MTG-Link: leveraging barcode information from linked-reads to assemble specific loci.

BACKGROUND: Local assembly with short and long reads has proven to be very useful in many applications: reconstruction of the sequence of a locus of interest, gap-filling in draft assemblies, as well as alternative allele reconstruction of large Structural Variants. Whereas linked-read technologies have a great potential to assemble specific loci as they provide long-range information while maintaining the power and accuracy of short-read sequencing, there is a lack of local assembly tools for linked-read data. RESULTS: We present MTG-Link, a novel local assembly tool dedicated to linked-reads. The originality of the method lies in its read subsampling step which takes advantage of the barcode information contained in linked-reads mapped in flanking regions. We validated our approach on several datasets from different linked-read technologies. We show that MTG-Link is able to assemble successfully large sequences, up to dozens of Kb. We also demonstrate that the read subsampling step of MTG-Link considerably improves the local assembly of specific loci compared to other existing short-read local assembly tools. Furthermore, MTG-Link was able to fully characterize large insertion variants and deletion breakpoints in a human genome and to reconstruct dark regions in clinically-relevant human genes. It also improved the contiguity of a 1.3 Mb locus of biological interest in several individual genomes of the mimetic butterfly Heliconius numata. CONCLUSIONS: MTG-Link is an efficient local assembly tool designed for different linked-read sequencing technologies. MTG-Link source code is available at https://github.com/anne-gcd/MTG-Link and as a Bioconda package.

Selection following Gene Duplication Shapes Recent Genome Evolution in the Pea Aphid Acyrthosiphon pisum.

Ecology of insects is as wide as their diversity, which reflects their high capacity of adaptation in most of the environments of our planet. Aphids, with over 4,000 species, have developed a series of adaptations including a high phenotypic plasticity and the ability to feed on the phloem sap of plants, which is enriched in sugars derived from photosynthesis. Recent analyses of aphid genomes have indicated a high level of shared ancestral gene duplications that might represent a basis for genetic innovation and broad adaptations. In addition, there are a large number of recent, species-specific gene duplications whose role in adaptation remains poorly understood. Here, we tested whether duplicates specific to the pea aphid Acyrthosiphon pisum are related to genomic innovation by combining comparative genomics, transcriptomics, and chromatin accessibility analyses. Consistent with large levels of neofunctionalization, we found that most of the recent pairs of gene duplicates evolved asymmetrically, showing divergent patterns of positive selection and gene expression. Genes under selection involved a plethora of biological functions, suggesting that neofunctionalization and tissue specificity, among other evolutionary mechanisms, have orchestrated the evolution of recent paralogs in the pea aphid and may have facilitated host-symbiont cooperation. Our comprehensive phylogenomics analysis allowed us to tackle the history of duplicated genes to pave the road toward understanding the role of gene duplication in ecological adaptation.

Genomic architecture of endogenous ichnoviruses reveals distinct evolutionary pathways leading to virus domestication in parasitic wasps.

BACKGROUND: Polydnaviruses (PDVs) are mutualistic endogenous viruses inoculated by some lineages of parasitoid wasps into their hosts, where they facilitate successful wasp development. PDVs include the ichnoviruses and bracoviruses that originate from independent viral acquisitions in ichneumonid and braconid wasps respectively. PDV genomes are fully incorporated into the wasp genomes and consist of (1) genes involved in viral particle production, which derive from the viral ancestor and are not encapsidated, and (2) proviral segments harboring virulence genes, which are packaged into the viral particle. To help elucidating the mechanisms that have facilitated viral domestication in ichneumonid wasps, we analyzed the structure of the viral insertions by sequencing the whole genome of two ichnovirus-carrying wasp species, Hyposoter didymator and Campoletis sonorensis. RESULTS: Assemblies with long scaffold sizes allowed us to unravel the organization of the endogenous ichnovirus and revealed considerable dispersion of the viral loci within the wasp genomes. Proviral segments contained species-specific sets of genes and occupied distinct genomic locations in the two ichneumonid wasps. In contrast, viral machinery genes were organized in clusters showing highly conserved gene content and order, with some loci located in collinear wasp genomic regions. This genomic architecture clearly differs from the organization of PDVs in braconid wasps, in which proviral segments are clustered and viral machinery elements are more dispersed. CONCLUSIONS: The contrasting structures of the two types of ichnovirus genomic elements are consistent with their different functions: proviral segments are vehicles for virulence proteins expected to adapt according to different host defense systems, whereas the genes involved in virus particle production in the wasp are likely more stable and may reflect ancestral viral architecture. The distinct genomic architectures seen in ichnoviruses versus bracoviruses reveal different evolutionary trajectories that have led to virus domestication in the two wasp lineages.

Functional insights from the GC-poor genomes of two aphid parasitoids, Aphidius ervi and Lysiphlebus fabarum.

BACKGROUND: Parasitoid wasps have fascinating life cycles and play an important role in trophic networks, yet little is known about their genome content and function. Parasitoids that infect aphids are an important group with the potential for biological control. Their success depends on adapting to develop inside aphids and overcoming both host aphid defenses and their protective endosymbionts. RESULTS: We present the de novo genome assemblies, detailed annotation, and comparative analysis of two closely related parasitoid wasps that target pest aphids: Aphidius ervi and Lysiphlebus fabarum (Hymenoptera: Braconidae: Aphidiinae). The genomes are small (139 and 141 Mbp) and the most AT-rich reported thus far for any arthropod (GC content: 25.8 and 23.8%). This nucleotide bias is accompanied by skewed codon usage and is stronger in genes with adult-biased expression. AT-richness may be the consequence of reduced genome size, a near absence of DNA methylation, and energy efficiency. We identify missing desaturase genes, whose absence may underlie mimicry in the cuticular hydrocarbon profile of L. fabarum. We highlight key gene groups including those underlying venom composition, chemosensory perception, and sex determination, as well as potential losses in immune pathway genes. CONCLUSIONS: These findings are of fundamental interest for insect evolution and biological control applications. They provide a strong foundation for further functional studies into coevolution between parasitoids and their hosts. Both genomes are available at https://bipaa.genouest.org.

Genetic control of contagious asexuality in the pea aphid.

Although evolutionary transitions from sexual to asexual reproduction are frequent in eukaryotes, the genetic bases of such shifts toward asexuality remain largely unknown. We addressed this issue in an aphid species where both sexual and obligate asexual lineages coexist in natural populations. These sexual and asexual lineages may occasionally interbreed because some asexual lineages maintain a residual production of males potentially able to mate with the females produced by sexual lineages. Hence, this species is an ideal model to study the genetic basis of the loss of sexual reproduction with quantitative genetic and population genomic approaches. Our analysis of the co-segregation of ∼ 300 molecular markers and reproductive phenotype in experimental crosses pinpointed an X-linked region controlling obligate asexuality, this state of character being recessive. A population genetic analysis (>400-marker genome scan) on wild sexual and asexual genotypes from geographically distant populations under divergent selection for reproductive strategies detected a strong signature of divergent selection in the genomic region identified by the experimental crosses. These population genetic data confirm the implication of the candidate region in the control of reproductive mode in wild populations originating from 700 km apart. Patterns of genetic differentiation along chromosomes suggest bidirectional gene flow between populations with distinct reproductive modes, supporting contagious asexuality as a prevailing route to permanent parthenogenesis in pea aphids. This genetic system provides new insights into the mechanisms of coexistence of sexual and asexual aphid lineages.

De novo transcriptome assembly of the grapevine phylloxera allows identification of genes differentially expressed between leaf- and root-feeding forms.

BACKGROUND: Grapevine phylloxera, an insect related to true aphids, is a major historic pest of viticulture only controlled through the selection of resistant rootstocks or through quarantine regulations where grapevine is cultivated own-rooted. Transcriptomic data could help understand the bases of its original life-traits, including a striking case of polyphenism, with forms feeding on roots and forms feeding in leaf-galls. Comparisons with true aphids (for which complete genomes have been sequenced) should also allow to link differences in life-traits of the two groups with changes in gene repertoires or shifts in patterns of expression. RESULTS: We sequenced transcriptomes of the grapevine phylloxera (Illumina technology), choosing three life-stages (adults on roots or on leaf galls, and eggs) to cover a large catalogue of transcripts, and performed a de novo assembly. This resulted in 105,697 contigs, which were annotated: most contigs had a best blastx hit to the pea aphid (phylogenetically closest complete genome), while very few bacterial hits were recorded (except for Probionibacterium acnes). Coding sequences were predicted from this data set (17,372 sequences), revealing an extremely high AT-bias (at the third codon position). Differential expression (DE) analysis among root-feeding and gall-feeding showed that i) the root-feeding form displayed a much larger number of differentially expressed transcripts ii) root-feeding biased genes were enriched in some categories, for example cuticular proteins and genes associated with cell-cell signaling iii) leaf-galling-biased genes were enriched in genes associated with the nucleus and DNA-replication, suggesting a metabolism more oriented towards fast and active multiplication. We also identified a gene family with a very high expression level (copies totaling nearly 10% of the reads) in the grapevine phylloxera (both in root and leaf galling forms), but usually expressed at very low levels in true aphids (except in sexual oviparous females). These transcripts thus appear to be associated with oviparity. CONCLUSIONS: Our study illustrated major intraspecific changes in transcriptome profiles, related with different life-styles (and the feeding on roots versus in leaf-galls). At a different scale, we could also illustrate one major shift in expression levels associated with changes in life-traits that occurred along evolution and that respectively characterize (strictly oviparous) grapevine phylloxera and (mostly viviparous) true aphids.

Masculinization of the x chromosome in the pea aphid.

Evolutionary theory predicts that sexually antagonistic mutations accumulate differentially on the X chromosome and autosomes in species with an XY sex-determination system, with effects (masculinization or feminization of the X) depending on the dominance of mutations. Organisms with alternative modes of inheritance of sex chromosomes offer interesting opportunities for studying sexual conflicts and their resolution, because expectations for the preferred genomic location of sexually antagonistic alleles may differ from standard systems. Aphids display an XX/X0 system and combine an unusual inheritance of the X chromosome with the alternation of sexual and asexual reproduction. In this study, we first investigated theoretically the accumulation of sexually antagonistic mutations on the aphid X chromosome. Our results show that i) the X is always more favourable to the spread of male-beneficial alleles than autosomes, and should thus be enriched in sexually antagonistic alleles beneficial for males, ii) sexually antagonistic mutations beneficial for asexual females accumulate preferentially on autosomes, iii) in contrast to predictions for standard systems, these qualitative results are not affected by the dominance of mutations. Under the assumption that sex-biased gene expression evolves to solve conflicts raised by the spread of sexually antagonistic alleles, one expects that male-biased genes should be enriched on the X while asexual female-biased genes should be enriched on autosomes. Using gene expression data (RNA-Seq) in males, sexual females and asexual females of the pea aphid, we confirm these theoretical predictions. Although other mechanisms than the resolution of sexual antagonism may lead to sex-biased gene expression, we argue that they could hardly explain the observed difference between X and autosomes. On top of reporting a strong masculinization of the aphid X chromosome, our study highlights the relevance of organisms displaying an alternative mode of sex chromosome inheritance to understanding the forces shaping chromosome evolution.

Genomic data integration for ecological and evolutionary traits in non-model organisms.

Why is it needed to develop system biology initiatives such as ENCODE on non-model organisms?

Contrasting Evolutionary Patterns Between Sexual and Asexual Lineages in a Genomic Region Linked to Reproductive Mode Variation in the pea aphid.

Although asexual lineages evolved from sexual lineages in many different taxa, the genetics of sex loss remains poorly understood. We addressed this issue in the pea aphid Acyrthosiphon pisum, whose natural populations encompass lineages performing cyclical parthenogenesis (CP) and producing one sexual generation per year, as well as obligate parthenogenetic (OP) lineages that can no longer produce sexual females but can still produce males. An SNP-based, whole-genome scan of CP and OP populations sequenced in pools (103 individuals from 6 populations) revealed that an X-linked region is associated with the variation in reproductive mode. This 840-kb region is highly divergent between CP and OP populations (FST = 34.9%), with >2,000 SNPs or short Indels showing a high degree of association with the phenotypic trait. In OP populations specifically, this region also shows reduced diversity and Tajima's D, consistent with the OP phenotype being a derived trait in aphids. Interestingly, the low genetic differentiation between CP and OP populations at the rest of the genome (FST = 2.5%) suggests gene flow between them. Males from OP lineages thus likely transmit their op allele to new genomic backgrounds. These genetic exchanges, combined with the selection of the OP and CP reproductive modes under different climates, probably contribute to the long-term persistence of the cp and op alleles.

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.

Sexual and asexual oogenesis require the expression of unique and shared sets of genes in the insect Acyrthosiphon pisum.

BACKGROUND: Although sexual reproduction is dominant within eukaryotes, asexual reproduction is widespread and has evolved independently as a derived trait in almost all major taxa. How asexuality evolved in sexual organisms is unclear. Aphids, such as Acyrthosiphon pisum, alternate between asexual and sexual reproductive means, as the production of parthenogenetic viviparous females or sexual oviparous females and males varies in response to seasonal photoperiodism. Consequently, sexual and asexual development in aphids can be analyzed simultaneously in genetically identical individuals. RESULTS: We compared the transcriptomes of aphid embryos in the stages of development during which the trajectory of oogenesis is determined for producing sexual or asexual gametes. This study design aimed at identifying genes involved in the onset of the divergent mechanisms that result in the sexual or asexual phenotype. We detected 33 genes that were differentially transcribed in sexual and asexual embryos. Functional annotation by gene ontology (GO) showed a biological signature of oogenesis, cell cycle regulation, epigenetic regulation and RNA maturation. In situ hybridizations demonstrated that 16 of the differentially-transcribed genes were specifically expressed in germ cells and/or oocytes of asexual and/or sexual ovaries, and therefore may contribute to aphid oogenesis. We categorized these 16 genes by their transcription patterns in the two types of ovaries; they were: i) expressed during sexual and asexual oogenesis; ii) expressed during sexual and asexual oogenesis but with different localizations; or iii) expressed only during sexual or asexual oogenesis. CONCLUSIONS: Our results show that asexual and sexual oogenesis in aphids share common genetic programs but diverge by adapting specificities in their respective gene expression profiles in germ cells and oocytes.

Predicted effector molecules in the salivary secretome of the pea aphid (Acyrthosiphon pisum): a dual transcriptomic/proteomic approach.

The relationship between aphids and their host plants is thought to be functionally analogous to plant-pathogen interactions. Although virulence effector proteins that mediate plant defenses are well-characterized for pathogens such as bacteria, oomycetes, and nematodes, equivalent molecules in aphids and other phloem-feeders are poorly understood. A dual transcriptomic-proteomic approach was adopted to generate a catalog of candidate effector proteins from the salivary glands of the pea aphid, Acyrthosiphon pisum. Of the 1557 transcript supported and 925 mass spectrometry identified proteins, over 300 proteins were identified with secretion signals, including proteins that had previously been identified directly from the secreted saliva. Almost half of the identified proteins have no homologue outside aphids and are of unknown function. Many of the genes encoding the putative effector proteins appear to be evolving at a faster rate than homologues in other insects, and there is strong evidence that genes with multiple copies in the genome are under positive selection. Many of the candidate aphid effector proteins were previously characterized in typical phytopathogenic organisms (e.g., nematodes and fungi) and our results highlight remarkable similarities in the saliva from plant-feeding nematodes and aphids that may indicate the evolution of common solutions to the plant-parasitic lifestyle.

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.

Revisiting the anatomy of the central nervous system of a hemimetabolous model insect species: the pea aphid Acyrthosiphon pisum.

Aphids show a marked phenotypic plasticity, producing asexual or sexual and winged or wingless morphs depending on environmental conditions and season. We describe here the general structure of the brain of various morphs of the pea aphid Acyrthosiphon pisum. This is the first detailed anatomical study of the central nervous system of an aphid by immunocytochemistry (synapsin, serotonin, and several neuropeptides), ethyl-gallate staining, confocal laser scanning microscopy, and three-dimensional reconstructions. The study has revealed well-developed optic lobes composed of lamina, medulla, and lobula complex. Ocelli are only present in males and winged parthenogenetic females. The central complex is well-defined, with a central body divided into two parts, a protocerebral bridge, and affiliated lateral accessory lobes. The mushroom bodies are ill-defined, lacking calyces, and only being visualized by using an antiserum against the neuropeptide orcokinin. The antennal lobes contain poorly delineated glomeruli but can be clearly visualized by performing antennal backfills. On the basis of our detailed description of the brain of winged and wingless parthenogenetic A. pisum females, an anatomical map is now available that should improve our knowledge of the way that these structures are involved in the regulation of phenotypic plasticity.

Genomics of environmentally induced phenotypes in 2 extremely plastic arthropods.

Understanding how genes and the environment interact to shape phenotypes is of fundamental importance for resolving important issues in adaptive evolution. Yet, for most model species with mature genetics and accessible genomic resources, we know little about the natural environmental factors that shape their evolution. By contrast, animal species with deeply understood ecologies and well characterized responses to environmental cues are rarely subjects of genomic investigations. Here, we preview advances in genomics in aphids and waterfleas that may help transform research on the regulatory mechanisms of phenotypic plasticity. This insect and crustacean duo has the capacity to produce extremely divergent phenotypes in response to environmental stimuli. Sexual fate and reproductive mode are condition-dependent in both groups, which are also capable of altering morphology, physiology and behavior in response to biotic and abiotic cues. Recently, the genome sequences for the pea aphid Acyrthosiphon pisum and the waterflea Daphnia pulex were described by their respective research communities. We propose that an integrative study of genome biology focused on the condition-dependent transcriptional basis of their shared plastic traits and specialized mode of reproduction will provide broad insight into adaptive plasticity and genome by environment interactions. We highlight recent advances in understanding the genome regulation of alternative phenotypes and environmental cue processing, and we propose future research avenues to discover gene networks and epigenetic mechanisms underlying phenotypic plasticity.

Bioinformatic prediction, deep sequencing of microRNAs and expression analysis during phenotypic plasticity in the pea aphid, Acyrthosiphon pisum.

BACKGROUND: Post-transcriptional regulation in eukaryotes can be operated through microRNA (miRNAs) mediated gene silencing. MiRNAs are small (18-25 nucleotides) non-coding RNAs that play crucial role in regulation of gene expression in eukaryotes. In insects, miRNAs have been shown to be involved in multiple mechanisms such as embryonic development, tissue differentiation, metamorphosis or circadian rhythm. Insect miRNAs have been identified in different species belonging to five orders: Coleoptera, Diptera, Hymenoptera, Lepidoptera and Orthoptera. RESULTS: We developed high throughput Solexa sequencing and bioinformatic analyses of the genome of the pea aphid Acyrthosiphon pisum in order to identify the first miRNAs from a hemipteran insect. By combining these methods we identified 149 miRNAs including 55 conserved and 94 new miRNAs. Moreover, we investigated the regulation of these miRNAs in different alternative morphs of the pea aphid by analysing the expression of miRNAs across the switch of reproduction mode. Pea aphid microRNA sequences have been posted to miRBase: http://microrna.sanger.ac.uk/sequences/. CONCLUSIONS: Our study has identified candidates as putative regulators involved in reproductive polyphenism in aphids and opens new avenues for further functional analyses.

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.

pH effect on strain-specific transcriptomes of the take-all fungus.

The soilborne fungus Gaeumannomyces tritici (G. tritici) causes the take-all disease on wheat roots. Ambient pH has been shown to be critical in different steps of G. tritici life cycle such as survival in bulk soil, saprophytic growth, and pathogenicity on plants. There are however intra-specific variations and we previously found two types of G. tritici strains that grow preferentially either at acidic pH or at neutral/alkaline pH; gene expression involved in pH-signal transduction pathway and pathogenesis was differentially regulated in two strains representative of these types. To go deeper in the description of the genetic pathways and the understanding of this adaptative mechanism, transcriptome sequencing was achieved on two strains (PG6 and PG38) which displayed opposite growth profiles in two pH conditions (acidic and neutral). PG6, growing better at acidic pH, overexpressed in this condition genes related to cell proliferation. In contrast, PG38, which grew better at neutral pH, overexpressed in this condition genes involved in fatty acids and amino acid metabolisms, and genes potentially related to pathogenesis. This strain also expressed stress resistance mechanisms at both pH, to assert a convenient growth under various ambient pH conditions. These differences in metabolic pathway expression between strains at different pH might buffer the effect of field or soil variation in wheat fields, and explain the success of the pathogen.

Large gene family expansion and variable selective pressures for cathepsin B in aphids.

Aphids exclusively feed on plant phloem sap that contains much sugar and some nonessential amino acids but is poor in lipids and proteins. Conventionally, it has been believed that aphids substantially have no intestinal digestion of proteins. However, we here report an unexpected finding that cysteine protease genes of the family cathepsin B are massively amplified in the lineage of aphids and that many of the protease genes exhibit gut-specific overexpression. By making use of expressed sequence tag data, sequenced cDNAs, and genomic trace sequences of the pea aphid Acyrthosiphon pisum, we identified a total of 28 cathepsin B-like gene copies in the genome of A. pisum. Phylogenetic analyses of all the cathepsin B genes in aphids revealed that genic expansion has continuously proceeded with basal, intermediary, and recent duplications. Estimation of molecular evolutionary rates indicated that major alterations of the rates often occurred after duplications. For example, a gene copy ("348") was shown to be slow evolving and close to genes of other insects like Drosophila melanogaster, whereas the other gene copies appeared to have evolved faster with higher ratios of nonsynonymous to synonymous substitutions. We identified a number of gene copies (16 in A. pisum) that contained a replacement at the site required for catalytic activity of the protease. Among these, 2 copies were pseudogenes, whereas the remaining copies were structurally intact and possibly acquired new functions. For example, a cluster of such gene copies ("1674") has been subjected to positive selection. Quantitative reverse transcriptase-polymerase chain reaction analyses revealed that the more conserved gene copy ("348") showed a constitutive expression, whereas 5 other forms ("84," "16," "16D," "1874," and "2744") were preferentially expressed in the gut of A. pisum. Putative biological roles of the diversified cathepsin B-like gene copies in aphids are discussed in relation to their nutritional physiology specialized for plant sap feeding lifestyle.