Immunity and other defenses in pea aphids, Acyrthosiphon pisum

Abstract

Background: Recent genomic analyses of arthropod defense mechanisms suggest conservation of key elements underlying responses to pathogens, parasites and stresses. At the center of pathogen-induced immune responses are signaling pathways triggered by the recognition of fungal, bacterial and viral signatures. These pathways result in the production of response molecules, such as antimicrobial peptides and lysozymes, which degrade or destroy invaders. Using the recently sequenced genome of the pea aphid (Acyrthosiphon pisum), we conducted the first extensive annotation of the immune and stress gene repertoire of a hemipterous insect, which is phylogenetically distantly related to previously characterized insects models.

Results: Strikingly, pea aphids appear to be missing genes present in insect genomes characterized to date and thought critical for recognition, signaling and killing of microbes. In line with results of gene annotation, experimental analyses designed to characterize immune response through the isolation of RNA transcripts and proteins from immune-challenged pea aphids uncovered few immune-related products. Gene expression studies, however, indicated some expression of immune and stress-related genes.

Conclusions: The absence of genes suspected to be essential for the insect immune response suggests that the traditional view of insect immunity may not be as broadly applicable as once thought. The limitations of the aphid immune system may be representative of a broad range of insects, or may be aphid specific. We suggest that several aspects of the aphid life style, such as their association with microbial symbionts, could facilitate survival without strong immune protection.

Figure 1

Some key insect recognition, signaling and response genes are missing in the pea aphid. Previously sequenced genomes of other insects (flies, mosquitoes, bees, beetles) have indicated that immune signaling pathways, seen here, are conserved across insects. In aphids, missing IMD pathway members (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 most genes central to the Toll, JNK and JAK/STAT signaling pathways.

Figure 2

Gene families implicated in arthropod immunity suggest unique features of the pea aphid immune system. Black indicates present (copy number is indicated, when known), white indicates absent, and gray indicates equivocal or unknown. Values for D. melanogaster, A. gambiae, T. castanateum, A. mellifera, and some D. pulex genes are based on published analyses [13,14,16,17,40]. For previously unannotated D. pulex genes, as well as for I. scapularis and P. humanus genes, we determined presence via cursory BLAST searches against available genome databases [127,128] (wfleabase.org, vectorbase.org) using both D. melanogaster and A. pisum protein sequences as queries. Gene presence for Ixodes was confirmed based on previous studies [129]. Future comprehensive annotation of the Pedicularis and Ixodes immune gene sets may reveal the presence of additional genes and lack of functionality of others. PPO, prophenoloxidase.

Figure 3

Evolutionarily conserved thaumatins are present in pea aphids and plants. (a) The three-dimensional structure of the pea aphid thaumatin ACYPI009605 (top) was calculated using the published crystallographic structure of a sweet cherry (plant) thaumatin 2AHN_A (bottom) [130] and Swissmodel [131], revealing that both thaumatins are similar in structure. However, one exposed loop, encircled by a dotted line, shows a significant difference in structure, suggesting possible adaptation to different targets. (b) Similarities are also revealed in the alignment of the pea aphid thaumatin with the plant thaumatin. A predicted signal sequence of the pea aphid thaumatin is underlined. Identical amino acids are highlighted in red. (c) Maximum likelihood phylogeny of thaumatins, indicating branches leading to nematode, plant, insect and bacteria-specific clades. Red highlights the sweet cherry thaumatin. Blue highlights the pea aphid thaumatins. Asterisks indicate approximate likelihood ratio test support >80. Abbreviations: Api, A. pisum; Cac, Catenulispora acidiphila; Cel, Caenorhabditis elegans; Mtr, Medicago truncatula; Pav, Prunus avium; Tca, Tribolium castaneum; Tpr, Trifolium pretense.

Figure 4

HPLC traces of inducible hemolymph peptides in the pea aphid compared to the rice weevil. Representative traces (solid, red lines) are from insects 18 hours after microbial challenge; traces generated from 18 hour control insects are overlaid (dashed, black lines). Phenylthiourea (PTU) served as an internal standard. Arrows indicate peaks that are significantly upregulated (solid, red arrows) or downregulated (dashed, black arrows). (a) Profile from pea aphids challenged with E. coli, showing no upregulated response. (b) Profile from pea aphids challenged with the fungus A. fumigatus, showing some differential peaks. (c) For comparison, profile from rice weevils (Sitophilus oryzae) challenged with E. coli, showing several differentials peaks at multiple retention times.