricultural problems.

Figure 1. The pea aphid life cycle.

During the spring and summer months, asexual females give birth to live clonal offspring (see photo). These offspring undergo four molts during larval development to become (A) unwinged or (B) winged asexually reproducing adults. Winged individuals, capable of dispersing to new plants, are induced by crowding or stress during prenatal stages. After repeated cycles of asexual reproduction, shorter autumn day lengths trigger the production of (C) unwinged sexual females and (D) males, which can be winged or unwinged in pea aphids, depending on genotype. After mating, oviparous sexual females deposit (E) overwintering eggs, which hatch in the spring to produce (F) wingless, asexual females. In some populations, especially in locations without a cold winter, the sexual and egg-producing portions of the life cycle are eliminated, leading to continuous cycles of asexual reproduction (photo by N. Gerardo; illustration by N. Lowe).

Figure 2. Buchnera aphidicola and Regiella insecticola within a pea aphid embryo.

(A) Transmission electron micrograph showing elongate Regiella cells within a bacteriocyte (pink arrows) and nearby bacteriocytes containing Buchnera (green arrows). Black arrows indicate the bacteriome cell membrane (photo by J. White and N. Moran). Scales are in microns. (B) Position of symbiont-containing bacteriocytes within the abdomen as revealed by fluorescent in situ hybridization using diagnostic probes. Blue is a general DNA stain, highlighting aphid nuclei, red indicates Regiella, and green indicates Buchnera (photo by R. Koga).

Figure 3. Comparative genomics across the insects.

The phylogeny is based on maximum likelihood analyses of a concatenated alignment of 197 widespread, single-copy proteins. The tree was rooted using chordates as the most external out group. Bars represent a comparison of the gene content of all species included in the analysis (scale on the top). Bars are subdivided to indicate different types of homology relationships; black: widespread genes that are found with a one-to-one orthology in at least 16 of the 17 species; blue: widespread genes that can be found in at least 16 of the 17 species and are sometimes present in more than one copy; red: widespread but insect-specific genes present in at least 12 of the 13 insect species; yellow: non-widespread insect-specific genes (present in less than 12 insect species); green: genes present in insects and other groups but with a patchy distribution; white: species-specific genes with no (detectable) homologs in other species (striped fraction corresponds to species-specific genes present in more than one copy). The thin red line under each bar represents the percentage of A. pisum genes that have homologs in the given species (scale across the bottom of the figure). The fractions of single genes (grey) and duplicated genes (black) for some of the species are represented as pie charts.

Figure 4. Lineage-specific gene expansions in the pea aphid.

(A) Size distribution of the major lineage-specific groups of in-paralogs (i.e., paralogs resulting from duplications occurring after the split of the lineages leading to the pea aphid and the louse Pediculus humanus). The y-axis (logarithmic scale) represents the number of gene families with lineage-specific expansions of a given size (x-axis), as inferred from the pea aphid phylome. (B) Maximum likelihood phylogenetic tree showing lineage-specific expansion of a family coding for Acetyl-CoA transporter. This expansion has resulted in 19 paralogs in the pea aphid, whereas other insects and out groups included in the analysis possess only a single ortholog.

Figure 5. Widespread gene duplication in an ancestor of the pea aphid, as suggested by the frequency distribution of synonymous divergence (dS) between pairs of recent paralogs (Reciprocal Best Hits) within pea aphid, honey bee, and Drosophila.

Vertical dotted lines show the estimated average dS between orthologs from different aphid species. 1: A. pisum and Myzus persicae (two species of the tribe Macrosiphini), mean dS = 0.25; 2: A. pisum and Aphis gossypii (tribe Aphidini), mean dS = 0.35 (estimates from [128]). Paralogs resulting from ancient duplications (dS>1.5) are also abundant in all three genomes (1,449 pairs in aphid, 1,726 in drosophila, 1,010 in bee; not shown).

Figure 6. Transposable element copy identity distribution.

We show the mean identities of (A) TE copies in the pea aphid genome to their consensus reference sequence, (B) LTR super-families, and (C) TIR super-families. The consensus reference TE sequences contain the most frequent nucleotide at each base position and are thus approximations of the ancestral TE sequences, correcting for mutations affecting a small number of copies. Hence, the identity here is a proxy for TE family ages, with recent family having high identity (few differences with the ancestral state), and allows the ordering of transposable element invasions of the pea aphid genome. Note that the repeat order “Others” (Table 1) is not shown here, and the y-axis is a log scale that emphasizes recent families.

Figure 7. CpG ratios in the coding sequence of selected insects.

CpG ratios were calculated using RefSeq data for each insect species. For each sequence the observed (obs) CpG frequency and the expected (exp) CpG frequency were calculated. The expected CpG frequency was calculated based on the GC content of each sequence and the CpG ratio was calculated as obs/exp. The frequency of each CpG ratio was plotted against the observed/expected ratio. A bimodal distribution was observed for A. pisum and A. mellifera, both of which show DNA methylation within the coding sequence of genes ,. D. melanogaster and T. castaneum both show a unimodal distribution, and there is only limited evidence of methylation in both of these species. In addition A. pisum and A. mellifera have all the DNA methyltransferases while D. melanogaster only has Dnmt2 and T. castaneum has Dnmt1 and Dnmt2.

Figure 8. Expansion of the miRNA pathway in the pea aphid.

miRNA biogenesis is initiated in the nucleus by the Drosha-Pasha complex, resulting in precursors of around 60–70 nucleotides named pre-miRNAs. Pre-miRNAs are exported from the nucleus to the cytoplasm by Exportin-5. In the cytoplasm, Dicer-1 and its cofactor Loquacious (Loq) cleave these pre-miRNAs to produce mature miRNA duplexes. A duplex is then separated and one strand is selected as the mature miRNA whereas the other strand is degraded. This mature miRNA is integrated into the multiprotein RISC complex, which includes the key protein Argonaute 1 (Ago1). Integration of miRNAs into RISC will lead to the inhibition of targeted genes either by the degradation of the target mRNA or by the inhibition of its translation. All components of the miRNA pathway have been identified in the pea aphid. Shown are the number of homologs in A. pisum (Ap) as well as Drosophila melanogaster (Dm), Anopheles gambiae (Ag), Tribolium castaneum (Tc), and Apis mellifera (Am). While all these genes are monogenic in these insect species, the pea aphid possesses two copies of dicer-1, loquacious, and argonaute-1 and four copies of pasha (red font). The second loquacious copy is degraded and probably corresponds to a pseudogene.

Figure 9. Amino acid relations of the pea aphid Acyrthosiphon pisum and its symbiotic bacterium Buchnera aphidicola.

The schematic shows hypothetical relations based on the annotation of amino acid biosynthesis genes in the two organisms. Buchnera cells are located in the cytoplasm of specialized aphid cells, known as bacteriocytes. Each Buchnera cell is bound by three membranes, interpreted as the inner bacterial membrane (brown), outer bacterial membrane (green), and a membrane of insect origin known as the symbiosomal membrane (purple). The predicted biosynthesis (dark arrows) of essential amino acids (purple) and nonessential amino acids (green) and transport (light arrows) of metabolites between the partners are shown. The thickness of dark arrows indicates the number of metabolic reactions represented; thin arrows represent a single reaction and thick arrows more than one reaction. *The amino acid Gly appears twice in the Buchnera cell because it is synthesized by both Buchnera and the aphid (and possibly taken up by Buchnera). Metabolite abbreviations appear as follows: 2obut, 2-oxobutanoate; 3mob, 3-methyl-2-oxobutanoate; 3mop, (S)-3-methyl-2-oxopentanoate; 4mop, 4-methyl-2-oxopentanoate; e4p, D-erythrose 4-phosphate; hcys-L, homocysteine; pep, phosphoenolpyruvate; phpyr, phenylpyruvate; prpp, phosphoribosyl pyrophosphate; pyr, pyruvate.

Figure 10. The IMD immune pathway is missing in the pea aphid.

Previously sequenced insect genomes (fly, mosquitoes, honeybee, red flour beetle) have indicated that the immune signaling pathways, including IMD and Toll pathways shown here, are conserved across insects. In Drosophila, response to many Gram-negative bacteria and some Gram-positive bacteria and fungi relies on the IMD pathway. In aphids, missing IMD pathway genes (dashed lines) include those involved in recognition (PGRPs) and signaling (IMD, dFADD, Dredd, REL). Genes encoding antimicrobial peptides common in other insects, including defensins and cecropins, are also missing. In contrast, we found putative homologs for all genes central to the Toll signaling pathway, which is key to response to bacteria, fungi, and other microbes in Drosophila.

Figure 11. Kinases important in the regulation of mitosis have expanded in the pea aphid genome.

The cell division cycle typically consists of four phases: two growth phases (G1 and G2), a DNA synthesis or replication phase (S), and mitosis (M). Distinct and overlapping sets of regulatory genes are required for orderly progression through these phases. (A) Genes important for G1 and S phase progression are similar in number to other insects (orange box). G1/S Cyclin/Cyclin-dependent kinase (Cdk) protein complexes, along with E2F transcription factors, are critical for entry into G1 and progression into DNA replication and are opposed by cell cycle inhibitors such as p21/p27 family members and pRb/p107 family (Rbf) members, respectively. (B) Genes important for G2 and M phases have expanded in pea aphids (blue box). Polo kinases, Aurora kinases, Cdc25 phosphatases, and G2/M Cyclin/Cdk protein complexes are all critical for promoting entry into and progression through mitosis and meiosis. Negative regulators of Cdk1 and entry into mitosis include the Wee1/Myt1 kinase family. However, while Cdk1 has undergone aphid-specific duplication, no expansion of its activation subunits, Cyclins A and B, has been observed. Expanded gene families are in bold italics. Copy number was compared to that in Drosophila melanogaster, Tribolium castaneum, Pediculus humanas, Nasonia vitripennis, Culex quinquefasciatus, Anopheles gambiae, Aedes aegyptii, Bombyx mori, and Apis mellifera. ^aNo Myt1 orthologs were identified in the A. pisum genome. ^bAmong sequenced insects other than the pea aphid, Cdc25 is duplicated only in Drosophilids. ^cThree Aurora kinase orthologs are also present in Nasonia and Aedes while other insects possess two orthologs.

Figure 12. Orthologs of circadian clock genes, some significantly diverged, are found in the pea aphid genome.

Shown is a schematic representation of pea aphid orthologs of the circadian clock genes arranged in a two-loop model, as proposed for Drosophila ,. Genes constituting the core of the clockwork in Drosophila are in filled shapes; other genes relevant to the clock mechanism in Drosophila are in empty ovals. In Drosophila, the per/tim feedback loop is centered on the transcription factors PER and TIM encoded by the genes period (per) and timeless (tim). Kinase 2 (CK2) and Shaggy (SGG), the Protein phosphotase 2a (PP2A), and the degradation signaling proteins Supernumerary limbs (SLMB) and jetlag (JET) participate in this loop either by stabilizing or destabilizing PER and TIM. Light entrainment is mediated through the participation of Cryptochrome 1 (CRY1) and JET, which promote the degradation of TIM. Absence of JET in A. pisum is indicated by a dashed cross. The positive feedback loop in Drosophila is centered on the gene Clock (Clk), whose expression is regulated by the products of the genes vrille (VR1) and Pdp1 (PDP1). In addition to all these genes, the pea aphid genome contains two copies of a mammalian-type cryptochrome, CRY2, which is present in all other insects examined except Drosophila. CRY2 has been proposed to be part of the core mechanism , acting as a repressor of CLK/CYC (indicated by a question mark). Some pea aphid orthologs have diverged significantly compared with orthologs in other insects (dashed outlines). This is most dramatic for PER and TIM proteins (double dashed outlines), whose sequences differ significantly from those of other insects. Wavy lines indicate rhythmic transcription in Drosophila. Thick arrows and lines ending in bars indicate positive and negative regulation, respectively.