# Immunity and other defenses in pea aphids, Acyrthosiphon pisum.

Nicole M Gerardo, Boran Altincicek, Caroline Anselme, Hagop Atamian, Seth

M Barribeau, Martin de Vos, Elizabeth Duncan, Jay D Evans, Toni

Gabaldon, Murad Ghanim, et al.

► To cite this version:

Nicole M Gerardo, Boran Altincicek, Caroline Anselme, Hagop Atamian, Seth M Barribeau, et al. "Immunity and other defenses in pea aphids, Acyrthosiphon pisum." Genome Biology, 2010, 11 (2), pp.R21. 10.1186/gb-2010-11-2-r21. [DOI: 10.1186/gb-2010-11-2-r21](https://doi.org/10.1186/gb-2010-11-2-r21). [HAL Id:](https://hal.science/hal-00459961v1)

Submitted on 25 Feb 2010

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<figure><img src="image_1.png" /></figure>
**Immunity and other defenses in pea aphids,**

*Acyrthosiphon pisum*

N.M. Gerardo<sup>1,§</sup>, B. Altincicek<sup>2</sup>, C. Anselme<sup>3,4</sup>, H. Atamian<sup>5</sup>, S.M. Barribeau<sup>1</sup>, M. de Vos<sup>6</sup>, E.J. Duncan<sup>7</sup>, J.D. Evans<sup>8</sup>, T. Gabaldón<sup>9</sup>, M. Ghanim<sup>10</sup>, A. Heddi<sup>3</sup>, I.

Kaloshian<sup>5</sup>, A. Latorre<sup>11</sup>, A. Moya<sup>11</sup>, A. Nakabachi<sup>12</sup>, B.J. Parker<sup>1</sup>, V. Pérez-Brocal<sup>3,11</sup>, M. Pignatelli<sup>11</sup>, Y.Rahbé<sup>3</sup>, J. S. Ramsey<sup>6</sup>, C. Spragg<sup>1</sup>, J., Tamames<sup>11</sup>, D. Tamarit<sup>11</sup>, C. Tamborindeguy<sup>13,14</sup>, C. Vincent-Monegat<sup>3</sup>, A. Vilcinskas<sup>2</sup>

<sup>1</sup>Department of Biology, Emory University, O. Wayne Rollins Research Center, 1510 E. Clifton Rd. N.E., Atlanta, GA, 30322, USA.

<sup>2</sup>Interdisciplinary Research Center, Institute of Phytopathology and Applied Zoology, Justus-Liebig-University of Giessen, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany.

<sup>3</sup>Université de Lyon, INRA, INSA-Lyon, IFR41 BioEnvironnement et Santé, UMR203 BF2I, Biologie Fonctionnelle Insectes et Interactions, Bat. Louis-Pasteur 20 ave. Albert-Einstein, F-69621 Villeurbanne, France.

<sup>4</sup>UMR Interactions Biotiques et Santé Végétale, INRA 1301-CNRS 6243-Université de Nice-Sophia Antipolis, 400 routes des Chappe, F-06903 Sophia-Antipolis cedex, France.
⁵Department of Nematology, Graduate Program in Genetics, Genomics and Bioinformatics, University of California, 900 University Ave., Riverside, CA, 92521, USA.

⁶Boyce Thompson Institute for Plant Research, Ithaca, New York, 14853, USA.

⁷Genetics Otago and The Laboratory for Evolution and Development, Department of Biochemistry, University of Otago, Box 56, Dunedin 9054, New Zealand

⁸USDA-ARS Bee Research Lab, BARC-East Bldg 476 Beltsville, MD 20705 USA.

⁹Bioinformatics and Genomics Programme. Centre for Genomic Regulation (CRG). Doctor Aiguader, 88. 08003 Barcelona, Spain.

¹⁰Department of Entomology, The Volcani Center, Bet Dagan 50250, Israel,

¹¹Instituto Cavanilles de Biodiversidad y Biología Evolutiva, Universitat de València, Avenida Blasco Ibañez 13, 46071 València (Spain), CIBER en Epidemiología y Salud Pública (CIBEResp) and Centro Superior de Investigación en Salud Pública (CSISP), Conselleria de Sanidad (Generalitat Valenciana), Avenida de Cataluña, 21. 46020 València (Spain)

¹²Advanced Science Institute. RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
¹³Plant Pathology and Plant-Microbe Biology Department, Cornell University, Tower Road, Ithaca, NY, 14853, USA.

¹⁴Department of Entomology, Texas A&M, College Station, TX 77843-2475, USA.

§Corresponding author

Email addresses:

NMG: nicole.gerardo@ memory.edu

BA: boran.altincicek@ agrar.uni-giessen.de

CA: caroline.anselme@sophia.inra.fr

HA: hagop.atamian@ ucr.edu

SB: seth.barribeau@gmail.com

MD: md285@ cornell.edu

EJD: elizabeth.duncan@ otago.ac.nz

JE: jayEvans@ars.usda.gov

TG: tgabaldon@ crg.es

MG: ghanim@agri.gov.il

AH: abdelaziz.heddi@ insa-lyon.fr

IK: isgouhi.kaloshian@ ucr.edu

AL: amparo.latorre@ uv.es

AM: andres.moya@ uv.es

AN: bachi@riken.jp

BJP: bparke4@ memory.edu

VP: vicente.perez-brocal@ uv.es
MP: miguel.pignatelli@uv.es

YR: yvan.rahbe@jouy.inra.fr

JSR: jsr47@cornell.edu

CS: cspragg@emory.edu

JT: javier.tamames@uv.es

DT: datachu@alumni.uv.es

CT: ctamborindeguy@ag.tamu.edu

CVM: carole.monegat@insa-lyon.fr

AV: Andreas.Vilcinskas@agrar.uni-giessen.de
# 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 response 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.

## Background

Aphids face numerous environmental challenges, including infection by diverse pathogens and parasites. These pressures include parasitoid wasps, which consume their hosts as they develop inside, and a variety of viral, bacterial and fungal pathogens. Both parasitoid wasp and fungal pathogens cause significant decline of natural aphid populations [1, 2], and have been suggested as potential agents for biocontrol of these agriculturally destructive pests. While facing such challenges, aphids also cope with predators and abiotic stresses, such as extreme temperature fluctuations. Thus, like most insects, aphids must attempt to survive in a harsh, complex environment.

Insects have a number of defense mechanisms. First, many insects, including aphids, behaviorally avoid predators, pathogens, and environmental stressors [3-6]. When stressors cannot be avoided, insects have a protective cuticle and gut pH inhospitable to many foreign organisms. If these barriers fail, immunological defense mechanisms recognize the invader, triggering a signaling cascade and response. While insects do not have adaptive, antigen-based responses typical of vertebrates, insects do have innate immune responses, which include clotting, phagocytosis, encapsulation, and production of antimicrobial substances [7, 8]. Phagocytosis and encapsulation, are referred to as cellular responses as they are mediated by blood cells [9]. Responses vary depending on the invader, with antimicrobial peptides being
central to combating microbes and encapsulation being central to combating larger
invaders, such as parasitoids. Until recently, it was presumed that insects were limited
to these non-specific innate immune responses and had no specific immunity (e.g., the
antigen based immune response of humans). There is, however increasing evidence
for the ability of insects to mount specific immune responses [10].

Here we focus on the identification of aphid genes that are known to play a role in
the recognition and degradation of microbial pathogens in other insects, as these are
the invertebrate defense processes that are best understood. In the fruit fly *Drosophila melanogaster*, recognition of an invasive microbe leads to signal production via four
pathways (Toll, IMD, JNK, and JAK/STAT) [11]. Each pathway is activated in
response to particular pathogens [12]. Signaling triggers the production of a multitude
of effectors, including, most notably, antimicrobial peptides (AMPs). Insect AMPs
may be 1000-fold induced in microbe-challenged insects compared to basal levels. In
insect genomes annotated to date, these pathways appear well conserved, with most of
the key components found across flies (*Drosophila* spp.), mosquitoes (*Aedes aegypti*,
*Anopheles gambiae*), bees (*Apis mellifera*) and beetles (*Tribolium castaneum*) [13-17].

Because aphids and other insects face diverse challenges, we propose models for
several genes critical to other elements of insect stress responses. These include genes
encoding heat shock proteins (HSPs), which are synthesized in almost all living
organisms when exposed to high temperatures or stress [18]. We also suggest models
for genes involved in the synthesis of the alarm pheromone (E)-β farnesene (EBF),
which aphids release in the presence of predators [19]. While there are undoubtedly
many other genes involved in stress and immunological responses, our selection of
genes for exploration provides a broad survey of the known insect immune and stress
repertoire and will serve as a basis for future exploration of more specific responses.

The pea aphid genome provides novel insights into arthropod immunity for two reasons. First, most of our understanding of insect immune and stress responses comes from holometabolous insects, the group of insects with complete metamorphosis, such as flies, butterflies, beetles and bees. The genome of the hemimetabolous pea aphid, *Acyrthosiphon pisum*, may thus provide novel insight into immunity and defense in more basal, non-holometabolous insects, which have incomplete metamorphosis. Second, aphids are unique amongst the arthropods sequenced to date in that they are intimately dependent on both obligate and facultative bacterial symbionts for their survival. The aphid symbiont community includes *Buchnera aphidicola*, obligate and intracellular Gram-negative bacteria that have the ability to synthesize required amino acids not readily available in the aphid diet. Beyond this obligate symbiosis, aphids frequently host one of more other Gram-negative bacterial symbionts, including most notably *Hamiltonella defensa*, *Serratia symbiotica* and *Regiella insecticola* [20, 21]. Unlike *Buchnera*, which is present in all aphids and is thus considered a *primary* symbiont, these bacteria are considered to be facultative, *secondary* symbionts, because their presence varies within an aphid species [22]. Secondary symbiotic bacteria have been shown to influence several aspects of aphid ecology, including heat tolerance and resistance to parasites and pathogens [23-26]. Specifically, both *H. defensa* and *S. symbiotica* confer protection against parasitoid wasp development [27, 28], and *R. insecticola* decreases *A. pisum* mortality after exposure to the fungal pathogen *Pandora neoaphidis* [29]. These are some of the best-studied examples of symbiont-conferred protection [30].
Aphids thus provide an excellent opportunity to study the immune system of an
organism that is dependent on microbial symbionts but is hampered by parasites and
pathogens. Despite this, little work has been done to characterize the aphid immune
response. Altincicek et al. (2008a) found that compared to other insects, stabbing a
pea aphid with bacteria elicits reduced lysozyme-like (muramidase) activity, and no
detectable activity against live bacteria in hemolymph assays. Furthermore,
suppression subtraction hybridization (SSH) of bacterial-challenged aphids uncovered
no antimicrobial peptides and few genes of known immune function [31]. These
results are surprising given that similar studies in other insects demonstrate that
antimicrobial peptide production and upregulation of immune-related genes is a
common feature of the insect immune response that can be captured in functional
assays such as SSH [32-35]. This suggests that aphids have a significantly reduced or
altered immune repertoire.

Using the recently sequenced genome of the pea aphid clone LSR1, in this study,
we take two approaches to study immunity and stress in pea aphids. First, we assay
presence/absence of a subset of known immune and stress-related genes. Second, we
combine functional assays targeting the production of RNA and proteins to gain
insight into how pea aphids respond to various challenges. Overall, our results suggest
that pea aphids are missing many genes central to immune function in other insects,
and that, although pea aphids do mount some response to challenges, the overall
immune-response of pea aphids is more limited than that of other insects studied to
date.
# Results and Discussion

## Overview of Annotation

We focused our manual annotation efforts on a subset of genes involved in the innate, humoral immune response contributing to recognition, signaling and response to bacteria and fungi in arthropods. We also manually annotated some genes involved in more general stress responses (e.g., heat shock proteins). All annotations are based on the recently completed sequencing of pea aphid clone LSR1 [36]. All genes manually annotated, as well as those genes that we found to be missing in the pea aphid genome, are listed in Supplemental Table S1. Also in this table, BLAST-based searches revealed that another aphid, *Myzus persicae* (green peach aphid), has putative homologs for many immune and stress related genes identified in the pea aphid.

## Annotation of Microbial Recognition Genes

**PGRPs.** Upon microbial invasion, *Drosophila* utilize several pathogen recognition receptors (PRRs) to detect pathogen-specific molecular patterns (e.g., cell-surface motifs) [37]. PRRs include peptidoglycan receptor proteins (PGRPs), which recognize peptidoglycans present in cell walls of Gram-positive and Gram-negative bacteria. PGRP-based recognition activates both the Toll and IMD/JNK pathways. PGRPs are highly conserved, with mammals and insect PGRPs sharing a 160 amino acid domain [38, 39]. Thus, it is surprising that pea aphids, in contrast to all other sequenced insects, appear to have no PGRPs. One other sequenced arthropod, the crustacean *Daphia pulex*, is also missing PGRPs [40].

**GNBPs.** GNBPs (Gram-Negative Binding Proteins, a historical misnomer) are thought to detect Gram-positive bacteria [41]. GNBPs and PGRPs are suspected to
form a complex. GNBPs then hydrolyze Gram-positive peptidoglycans into small
fragments, which are detected by PGRPs [41, 42]. Aphids have two *GNBP* paralogs,
*GNBP1* and *GNBP2* (see Figure S1a in Additional data file 1). Because GNBPs are
thought to form a complex with PGRPs, the presence of GNBPs without PGRPs in
aphids, as well as in the crustacean *D. pulex* [40], calls into question whether GNBPs
play a role in bacterial detection in these organisms. Some GNBPs and similar
proteins are known to function in fungal recognition [42], which may be the primary
function of these molecules in aphids.

**Lectins.** Lectins are a diverse group of sugar binding proteins. Many lectins function in insect immune recognition by binding to polysaccharide chains on the surface of pathogens [43]. *Drosophila* c-type lectins also appear to facilitate encapsulation of parasitoid invaders, by marking surfaces for hemocyte recruitment [44]. Aphids have five c-type *lectin* paralogs.

Galectins are another widely-distributed group of lectins [45]. In mosquitoes,
*galectins* are upregulated in response to both bacterial and malaria parasite infection
[46, 47]. Insect galectins are thought to be involved in either pathogen recognition, via
recognition of β-galactoside, or in phagocytosis [45]. Aphids have two *galectin*
paralogs.

**Class C scavenger receptors.** Scavenger receptors exhibit broad affinity
towards both Gram-positive and Gram-negative bacteria, but not yeast [48]. Pathogen
recognition by class C scavenger receptors in *Drosophila* facilitates phagocytosis, and
natural genetic variation of *Drosophila* scavenger receptors is correlated with
variation in the ability to suppress bacterial infection [49]. While *D. melanogaster* has
four class C scavenger receptor homologs, *A. gambiae* and *A. mellifera* have only one.
Pea aphids appear to have no class C scavenger receptors.
**The Nimrod superfamily and Dscam.** Several members of the Nimrod superfamily appear to function as receptors in phagocytosis and bacterial-binding [50, 51]. Such insect genes include *eater* and *nimrod*. Many of these genes are characterized by a specific EGF-repeat (Epidermal Growth Factor-repeat), and are duplicated in the genomes of *D. melanogaster*, *T. castaneum* and *A. mellifera* [52]. We were unable to identify any EGF-motif genes in the pea aphid genome.

Complex alternative splicing of Dscam (Down Syndrome Cell Adhesion Molecule) generates diverse surface receptors sometimes employed in arthropod innate immune defenses [53-55]. Though we did not manually annotate this complex gene as a part of this initial aphid immune gene project, we did identify multiple predicted proteins sequences in the aphid genome with strong similiarity to Dscam in other insects (GenBank: XP_001951010, XP_001949262, XP_001945921, XP_001951684, XP_001942542). Further investigations will be necessary to determine the activity and hypervariability of these genes and their transcripts in aphids.

**Annotation of Signaling Pathways**

**The toll signaling pathway.** The toll pathway is a signaling cascade involved in both development and innate immunity. In *Drosophila*, deletion of many of the component genes leads to increased susceptibility to many Gram-positive bacteria and fungal pathogens [11], and some Gram-negative bacteria and viruses [12]. In addition, upregulation of many components of the toll pathway is observed following parasitoid wasp invasion [56]. The toll pathway appears to be intact in pea aphids. We found convincing matches for genes encoding the extracellular cytokine spätzle, the transmembrane receptor Toll, the tube and MyD88 adaptors, the kinase pelle, the
inhibitor molecule cactus (a homolog of IkB), cactin, Pellino, Traf, and the
transactivator dorsal (Figure 1). The latter two genes are duplicated.

As in other insects, there are several gene families associated with the toll
pathway that are represented in aphids. First, aphids seem to have multiple spätzles
that segregate with *Drosophila spätzles* 1,2,3,4 and 6 in phylogenetic analyses (see
Figure S1b in Additional data file 1). Second, aphids also have a suite of serine
proteases and serine protease inhibitors (serpins). Though we did not manually
annotate serine proteases and serine protease inhibitors (serpins) as a part of this
initial aphid immune gene project, we did identify multiple predicted protein
sequences in the aphid genome with strong similarity to serine proteases and serpins
in other insects. In insects, these molecules function in digestion, embryonic
development and defense responses towards both microbial and parasitoid wasp
invaders [57-59]. In the absence of microbial challenge, the serpin necrotic prevents
activation of the toll pathway, but upon immunological challenge, the toll pathway is
triggered by a cascade of serine proteases, including persephone, which is thought to
be specific to fungal challenge [41]. Though it is not clear which of the many aphid
serine proteases is homologous to persephone, it is likely that pea aphids have serine
proteases capable of triggering the Toll pathway. Finally, aphids also have multiple
genes encoding Toll receptors, which function as transmembrane receptors in both
mammals and insects. While nine single-copy Toll genes have been identified in *D.
melanogaster* (*Toll1* to *Toll9*), it seems that pea aphids, like other insects, lack some
of these genes, but have multiple copies of others (see Figure S1c in Additional data
file 1). In other organisms, some, but not all Tolls serve a role in immune function,
while others function in developmental processes [60-62]. For aphids, it is not yet
clear what role each Toll serves.
**The JAK/STAT signaling pathway.** Like the toll pathway, in *Drosophila*, the JAK/STAT (Janus Kinase / Signal Transducers and Activators of Transcription) pathway is involved in both development and immunity. The JAK/STAT pathway is the least understood of the core insect immune pathways. JAK/STAT pathway induction appears to lead to overproliferation of hemocytes, upregulation of thiolester-containing proteins (TEPs), and an antiviral response [63]. Changes in gene expression following parasitoid wasp invasion of *Drosophila* larvae suggest a role for the JAK/STAT pathway in parasitoid response [56]. Pea aphids have homologs of all core JAK/STAT genes, including genes encoding for the cytokine receptor domeless, JAK tyrosine kinase (a.k.a Hopscotch), and the STAT92E transcription factor (Figure 1). *STAT92E* appears to be duplicated. No homologs were found for *upd* (unpaired), considered a key ligand in *Drosophila* JAK/STAT induction. This ligand is also missing in other insects (e.g. *A. mellifera*) [14].

**IMD and JNK signaling pathways.** Surprisingly, pea aphids appear to be missing many crucial components of the IMD (immunodeficiency) signaling pathway. This pathway is critical for fighting Gram-negative bacteria in *Drosophila* [11, 64], and IMD pathway member knockouts influence susceptibility to some Gram-positive bacteria and fungi as well [12]. IMD-associated genes missing in pea aphids include *PGRPs* (see above), *IMD*, *dFADD*, *Dredd* and *Relish* (Rel) (Figure 1). In contrast, conserved one to one orthologs of these same genes are found across *Drosophila*, *Apis*, *Aedes*, *Anopheles* and *Tribolium* [13]. Cursory BLAST-based searches for these genes in other arthropods, suggest that some may be missing (Figure 2). Pea aphids do have homologs for a few pathway members (*TAB*, *TAK*, *kenny*, *Iap2* and *IRD5*) (Figure 1).

While missing IMD-associated genes, pea aphids have plausible orthologs for
most components of the JNK pathway (Figure 1). In *Drosophila*, the JNK pathway regulates many developmental processes, as well as wound healing [65], and has been proposed to play a role in antimicrobial peptide gene expression and cellular immune responses [11, 66]. Genes present include *hep*, *basket*, and *JRA*. Searchers for homologs to the *Drosophila* *kayak* (*kay*) gene found an apparently similar transcription factor encoding gene in the *A. pisum* genome [GenBank: XP_001949014], but this match was largely restricted to the leucine zipper region, and failed tests of reciprocity.

The absence of IMD but presence of JNK in pea aphids is surprising as, in *Drosophila*, the IMD signaling pathway leads to activation of components of the JNK signaling pathway [11]. Specifically, when TAK, a protein kinase of the IMD pathway, is activated, it triggers the JNK pathway. Whether TAK can be activated without the rest of the IMD pathway is unknown. An alternative IMD-independent activation of JNK, via the inducer Eiger [67], has been proposed in *Drosophila* [66]. As Eiger is present in the pea aphid, this mode of activation may serve a critical role in any aphid JNK-based immune response.

## Annotation of Recognition Genes

**Antimicrobial peptides.** Introduction of microbes into most insects leads to the production of antimicrobial peptides (AMPs) by the fat body, an insect immune-response tissue, and occasionally by hemocytes and other tissues [68-71]. These peptides are secreted into the hemolymph, where they exhibit a broad range of activities against fungi and bacteria. The mechanisms of AMP action are poorly understood, but at least in some cases (e.g., drosomycin in *Drosophila*), AMPs destroy invading microbes by disrupting microbial cell membranes, leading to cell lysis [71].
Antimicrobial peptides are diverse and ubiquitous. They tend to be small molecules (<30 kDa) specialized at attacking particular microbial classes (i.e., Gram-positive bacteria, fungi, etc.) [68, 69]. While some antimicrobial peptides are found in only a single insect group (e.g., metchnikowin is found only in *Drosophila*), others are widely dispersed across eukaryotes (e.g., defensins are present in fungi, plants and animals). Genomics, coupled with proteomics, has revealed that all sequenced insects, and many other insects, have multiple types of antimicrobial peptides (Figure 2). Pea aphids, surprisingly, are missing many of the antimicrobial peptides common to other insects. For example, while all insect genomes annotated thus far have genes encoding for defensins [13], homology-based searches, phylogenetic-based analyses, transcriptomics (see below), and proteomics (see below) failed to find any signatures of defensins in the pea aphid genome. The presence of defensins in the human louse *Pediculus humanus* (Figure 2), and in the ancient apterygote insect, the fire brat *Thermobia domestica* [34], suggests that defensins have been lost during aphid evolution.

Extensive searches for genes encoding for insect cecropins, drosocin (and other proline-rich arthropod AMPs), dipticicin (and other glycine-rich AMPs), drosomycin, metchnikowin, formicin, moricin, spingerin, gomesin, tachyplesin, polyphemusin, andropin, gambicin, and virescein also revealed no hits. Weak hits were found for genes that encode for two antimicrobial peptides in other invertebrates: megourin [UniProtKB: P83417], originally isolated from another aphid species, the vetch aphid *Megoura viciae* (Bulet et al., unpublished) and penaeidin [UniProtKB: P81058], originally isolated from the shrimp *Penaeus vannamei*. The putative pea aphid *megourin* (scaffold EQ11086, positions 45752 – 45892), however, is highly diverged from that of *M. viciae* (31% identity) and, as compared to its *M.*
*viciae* counterparts, seems to have a shorter C-terminal region containing a stop-codon (see Figure S2 in Additional data file 1). Using three different primer pairs, we were unable to amplify products of this putative *Megourin* from cDNA generated for expression analyses (see below). The highly divergent *Penaeidin* [GenBank: ACYPI37769] (see Figure S2 in Additional data file 1) also did not amplify from cDNA.

We found six *Thaumatin* homologs in the *A. pisum* genome that show overall sequence and predicted structure similarities to plant thaumatins (Figure 3a,b).

Thaumatin-like proteins are disulfide-bridged polypeptides of about 200 residues.

Some thaumatins possess antifungal activity in plant tissues after infection [72].

Recently, a thaumatin found in the beetle *Tribolium castaneum* was shown to inhibit spore germination of filamentous fungi *Beauveria bassiana* and *Fusarium culmorum* [32]. Phylogenetic analyses revealed that *A. pisum* thaumatins form a monophyletic group closely related to beetle thaumatins (Figure 3c). Since thaumatin-like genes are conspicuously absent from the genomes of *Drosophila*, *Apis*, *Anopheles*, *Pediculus* and *Ixodes* (Figure 2), our findings indicate that thaumatins may represent ancient defense molecules that have been lost in several insect species, or have been independently acquired in aphids and beetles. The monophyly of aphid and beetle thaumatins provides no indication of an origin of novel acquisition (Figure 3c).

**Lysozymes.** Lysozymes represent a family of enzymes that degrade bacterial cell walls by hydrolyzing the 1,4-beta-linkages between N-acetyl-D-glucosamine and N-acetylmuramic acid in peptidoglycan heteropolymers [73]. They are ubiquitously distributed among living organisms and are believed to be essential for defense against bacterial infection. Lysozymes are classified into several types (i.e., c (chicken), g (goose), i (invertebrate), plant, bacteria and phage types). C-type
lysozymes are the most common for metazoa, being found in all vertebrates examined
thus far and many invertebrates, including all the previously sequenced insects. For
example, *D. melanogaster* and *A. gambiae* have at least seven and nine loci for c-type
lysozymes, respectively [74, 75]. Insects also have i-type homologs, but their
bacteriolytic activities are unclear [76].

Unlike other insects sequenced thus far, similarity searches demonstrated that
*A. pisum* lacks genes for c-type lysozymes. The analysis further verified that the
genome also lacks genes for g-type, plant-type, and phage-type lysozymes. Only three
genes for i-type homologs were detected in the genome (see Figure S1d in Additional
data file 1). One of them, *Lys1*, is highly expressed in the bacteriocyte [77]. Two
others, *Lys2* and *Lys3*, are located adjacent to *Lys1*.

Notably, two genes that appear to have been transferred from bacterial
genomes to the *A. pisum* genome encode bacteriolytic enzymes [36]. One is for a
chimeric protein that consists of a eukaryotic carboxypeptidase and a bacterial
lysozyme. The other (*AmiD*) encodes *N*-acetylmuramoyl-L-alanine amidase, which is
not a true lysozyme (1,4-beta-N-acetylmuramidase) but similarly degrades bacterial
cell walls. While some of these bacteriolotyic-related genes are highly expressed in
the bacteriocyte, and lysozymes appear to be upregulated in response to some
challenges (see gene expression study, below), assays of bacterioltyic activity of
hemolymph from immune-challenged aphids suggest that aphid hemolymph has weak
to no lysozyme-like activity [31]. Further studies will determine the role of these gene
products.

**Chitinases.** Chitinases are enzymes that degrade chitin (a long-chain polymer of *N*-acetyl-D-glucosamine), hydrolyzing 1,4-beta-linkages between *N*-acetyl-D-glucosamines. Chitinases and lysozymes represent a superfamily of hydrolases, and
their catalytic activities are similar. Indeed, some chitinases show lysozyme activity
and vice versa [73]. In insects, chitinases are used to degrade the chitin in the
exoskeleton and peritrophic membrane during molting, and some are suspected to
have antifungal activity, as fungal cell walls also consists of chitin [78]. Similarity
searches followed by phylogenetic analyses demonstrated that the genome of *A. pisum*
encodes seven genes for putative chitinase-like proteins [79]. Further studies are
required to determine the biochemical properties and substrate specificity of these
chitinase-like proteins.

**TEPs and Tots.** Some thiolester-containing proteins (TEPs) can covalently attach to pathogens and parasites in order to ‘mark’ them for phagocytosis [80]. Like other insects, aphids have multiple *TEP* paralogs. Both are homologous to *TEPIII* (see Figure S1e in Additional data file 1). Homologs of *TepI*, *TepII* and *TepIV* were not found. In contrast, no *Turandot (Tot)* genes, which encode small peptides induced by severe stress and septic injury in *Drosophila* [81-83], have been found in aphids or in other insects other than *Drosophila* spp.. Both TEPs and Tots are thought to be regulated by the JAK/STAT pathway.

**Prophenoloxidase (ProPO).** Phenoloxidase-mediated melanin formation characteristically accompanies wound clotting, phagocytosis and encapsulation of pathogens and parasites [84]. In insects, the inactive enzyme prophenoloxidase (ProPO) is activated by serine proteases to yield phenoloxidase [85]. Aphids appear to have two prophenoloxidase homologs (*ProPO1*, *ProPO2*) (see Figure S1f in Additional data file 1), which are homologous to *D. melanogaster Diphenol oxidase A3* (Flybase: CG2952).

**Nitric oxide synthase.** Production of nitric oxide is mediated by the enzyme nitric oxide synthase (NOS). Nitric oxide is a highly unstable free radical gas that has
been shown to be toxic to both parasites and pathogens. In insects, *Nos* is upregulated
after both parasite and Gram-negative bacterial infection [86, 87]. Like other insects,
pea aphids have one *Nos* homolog.

**Heat shock proteins.** Though called heat shock proteins, HSPs are produced in response to a range of stresses in both eukaryotic and prokaryotic organisms [18]. They serve as chaperones, facilitating protein-folding and stabilization, and as proteases, mediating the degradation of damaged proteins. HSPs may also serve as signaling proteins during immune responses [18, 88]. In many insects, including aphids, *HSPs* have been shown to be upregulated after septic injury and microbial infection [31, 89-92]. We identified 15 *HSPs* of varying molecular weight in pea aphids (see Figure S1g in Additional data file 1).

**Gluthione-S-tranferases (GSTs).** GSTs are a diverse class of enzymes that detoxify stress-causing agents, including toxic oxygen free radical species. GSTs are upregulated in some arthropods upon oxidative stress [93] and microbial challenge [89, 94]. Pea aphids have at least 18 genes encoding GSTs and many other detoxification enzymes that likely play a role in stress responses [95]. Ramsey et al. (2009) identified many of the genes encoding detoxification enzymes in *A. pisum* and in *Myzus persicae*.

**Alarm pheromone production.** In response to predators, aphids release an alarm pheromone that causes neighboring aphids to become more mobile and to produce more winged than unwinged offspring [19, 96]. These winged offspring have the ability to disperse to enemy-free space. While many insects produce a suite of chemicals that constitute an alarm signal, the aphid alarm pheromone is dominated by a single compound, (*E*)-β farnesene (EBF) [97]. While the genes underlying alarm pheromone production have not been fully characterized, we have identified a
*Farnesyl diphosphate synthase (FPPS)* and an *Isoprenyl diphosphate synthase (IPPS)*, which may underlie alarm pheromone production [98].

## Functional Assays

**Gene expression.** We utilized real-time quantitative PCR to conduct a preliminary investigation of the expression of 23 recognition, signaling and response genes in aphids subjected to a number of infection and stress treatments, (see Supplementary Materials and Table S2 in Additional data file 1). While future studies with more biological replicates will be necessary to fully survey gene regulation in the face of stress and infection, this initial survey indicates that aphids do express these genes under both control and infection/stress conditions (see Tables S4 and S5 in Additional data file 1). This suggests that these genes are functional even in the absence of many other missing immune-related genes.

One expression pattern seen in this initial survey is of particular note. Unlike other insect immune expression studies, we found no strong upregulation of antimicrobial peptides, which frequently exhibit 10-fold or greater upregulation in the face of infection. For example, while Altincicek et al. (2008b) observed 20-fold upregulation of *Thaumatins* in tribolium beetles after stabbing with lipopolysaccaride endotoxin derived from *E. coli*, we saw modest upregulation (approximately 2-fold) of only one *Thaumatin* (*Thm2*) after stabbing aphids (Supplemental Table S5).

Furthermore, despite the fact that they are known to suppress fungal germination in beetles, the *Thaumatin* homologs were not upregulated after fungal infection at the time point included in this study, and were only approximately 2-fold upregulated at two additional time points and in a follow-up fungal infection experiment (data not shown) [32]. The role of thaumatins in fighting microbial infections, however, should
not be discounted, as they may function in the absence of significant upregulation
(i.e., they may be constitutively expressed).

**Exploration of ESTs from infected and uninfected aphids.** In the first of two EST-based experiments, we compared a cDNA library synthesized from the guts of *A.pisum* that had been fed a Gram-negative pathogen, *Dickeya dadantii* [99], to a cDNA library synthesized from uninfected guts. Strikingly, no standard immune-related genes, such as antimicrobial peptides, were identified in the infected sample. The main functional classes differentially expressed were the “*biopolymer metabolism*” class, many members of which were down-regulated in infected guts, and “*transport*” or “*establishment of localization*” classes, whose genes were upregulated in infected guts (Supplemental Table S6). The “*immune response*” class, in contrast, was only represented by five genes. Four of these five genes were in the uninfected library, while only one, a *leucyl-aminopeptidase*, was identified from the infected library; the immune function of leucyl-aminopeptidases is not well understood. Moreover, the “response to stress/external stimulus/biotic stimulus” classes were not overrepresented in the infected gut library.

In a separate experiment, to further identify aphid immune-relevant genes, we utilized SSH to compare cDNA from *E. coli*-infected aphids and cDNA from unchallenged aphids. To obtain genes expressed at different phases of the immune response, three RNA samples were extracted 3, 6 and 12 hours after *E. coli* infection and mixed prior to cDNA synthesis.

Among the 480 expressed sequence tags (ESTs) that were sequenced from the subtracted library [GenBank: GD185911 to GD186390], we found some genes with similarity to proteases and protease inhibitors but few other immune-related proteins. Interestingly, SSH-based EST analysis failed to identify any pathogen recognition
receptors (PRRs), such as PGRPs or GNBPs, or any antimicrobial peptides (Supplemental Table S7). It is noteworthy that this aphid experiment was conducted in parallel to a similar *Sitophilus weevil* experiment, where many immune-related genes (more than 18% of ESTs) were identified, including antibacterial peptides and PRRs [35]. This suggests that the paucity of immune genes identified in *A. pisum* is not a technical issue but may be a specific feature of aphids [31]. In addition, dot blot analysis demonstrated that only a few genes (less than 5%) were differentially expressed between *E.coli*-stabbed and unstabbed aphids. These findings indicate that, in contrast to other insects, either aphids respond only weakly to challenge with Gram-negative bacteria or aphid genes and pathways directed against these bacteria are expressed only constitutively.

**High Performance Liquid Chromotography.** HPLC peptide analyses targeting production of small peptides (e.g., antimicrobial peptides) were run on hemolymph samples from pea aphids challenged by three microorganisms: *E. coli* (Gram-negative bacteria), *Micrococcus luteus* (Gram-positive bacteria) and *Aspergillus fumigatus* (fungi). Profiles were compared between control, infected and sterile-stabbed aphids at 6, 12 and 18 hours after challenge. When identified, the production of small peptides was maximal at 18 hours. In *E. coli*-treated samples, no upregulation could be identified (Figure 4a), in *M. luteus*-treated samples, there was modest upregulation (data not shown), and in *A. fumigatus*-treated samples, there was a significant response, though few peaks (Figure 4b). In contrast, a response profile to *E.coli* from another obligate symbiotic insect (the weevil, *Sitophilus oryzae*) exhibited at least five well-distinguishable upregulated peaks (Figure 4c). Response being restricted to Gram-positive bacteria and fungi is consistent with previous identification of megourin, an antimicrobial peptide in the aphid *Megoura viciae*, which appears to
have activity against Gram-positive bacteria and fungi, but not against Gram-negative bacteria (Bulet, unpublished). Because so few distinguishable peaks were present in the aphid samples, we did not choose to identify the associated products, but overall the presence of few inducible peptides suggests a peculiar scarcity of antimicrobial peptides in aphids.

## Conclusions

Aphids are one of only a few genomic models for hemimetabolous insects, yet until recently, virtually nothing was known about aphid immune and stress response systems. Here, by coupling gene annotation with functional assays, we see evidence that aphids have some defense systems common to other arthropods (e.g., the Toll and Jak/STAT signaling pathways, heat shock proteins, prophenoloxidase). Surprisingly, however, several of the genes thought central to arthropod innate immunity are missing in aphids (e.g., PGRPs, the IMD signaling pathway, defensins, c-type lysozymes). This calls into question the generality of the current model of insect immunity, and it remains to be determined how aphids protect themselves from the diverse pathogens and parasites that they face.

The fact that we cannot find aphid homologs to many insect immune genes could be a consequence of the large evolutionary distance between aphids and the taxa (in most cases, flies, mosquitoes and bees) from which these genes are known (i.e., the split between the ancestors of aphids and these taxa occurred approximately 350 million years ago [100]), making it challenging to find divergent genes via homology-based searches, even when using highly sensitive methods as done here. Though we cannot preclude this possibility in all cases, in some cases, similar
homology-based methods are able to recover homologs in even more distantly-related
taxa. For example, querying genome databases with *Drosophila* genes via BLAST,
recovers putative homologs of PGRPs and defensins in *Pediculus humanus* (human
body louse) and in *Ixodes scapularis* (deer tick) (Figure 2). The divergence time
between *Drosophila* and these taxa is equal to or greater than that between
*Drosophila* and aphids. Moreover, for some cases, we could identify genomic regions
similar to functional genes in other species, but these regions contain large insertions
or stop codons (e.g., the putative antimicrobial peptide Megourin), indicating they are
the result of pseudogenization.

One potential explanation for the lack of known immune-related genes in pea
aphids is that aphids mount an alternative, but equal, immune-response. Our
functional analyses, as well as those of Altincicek et al. [31], found little evidence for
an alternative response. In EST and HPLC analyses, few novel ESTs or peptide
signals were recovered from immune-challenge aphids relative to their unchallenged
controls. It should be noted, however, that these challenges were primarily limited to
exposure to *E. coli* bacteria. When testing for expression of a few immune genes in
response to a wider array of challenges, we do see some evidence of an aphid immune
and stress response. Future expression studies, including large-scale transcriptional
and proteomic studies, will extend this work and allow for more comprehensive
characterization of the full complementation of aphid immune responses.

While we have focused mainly on the humoral component of the innate
immune response, it is interesting to note that there is some evidence that the cellular
component of pea aphids' innate immune response may also be different to that seen
in other insects. While many insects encapsulate parasitoid wasp larvae, smothering
them to death with hemocytes (insect immune cells), aphids appear not to have this
layer of protection [101, 102]. Aphids, however, appear to recruit some hemocytes to
parasitoid eggs, suggesting that cellular immunity may play an alternative, though
possibly more limited role [101]. Better insights into the capacity of the aphid
immune system will require further investigation of both the humoral and cellular
components of aphid immunity.

The lack of genomic and molecular data regarding immune systems of aphid
relatives makes it difficult to establish whether the pea aphid immune system is
unique. There are, however, a number of aspects of aphid ecology that could facilitate
ecological success without a strong immune defense. Altincicek et al. [31] proposed
three hypotheses to explain the apparent lack of antimicrobial defenses. First, they
suggested that contrary to *Drosophila*, whose natural environment consists of
decaying fruit that is colonized by microbes, aphids exploit phloem sap, which is
usually sterile [103]. Thus, the risk of encountering pathogens while feeding is
limited. This assumption, however, is only partly true. While probing plants, aphids
are capable of acquiring pathogenic bacteria from the surface of their host plants'
leaves [104], and aphids become host to a diverse assemblage of bacteria and fungi
under stressful conditions [105], some of which are pathogenic (Gerardo, unpublished
data). Furthermore, *Sitophilus weevils*, which when challenged with *E. coli*
significantly upregulate immune genes [35], spend their entire larval and nymph
stages within sterile cereal grains, indicating that a sterile diet is not likely to explain
the absence of antibacterial defenses in aphids.

Altincicek *et al.* [31] also suggest that aphids may invest in terminal
reproduction in response to an immune challenge, rather than in a costly immune
response. In their study, stabbed aphids produced significantly more offspring than
untreated aphids within 24 hours of injury. Such an increase in reproduction upon
challenge is not uncommon for invertebrates. *Biomphalaria* snails [106] [107], *Acheta*
crickets [108], *Daphnia* waterfleas [109], and *Drosophila* flies [110] have all been
shown to increase their investment in reproduction in response to infection. Yet,
*Drosophila* still mount a complex immune response. Furthermore, aphids do not
increase their reproductive effort in the face of all immune challenges: fungal
infection reduces the number of offspring *A. pisum* produce within 24 hours of
inoculation, and response to stabbing with bacteria seems to be specific to the aphid
genotype and to the location of the stab [111, 112]. Therefore, though aphids have the
capacity to reproduce many offspring prior to succumbing to some pathogens, it
seems that immune competence would still provide increased fitness.

Even without increased reproduction following infection, the prolific
reproductive capacity of aphids suggests these insects, in general, may invest most
resources towards rapid, early onset reproduction rather than towards fewer, though
better-protected offspring (a.k.a., in terms of classical ecological theory, aphids may
be r-selected rather k-selection organisms [113]). Recent theory of the evolution of
immunity suggests that such organisms may specifically invest less in costly immune
responses [114, 115]. Many characteristics of aphids, including their rapid generation
time, short life span and small body size all fit a model of r-selection [116].
*Drosophila* spp., however, also exhibit many of these characteristics and still invest in
a strong defense repertoire.

The third hypothesis proposed by Altincicek et al. (2008a) concerning the
evolution and maintenance of aphid defense relies on the presence of secondary
symbionts that can be found extracellularly in aphids [117]. *A. pisum* is protected
against fungal pathogens by one of these secondary symbionts, *Regiella insecticola*
[29] and also against the parasitoid wasp *Aphidius ervi* by another secondary
symbiont, *Hamiltonella defensa* [27]. Such symbiont-mediated host protection may
explain why aphids have a reduced (or specialized) antimicrobial defense. This
hypothesis seems plausible with regard to the cost of immune gene expression versus
the benefit of protection by the secondary endosymbionts. However, it does not
explain how the secondary endosymbionts (as Gram-negative bacteria), often present
in aphid hemolymph, are themselves perceived and controlled by the aphid immune
system. Thus, it is challenging to say whether the presence of secondary symbionts is
a cause or a consequence of reduced antimicrobial activity.

Potentially, all of these forces could shape the evolution of aphid stress and
immune responses. In order to test these hypotheses (e.g., reproductive investment,
symbiont-mediated host protection), we need more studies characterizing the global
aphid response under more conditions, and in more aphid species. Potential insight
from aphid relatives with different lifestyles (e.g., those not associated with secondary
symbionts, or those that live in soil or other microbe-rich habitats) may be particularly
helpful. More broadly, as the pea aphid is the first published genome of a
hemimetabolous insect, future analyses of the immune and stress related genes of
more insects in this group will facilitate the reconstruction of the evolutionary history
of innate immunity and other defenses.

**Materials and methods**

**Bioinformatic Screening of the Pea Aphid Genome**

Immune and stress gene candidates from other insects (e.g., *D. melanogaster*,
*A. aegypti*, *A. gambiae*, *A. mellifera*) were used to query the pea aphid genome. Most
searches utilized the blastp search function to search for hits against the predicted *A*.
*pisum* proteome [118]. For some gene families and putative paralogs, protein sequences were aligned to sequences from other insects and outgroups using ClustalW [119]. These alignments, as well as available EST and full length cDNA sequences, served to refine aphid gene models (exon/intron boundaries, *etc.*), and to facilitate phylogenetic analyses. In addition, a comprehensive database of all available EST sequences from the green peach aphid, *Myzus persicae*, was screened using tblastn to search for potential homologs to all immune and stress genes annotated in the pea aphid.

For genes that could not be found in the proteome, we also conducted a tblastn
search against all contigs and unassembled reads. Then, a final, more sensitive
profile-based search was performed for those immune defense proteins that produced
no hits with BLAST searches. For this analysis, insect and other species protein
sequences belonging to the family of interest were retrieved from NCBI and aligned
with MUSCLE [120]. A hidden Markov model for the alignment was built and
calibrated using HMMER [121]. This was used to perform a profile-based search
(hmmsearch) against the six-frame translated sequences of the assembled pea aphid
genome and the unassembled reads. Additionally, a similar search with PFAM
profiles [122] was also performed for those families encoding PFAM domains in their
sequences. Whenever a significant hit was found, the genomic region was analyzed to
discard the possibility that it encoded a pseudogene (presence of stop codons, absence
of relevant domains, *etc.*).

Phylogenetic analyses of selected protein families were performed using their
corresponding Maximum Likelihood phylogenetic trees from the pea aphid phylome
[36], deposited in PhylomeDB [123]. When necessary, additional sequences were
added to the original PhylomeDB alignment, realigned with MUSCLE and used to
reconstruct a Maximum Likelihood phylogenetic tree, using the JTT model as implemented in PhyML v2.4.4 [124], assuming a discrete gamma-distribution model with four rate categories and invariant sites, and estimating the gamma shape parameter and the fraction of invariant sites. Cladograms were edited using Dendrogram [125].

Exploration of ESTs from Infected and Uninfected Aphids

In the first experiment, two EST libraries (one control, one infected) were generated by standard procedures using a SMART cDNA kit (Clontech), starting from approximately 1000 dissected *A. pisum* midguts for each library. The aphids were clonal, young, reproducing asexuals, which were either fed on control diet or infected by feeding on artificial diet with the Gram-negative aphid pathogen *Dickeya dadantii* at 10⁶ bacteria per mL [99]. Twenty-four hours after infection, control and treated aphids were dissected, and complete guts were transferred immediately into RNeasy solution (Qiagen). ESTs were sequenced according to procedures in Sabater-Munoz et al. (2006) [126].

In another EST-based experiment utilizing Suppression Subtractive Hybridization (SSH) and dot-blot technology, we treated aphids (clone LL01) with rifampicin as described in Rahbé *et al.* [127] to reduce symbiont load. We challenged wingless fourth-instar aposymbiotic aphids by stabbing them with needles previously dipped into a pellet of overnight cultures of *E. coli* (TOP10, Invitrogen), and then maintained them on fava plants. At three, six, and twelve hours post-treatment, we stored surviving aphids at -80°C. To identify genes that are differentially expressed in response to septic injury, we performed SSH using RNAs from immune challenged (3, 6 and 12 hours post-treatment) and untreated aposymbiotic aphids, using the SMART PCR cDNA synthesis Kit and the PCR-Select cDNA subtraction kit
(Clontech laboratories) according to the manufacturer's instructions and as described
in Anselme *et al*. [35]. After transformation by electroporation, we recovered
approximately 1500 colonies from LB agar plates. We plasmid extracted and
sequenced 500 randomly picked colonies (NucleoSpin® Plasmid Kit, Macherey-
Nagel) utilizing the sequencing center at the University of Valencia (Spain). We
compared all sequences against UniProt using blastx. Immune-related gene sequences
(Supplemental Table S7) were then compared to the aphid genome using blastn.

To analyze the differential expression status of each expressed sequence tag
(EST) we conducted a dot-blot experiment. Briefly, we amplified 344 ESTs from the
SSH library by colony PCR with nested PCR primers 1 and 2R from the PCR-Select
cDNA subtraction Kit. We then spotted 10 µL from each PCR product onto two
different membranes (Hybon™-N, Amersham) using a *Bio-Dot* Microfiltration
System (Biorad). We hybridized membranes with radiolabeled cDNA probes
generated by reverse-transcription from RNA extracted from either aposymbiotic
aphids stabbed with *E. coli* or unstabbed aposymbiotic aphids. We synthesized these
probes using the Super Script™ First Strand synthesis (Invitrogen) system for RT-
PCR and [α-³²P]dCTP, and purified them using *Quick Spin* Column (Roche molecular
Biochemicals). After exposing blots for up to 24 hours to a Storm PhosphorImager
imaging plate (Amersham), we analyzed differential expression by comparison of
band intensities between the two membranes. We, however, did not normalize the
data, as we failed to see any signal from the *gapdh* gene, though the same amount of
each PCR product was loaded on both membranes.

HPLC

Aphids were challenged by abdominal puncture with triple-0 needles dipped in
a solution of Gram-negative bacteria (E. coli strain Top10), Gram-positive bacteria
(*M. luteus*) or fungal spores (*A. fumigatus*). For each microbial treatment, five hemolymph samples from 50 aphids each were collected at four times points (t = 0, 6, 12 and 18 hours).

Hemolymph was flash-extracted by centrifuging (1 min, 10,000g, 4°C) live aphids through a 1 mL pipette tip and directly into 40 µL 0.1% trifluoracetic acid (TFA) containing 10 µL of saturated phenylthiourea (PTU) for phenoloxidase inhibition. Resulting samples were highly similar to pure hemolymph samples obtained by leg bleeding (> 95% band identity by silver-stained SDS-PAGE).

After initial collection, tips were removed and the samples were centrifuged for 5 minutes at 15,000g. Following addition of 70 µL TFA 0.1%, the supernatant was left for 1 hour at 4°C to allow for protein precipitation prior to a final 10 min centrifugation at 15,000g to recover peptides. Samples were evaporated and stored at -20°C until use in HPLC. Chromatography was performed on standard peptide C18-300Å reverse phase columns using water acetonitrile gradients [128]. For retention time (RT) standardization, PTU served as an internal standard, and samples were analysed by area-normalization to unchallenged sample peaks (RT = 14 min, preceding PTU).

## Abbreviations

ALPV: aphid lethal paralysis virus; aLRT: approximate likelihood ratio test; AMP: antimicrobial peptide; Ct: comparative threshold cycle; DSCAM: Down Syndrome Cell Adhesion Molecule; EBF: E-β farnesene; EGF: epidermal growth factor; EST: expressed sequence tag; GNBP: gram-negative binding protein; HPLC: high performance liquid chromatography; HSP: heat shock protein; IMD:
immunodeficiency; JAK/STAT: janus kinase / signal transducers and activators of
transcription; JTT: Jones-Taylor-Thornton; PDA: potato dextrose agar; PGRP:
petidoglycan receptor protein; ProPO: prophenoloxidase; PRR: pathogen recognition
receptor; PTU: phenylthiourea; SSH: suppression subtractive hybridization; RT:
retention time; RQ: relative quantity; Tep: thiolester-containing protein; TFA:
trifluoracetic acid; Tot: turandot.

Authors' contributions

NMG, SMB, and MG were group leaders for the project. NMG, BA, HA, SMB,
MDV, EJD, JDE, AM, MG, IK, AN, BJP, MP, JSR, JT, DT, and CT designed and
performed manual gene annotation. TG and SMB conducted phylogenetic analyses.
BA and AV conceived of and conducted analyses of Thaumatin. SMB, NMG, CS and
BJP performed experiments and analyses for the gene expression study. CA, AH,
VPB, AM, and AL conceived of and conducted the SSH study, and CVM constructed
the aphid gut libraries. YR conducted the HPLC study. The manuscript was prepared
by NMG, SMB, CA, TG and YR with input from MDV, BA, AN, AV and AH. All
authors have read and approved the final version of the manuscript.

Acknowledgements

We thank Angela Douglas, Nancy Moran, Tom Little and members of the
International Aphid Genomics Consortium for insightful discussion, and Charles
Godfray and two anonymous reviewers for comments that enhanced this manuscript.
Cultures of *Z. occidentalis* were provided by the USDA ARS Collection of
Entomopathogenic Fungal Cultures. Samples of ALPV and virus-infection protocols
were provided kindly by Bryony Bonning and Liljana Georgievska. Comparison with
*M. persicae* was supported by USDA grant 2005-35604-15446 to Georg Jander.
<caption>**Figure 1** - Some key insect recognition, signaling and response genes are missing in the pea aphid.</caption>

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.

<caption>**Figure 2** - Gene families implicated in arthropod immunity suggest unique features of the pea aphid immune system.</caption>

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 (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.

**Figure 3 - Evolutionarily conserved thaumatins are present in pea aphids and plants.**

(a) The three-dimensional structure of the pea aphid thaumatin ACYPI009605 (left) was calculated using the published crystallographic structure of a sweet cherry (plant) thaumatin 2AHN_A (right) [130] and Swissmodel [131], revealing that both thaumatins are similar in structure. However, one exposed loop, indicated by a dashed circle, 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. * indicates approximate likelihood ratio test (aLRT) support > 80. (Api: *A. pisum*; Cac: *Catenulispora acidiphila*; Cel: *Caenorhabditis elegans*; Mtr: *Medicago truncatula*; Pav: *Prunus avium*; Tca: *Tribolium castaneum*; Tpr: *Trifolium pratense*)
<caption>**Figure 4** - HPLC traces of inducible hemolymph peptides in the pea aphid compared to the rice weevil.</caption>

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 (indicated as 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.

**Additional files**

**Additional data file 1 – Supplemental Material**

A single supplementary document includes: Supplementary methods for the gene expression study; Table S1, Pea aphid immune and stress gene list; Table S2, Samples for qPCR expression study; Table S3, Primers for qPCR expression study; Table S4, Relative expression of recognition and signalling genes; Table S5, Relative expression of response genes; Table S6, Gut EST library statistics; Table S7, List of selected ESTs from the subtracted library; Figure S1, Maximum likelihood phylogenies of selected immune and stress gene families; Figure S2, Alignments of putative antimicrobial peptides megourin and penaeidin; and Figure S3, Survival curves for experimental infections associated with qPCR study.
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## IMD/JNK PATHWAYS

### Bacteria

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

PGRP

PGRP

PGRPs
recruit IMD.

## TOLL PATHWAY

### Bacteria

<figure><img src="image_4.png" /></figure>

GNBP

PGRP

Serine proteases
cleave spatzle.

<figure><img src="image_5.png" /></figure>

Persephone

<figure><img src="image_6.png" /></figure>

Fungi cleave
persephone, a serine
protease.

### Fungi

## JAK/STAT PATHWAY

### Viruses

<figure><img src="image_7.png" /></figure>

Viruses bind to
unknown recognition
molecules, which trigger
JAK/STAT pathway.

<figure><img src="image_8.png" /></figure>

JAK

STAT

Domeless

STAT

JAK

JAKs become activated and
phosphorylate the domeless
receptor. Stat proteins then
bind to the receptor, and are
phosphorylated.

cell membrane

<figure><img src="image_9.png" /></figure>

Phosphorylated
STATs dimerize.

<figure><img src="image_10.png" /></figure>

Tep

The Stat complex
translocates to the
nucleus and activates
expression of thiolester
containing proteins.

Apoptosis

Rel

Dredd

dFADD

IMD

Tab

Tak

activation
of JNK pathway

<figure><img src="image_11.png" /></figure>

Kenny

Rel translocates
to nucleus and
activates expression of AMPs
(e.g., Defensin, Cecropin).

Def

Cec

Rel

Basket

Hep

Basket

Jra

Kay

Dor

Dor

Dor

Dor

After spz-binding, Toll
recruits MyD88, pelle, tube.

<figure><img src="image_12.png" /></figure>

Pelle

MyD88

Cactus is phosphorylated.
Dorsal is released.

<figure><img src="image_13.png" /></figure>

Dorsals translocate
to nucleus and
activate expression of AMPs,
lysozymes and prophenoloxidase.

nucleus
<figure><img src="image_14.png" /></figure>

Recognition

Signaling

Response
<figure><img src="image_15.png" /><figcaption>(a)</figcaption></figure>

aphid

<figure><img src="image_16.png" /></figure>

plant

<caption>(b)</caption>

**PLANT** ATISFKNNCPYMVWPGLTTSDQKPQLSTTGFEL
**APHID** MDFKFILFFVOMSGVTMAHMIRVTNNCPFTVWPGIQGNTGQQHLENGGFSV

**PLANT** ASQASFQLDTPVPWNGRFWARTGCSTDASGKFVCATADCASGQVMCNGNGA
**APHID** GAYKTHFILSSRNWAGRIWGRTNC--DSQGK--CETGDCGN-KIQCNGTLG

**PLANT** IPPATLAEFNIPAGGGQDFYDVSLVDGFNLPMSVTPQG------GTGDCKT
**APHID** VPPLTLAEIQFAESDNIDSYYVSLVDGFNLPIKIMPNKYPMTSKNSIDCKP

**PLANT** ASCPANVNAVCPSELQKKGSDGS-VVACLSACVKFGTPQYCCTPPQNTPET
**APHID** ADCVADLNSKCPDKLAVKAADGSSVVACKSACALFNTDSDCCQGVYTTPAT

**PLANT** CP----PTNYSEIFHNACPDAYSYAYDDKRGFTCTNGGP--NYAITFCP
**APHID** CNSSSWPQNYPPFFKKACPYAYSYPFDNTTSTFTCHGNSLTKFDIVFCP

<figure><img src="image_17.png" /><figcaption>(c)</figcaption></figure>
<figure><img src="image_18.png" /><figcaption>(b) pea aphids challenged with fungus</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>(c) weevils challenged with *E. coli*</figcaption></figure>

<figure><img src="image_20.png" /></figure>
**Supplemental Material: Immunity and defense in pea aphids, *Acyrthosiphon pisum***

**Supplementary Methods for Gene Expression Study**

**Gene expression overview.** We utilized real-time quantitative PCR to conduct a preliminary investigation of the expression of 23 recognition, signaling and response genes in aphids subjected to a number of infection and stress treatments (Tables S2, below). First, we verified expression of the immune and stress genes in aphids stabbed with needles inoculated with the natural commensal *Escherichia coli*, a natural Gram-positive bacterial pathogen, a natural Gram-negative bacterial pathogen, or with no needle (control). More closely mimicking the uptake of bacteria while feeding, we verified expression of the genes in aphids fed on artificial diet containing either *E. coli* or a Gram-negative pathogen relative to that of aphids fed on control diet with no bacteria. We also verified gene expression in entomopathogen fungus-infected to uninfected aphids, and in aphids stabbed with pathogenic virus-inoculated needles to unstabbed control aphids. All microbes used have been isolated from pea aphids in natural populations. Finally, to assess effects of environmental stress, we verified gene expression in aphids exposed to alarm pheromones, starvation, and heat-shock.

We conducted the five challenge experiments with aphids of clonal line LSR1, the clone sequenced in the genome sequencing project. This line was cured of its secondary symbiont, *R. insecticola*, more than two years ago using a standard antibiotic treatment [1], and has been continuously maintained as an asexual clone on fava beans at 16hr light: 8hr dark at 20°C. For each experiment, we used 5-6 day old, unwinged aphids, which were maintained on fava bean plants at 20°C unless otherwise noted.

**Bacterial stabbing experiment.** For the first experiment, we stabbed aphids with *E. coli*, Gram-negative pathogen Ng5B, in the genus *Enterobacter*, or Gram-positive pathogen 6B, in the genus *Staphylococcus*. *E. coli* has been previously shown to be a commensal that has little affect on aphid
survival when either injected into or fed to aphids as it is quickly cleared by the host (Figure S3, below) [2]. Ng5b and 6b are bacteria originally isolated from laboratory pea aphids. When fed to aphids, these pathogens kill most aphids in approximately 48 hours (Figure S3a, below). The night before the infection, we grew bacterial cultures from glycerol stocks on Luria broth (LB) agar at 37°C. The morning of the infection, we transferred bacterial colonies to LB and grew them at 37°C. We determined concentration of broth cultures by optical density (OD₆₀₀), and then standardized the cultures to OD₆₀₀ = 0.5. Next, we stabbed aphids with a minutin pin contaminated with the standardized bacterial cultures or with sterile LB and then transferred the stabbed aphids and unstabbed control aphids to fava bean plants. After eight hours, we froze five aphids per condition in liquid nitrogen and stored them at -80°C for subsequent RNA extraction. We monitored ten aphids per condition for survival (Figure S3, below).

**Bacterial feeding experiment.** For the second experiment, we fed aphids on AP3 artificial *A. pisum* diet [3] containing no bacteria (control), *E. coli*, or the Gram-negative pathogen Ng5b. We grew bacteria as above and then inoculated the treatment diets with 1 µL bacterial culture per 20 mL diet, and control diet with the same amount of bacteria-free LB. We plated the diets onto LB agar to confirm that the control diet was free of bacteria and that the final bacterial concentration in the treatment diet was approximately 2 x 10⁴ colony forming units (cfu) per mL. We added one drop of food-grade blue dye per 5 mL of diet to allow us to detect whether aphids had ingested the diet. We then filled 10 mm Petri dish bottoms with diet (either control or treatment) and covered the dish with stretched Parafilm. We affixed the feeding dishes to the bottom of 15 mm Petri dishes, and transferred 30 - 50 aphids directly from plants to each feeding dish. We maintained dishes upside down in a 20°C incubator.

Approximately 12 hours after being exposed to the diet, we froze five aphids from each treatment that had fed (as determined by the presence of dye in the aphid intestinal tract) in liquid nitrogen and maintained them at -80°C for subsequent RNA extraction. Eight hours later, we transferred the
remaining aphids that had fed (n = 30 per treatment) to fava bean plants and monitored them for survival (Figure S3a, below).

**Fungal shower experiment.** For the third experiment, we exposed aphids to a shower of spores of the fungus *Zoophthora occidentalis*, an aphid specific fungal entomopathogen [4]. We placed aphids in a 70 mm tall cylinder with a damp sponge at the bottom. We then inverted an approximately two-week-old culture of *Z. occidentalis* on potato dextrose agar (PDA) over the cylinder and allowed the spores to fall on the aphids for 2 hours. Control aphids were exposed to the same conditions but with a sterile PDA plate. After exposure, we transferred the aphids to fava plants. We froze five aphids per condition in liquid nitrogen 24 hours after exposure and monitored 22 aphids per condition for survival (Figure S3b, below). The frozen samples were maintained at -80°C for subsequent RNA extraction.

**Viral stabbing experiment.** For the fourth experiment, we stabbed aphids with a solution containing Aphid Lethal Paralysis Virus (ALPV), a RNA virus found in natural populations that is lethal to pea aphids (Georgievska, Miller & Bonning, unpublished data) [5, 6]. The solution was made by grinding up virus-killed aphid cadavers in 7.5 µL of water per cadaver. After stabbing, aphids were isolated for an hour and then kept on fava bean plants until 16 hours from initial stabbing, when five aphids per condition were removed from the plants, frozen in liquid nitrogen, and stored at -80°C prior to RNA extraction. Unstabbed control aphids, raised and treated under similar conditions, were frozen as well. In addition, 20 stabbed and 20 unstabbed aphids were monitored over a period of 96 hours for survival (Figure S3c, below). Though it is possible that responses to stabs with the virus-slurry would be a result of exposure to other pathogens in the ground-up cadaver, stabs with slurry from non-virus killed aphid cadavers do not lead to a significant increase in mortality (Parker, unpublished data), and thus we expect that responses can be largely attributed to virus exposure.
**Stress experiment.** For the fifth experiment, we exposed aphids to a number of stressors. We exposed one group of aphids on plants to 3 µL of (*E*)-β farnesene (EBF) (1 µg/µL in hexane), which we placed on a small piece of filter paper near the base of each plant. We enclosed the plants under a plastic cover with no ventilation. Within minutes of exposure, we froze five EBF-exposed aphids, and five control, unexposed aphids. Twelve hours later, we froze another five aphids that had been exposed to EBF. We heat shocked a second group of aphids following standard procedures [7]. After two hours at 36°C, we froze five heat-shocked aphids and then maintained another five heat-shocked aphids at 20°C for another eight hours prior to freezing. To assess the affects of starvation, we placed a third batch of aphids into sterile Petri dishes with moistened filter paper for 12 hours prior to freezing.

**Quantitative PCR.** For each sample, we extracted RNA from five whole aphids using a Qiagen RNA easy tissue kit and prepared cDNA from each sample using a Qiagen Quantitect reverse transcription kit. We carried out expression studies, utilizing the delta-delta CT method, on an Applied Biosystems Step One Plus machine. Each reaction contained 10 µL AB Power SYBR PCR master mix, 300 nM of each primer, approximately 100 ng cDNA, and water to a total volume of 20 µL. We designed primers utilizing Primer3 [8] or Primer Express (Applied Biosystems) to amplify approximately 100 bps of the gene of interest (Table S3, below). Primers spanned an exon-exon boundary where possible. For each sample, we carried out three separate reactions for each primer pair, and averaged the comparative threshold cycle (Ct) among the three values. We standardized all Ct values for the gene of interest relative to the Ct values for the endogenous control gene actin, yielding the delta Ct value. We then standardized relative to the appropriate control, yielding the delta-delta Ct value. Finally, these delta-delta Ct values were standardized such that each control treatment average was one, yielding the relative quantity (RQ) values, to allow for comparison with studies reporting fold-changes.
<table><caption>**Table S1. Pea aphid immune and stress gene list.** Genes are listed in the approximate order to which they are mentioned in the text. The last column indicates results of a tblastn search (contig ID and e-value) of all identified pea aphid genes against an EST sequence database for *Myzus persicae*, the green peach aphid. All pea aphid sequences are accessible at AphidBase Gbrowse [9]. All green peach aphid sequences can be downloaded at AphidBase Downloads (files Myzus454 and MyzusSanger) [10].</caption><thead><tr><th>Class</th><th>Role in Insect Immunity</th><th>Gene Symbol</th><th>Gene Name</th><th>Other Gene Names</th><th>ACYPI identifier</th><th>NCBI gene ID</th><th>NCBI transcript ID</th><th>NCBI protein ID</th><th>Myzus ID (e-value)</th></tr></thead><tbody><tr><td>recognition</td><td>bacterial recognition</td><td>PGRP</td><td>Peptidoglycan recognition protein</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>recognition</td><td>bacterial and fungal pattern recognition</td><td>GNBP1</td><td>Gram Negative Binding Protein 1</td><td></td><td>ACYPI005376</td><td>100164352</td><td>XM_001944438.1</td><td>XP_001944473.1</td><td>6372 (7e-134)</td></tr><tr><td>recognition</td><td>bacterial and fungal pattern recognition</td><td>GNBP2</td><td>Gram Negative Binding Protein 2</td><td></td><td>ACYPI006143</td><td>100165182</td><td>XM_001947795.1</td><td>XP_001947830.1</td><td>3883 (4e-178)</td></tr><tr><td>recognition</td><td>bacterial recognition, induction of phenoloxidase</td><td>Ctl1</td><td>C-type Lectin 1</td><td></td><td>ACYPI004676</td><td>100163601</td><td>XM_001944997.1</td><td>XP_001945032.1</td><td>26603 (3e-39)</td></tr><tr><td>recognition</td><td>bacterial recognition, induction of phenoloxidase</td><td>Ctl2</td><td>C-type Lectin 2</td><td></td><td>ACYPI005998</td><td>100165024</td><td>XM_001946121.1</td><td>XP_001946156.1</td><td>26603 (3e-38)</td></tr><tr><td>recognition</td><td>bacterial recognition, induction of phenoloxidase</td><td>Ctl3</td><td>C-type Lectin 3</td><td></td><td>ACYPI004682</td><td>100163607</td><td>XM_001943575.1</td><td>XP_001943610.1</td><td>60035 (3e-6)</td></tr><tr><td>recognition</td><td>bacterial recognition, induction of phenoloxidase</td><td>Ctl</td><td>C-type Lectin galactose-binding</td><td></td><td>ACYPI009411</td><td>100168734</td><td>XM_001951375.1</td><td>XP_001951410.1</td><td>3644 (3e-130)</td></tr><tr><td>recognition</td><td>bacterial recognition, induction of phenoloxidase</td><td>Ctl</td><td>C-type Lectin selectin-like</td><td></td><td>ACYPI003045</td><td>100161853</td><td>XM_001942942.1</td><td>XP_001942977.1</td><td>73637 (1e-31)</td></tr><tr><td>recognition</td><td>several roles have been hypothesized</td><td>gale1</td><td>galectin 1</td><td>Galactoside-binding soluble lectin</td><td>ACYPI001371</td><td>100160038</td><td>XM_001943734.1</td><td>XP_001943769.1</td><td>30445 (2e-6)</td></tr><tr><td>recognition</td><td>several roles have been hypothesized</td><td>gale2</td><td>galectin 2</td><td>Galactoside-binding soluble lectin</td><td>ACYPI000409</td><td>100158995</td><td>XM_001947578.1</td><td>XP_001947613.1</td><td>24692 (8e-32)</td></tr><tr><td>recognition</td><td>bind to lipoproteins, bacteria</td><td>sr-CI</td><td>Scavenger receptor class C, type I</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>recognition</td><td>bind to lipoproteins, bacteria</td><td>sr-CII</td><td>Scavenger receptor class C, type II</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>recognition</td><td>bind to lipoproteins, bacteria</td><td>sr-CIII</td><td>Scavenger receptor class C, type III</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>recognition</td><td>bind to lipoproteins, bacteria</td><td>sr-CIV</td><td>Scavenger receptor class C, type IV</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>recognition</td><td>receptor in phagocytosis and microbial binding</td><td>eater</td><td>eater</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>recognition</td><td>receptor in phagocytosis and microbial binding</td><td>nim-C1</td><td>nimrod C1</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>signaling</td><td>toll pathway</td><td>spz1-1</td><td>spätzle 1Bi</td><td>spatzle 1b</td><td>ACYPI004362</td><td>100163265</td><td>XM_001950373.1</td><td>XP_001950408.1</td><td>649 (1e-99)</td></tr><tr><td>signaling</td><td>toll pathway</td><td>spz1-2</td><td>spätzle 1Bii</td><td>spatzle 1b</td><td>ACYPI001858</td><td>100160571</td><td>XM_001947931.1</td><td>XP_001947966.1</td><td>649 (6e-119)</td></tr><tr><td>signaling</td><td>toll pathway</td><td>spz1-3</td><td>spätzle 1-3</td><td>spatzle 1b, spaetzle 1b</td><td>ACYPI41073</td><td></td><td></td><td></td><td>649 (4e-83)</td></tr><tr><td>signaling</td><td>toll pathway</td><td>spz1-4</td><td>spätzle 1-4</td><td>spatzle 1b, spaetzle 1b</td><td>ACYPI52992</td><td></td><td></td><td></td><td>649 (5e-71)</td></tr></tbody></table>
<table><thead><tr><th>Class</th><th>Role in Insect Immunity</th><th>Gene Symbol</th><th>Gene Name</th><th>Other Gene Names</th><th>ACYPI identifier</th><th>NCBI gene ID</th><th>NCBI transcript ID</th><th>NCBI protein ID</th><th>Myzus ID (e-value)</th></tr></thead><tbody><tr><td>signaling</td><td>toll pathway</td><td>spz1-5</td><td>spätzle 1-5</td><td>spatzle 1b, spaetzle 1b</td><td>ACYPI21155</td><td></td><td></td><td></td><td>649 (3e-65)</td></tr><tr><td>signaling</td><td>toll pathway</td><td>spz2</td><td>spätzle 2</td><td>spatzle 2</td><td>ACYPI003414</td><td>100162252</td><td>XM_001948424.1</td><td>XP_001948459.1</td><td>649 (2e-10)</td></tr><tr><td>signaling</td><td>toll pathway</td><td>spz3</td><td>spätzle 3</td><td>spatzle 3</td><td>ACYPI55738</td><td></td><td></td><td></td><td>649 (3e-4)</td></tr><tr><td>signaling</td><td>toll pathway</td><td>spz4</td><td>spätzle 4</td><td>spatzle 4</td><td>ACYPI006811</td><td>100165897</td><td>XM_001949337.1</td><td>XP_001949372.1</td><td>no hit</td></tr><tr><td>signaling</td><td>toll pathway</td><td>spz6</td><td>spätzle 6</td><td>spatzle 6</td><td>ACYPI001990</td><td>100160712</td><td>XM_001944011.1</td><td>XP_001944046.1</td><td>no hit</td></tr><tr><td>signaling</td><td>toll pathway</td><td>spz-like</td><td>spätzle-like, partial</td><td>spatzle, spaetzle</td><td>ACYPI009165</td><td>100168467</td><td>XM_001946778.1</td><td>XP_001946813.1</td><td>no hit</td></tr><tr><td>signaling</td><td>some tolls function in toll signaling pathway</td><td>18w</td><td>18 wheeler</td><td>Toll, Toll-2</td><td>ACYPI008698</td><td>100167952</td><td>XM_001946908.1</td><td>XP_001946943.1</td><td>6369 (6e-28)</td></tr><tr><td>signaling</td><td>some tolls function in toll signaling pathway</td><td>Toll</td><td>Toll-like</td><td></td><td>ACYPI000177</td><td>100158739</td><td>XM_001942698.1</td><td>XP_001942733.1</td><td>5712 (4e-115)</td></tr><tr><td>signaling</td><td>some tolls function in toll signaling pathway</td><td>Toll</td><td>Toll-like</td><td></td><td>ACYPI002340</td><td>100161089</td><td>XM_001946411.1</td><td>XP_001946446.1</td><td>5712 (4e-122)</td></tr><tr><td>signaling</td><td>some tolls function in toll signaling pathway</td><td>Toll</td><td>Toll-like, partial</td><td></td><td>ACYPI004287</td><td>100163187</td><td>XM_001949147.1</td><td>XP_001949182.1</td><td>5712 (2e-109)</td></tr><tr><td>signaling</td><td>some tolls function in toll signaling pathway</td><td>Toll-6</td><td>Toll-6</td><td></td><td>ACYPI005417</td><td>100164395</td><td>XM_001947289.1</td><td>XP_001947324.1</td><td>6369 (1e-34)</td></tr><tr><td>signaling</td><td>some tolls function in toll signaling pathway</td><td>Toll</td><td>Toll-like</td><td></td><td>ACYPI008268</td><td>100167471</td><td>XM_001950727.1</td><td>XP_001950762.1</td><td>6369 (5e-19)</td></tr><tr><td>signaling</td><td>some tolls function in toll signaling pathway</td><td>Tollo</td><td>Tollo</td><td>Toll-8</td><td>ACYPI002754</td><td>100161538</td><td>XM_001948531.1</td><td>XP_001948566.1</td><td>6369 (1e-44)</td></tr><tr><td>signaling</td><td>toll pathway</td><td>tub</td><td>tube</td><td>interleukin-1 receptor-associated kinase 4</td><td>ACYPI006580</td><td>100165647</td><td>XM_001950581.1</td><td>XP_001950616.1</td><td>4836 (2e-113)</td></tr><tr><td>signaling</td><td>toll pathway</td><td>Myd88</td><td>myeloid differentiation primary response gene</td><td></td><td>ACYPI001638</td><td>100160335</td><td>XM_001948285.1</td><td>XP_001948320.1</td><td>1295 (9e-168)</td></tr><tr><td>signaling</td><td>toll pathway</td><td></td><td>pelle</td><td></td><td>ACYPI009928</td><td>100169297</td><td>XM_001942995.1</td><td>XP_001943030.1</td><td>4836 (2e-38)</td></tr><tr><td>signaling</td><td>toll pathway</td><td>cact</td><td>cactus</td><td></td><td>ACYPI006820</td><td>100165906</td><td>XM_001950793.1</td><td>XP_001950828.1</td><td>259 (3e-135)</td></tr><tr><td>signaling</td><td>toll pathway</td><td>cactin</td><td>cactin</td><td></td><td>ACYPI006968</td><td>100166064</td><td>XM_001952252.1</td><td>XP_001952287.1</td><td>2493 (0)</td></tr><tr><td>signaling</td><td>toll pathway</td><td>Pli</td><td>Pellino</td><td></td><td>ACYPI001694</td><td>100160395</td><td>XM_001946247.1</td><td>XP_001946282.1</td><td>9336 (3e-71)</td></tr><tr><td>signaling</td><td>toll pathway</td><td>Traf1</td><td>TNF-receptor-associated factor 1</td><td></td><td>ACYPI000855</td><td>100159489</td><td>XM_001948320.1</td><td>XP_001948355.1</td><td>24857 (2e-7)</td></tr><tr><td>signaling</td><td>toll pathway</td><td>Traf2</td><td>TNF-receptor-associated factor 2</td><td>dTraf2, Traf6, TNF-receptor-associated factor 6</td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>signaling</td><td>toll pathway</td><td>dl</td><td>dorsal</td><td></td><td>ACYPI003588</td><td>100162436</td><td>XM_001947394.1</td><td>XP_001947429.1</td><td>6239 (2e-119)</td></tr><tr><td>signaling</td><td>toll pathway</td><td>dIB</td><td>dorsal B</td><td></td><td>ACYPI005133</td><td>100164092</td><td>XM_001949463.1</td><td>XP_001949498.1</td><td>6239 (3e-124)</td></tr><tr><td>signaling</td><td>jak/stat pathway</td><td>dome-1</td><td>domeless 1</td><td></td><td>ACYPI21995</td><td>100294629</td><td></td><td></td><td>57862 (9e-8)</td></tr></tbody></table>
<table><thead><tr><th>Class</th><th>Role in Insect Immunity</th><th>Gene Symbol</th><th>Gene Name</th><th>Other Gene Names</th><th>ACYPI identifier</th><th>NCBI gene ID</th><th>NCBI transcript ID</th><th>NCBI protein ID</th><th>Myzus ID (e-value)</th></tr></thead><tbody><tr><td>signaling</td><td>jak/stat pathway</td><td>dome-2</td><td>domeless 2</td><td></td><td>ACYPI004970</td><td>100163919</td><td></td><td></td><td>57862 (6e-8)</td></tr><tr><td>signaling</td><td>jak/stat pathway</td><td>dome-3</td><td>domeless 3</td><td></td><td>ACYPI21996</td><td>100294630</td><td></td><td></td><td>57862 (3e-8)</td></tr><tr><td>signaling</td><td>jak/stat pathway</td><td>dome-4</td><td>domeless 4, 5' partial</td><td></td><td>ACYPI40957</td><td>100294721</td><td></td><td></td><td>no hit</td></tr><tr><td>signaling</td><td>jak/stat pathway</td><td>Jak</td><td>Janus kinase</td><td>hopscotch</td><td>ACYPI008118</td><td>100167312</td><td>XM_001948012.1</td><td>XP_001948047.1</td><td>1739 (2e-158)</td></tr><tr><td>signaling</td><td>jak/stat pathway</td><td>Stat92E-1</td><td>Signal-transducer and activator of transcription 1</td><td>Stat</td><td>ACYPI002351</td><td>100161101</td><td>XM_001946610.1</td><td>XP_001946645.1</td><td>2173 (4e-114)</td></tr><tr><td>signaling</td><td>jak/stat pathway</td><td>Stat92E-2</td><td>Signal-transducer and activator of transcription 1, partial</td><td>Stat</td><td>ACYPI005642</td><td>100164649</td><td>XM_001943623.1</td><td>XP_001943658.1</td><td>65719 (5e-27)</td></tr><tr><td>signaling</td><td>jak/stat pathway</td><td>upd</td><td>unpaired</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>signaling</td><td>imd pathway</td><td>imd</td><td>immune deficiency</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>signaling</td><td>imd pathway</td><td>dFadd</td><td>dFadd</td><td>Drosophila BG4, FADD</td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>signaling</td><td>imd pathway</td><td>Dredd</td><td>Death related ced-3</td><td>caspase 8</td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>signaling</td><td>imd pathway</td><td>Rel</td><td>Relish</td><td>Nf-KB, REL</td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>signaling</td><td>imd pathway</td><td>Tab2</td><td>TAK1-associated Binding Protein2</td><td></td><td>ACYPI002796</td><td>100161584</td><td>XM_001950344.1</td><td>XP_001950379.1</td><td>5307 (5e-106)</td></tr><tr><td>signaling</td><td>imd pathway</td><td>Tak1</td><td>TGF-β activated kinase 1</td><td></td><td>ACYPI001063</td><td>100159713</td><td>XM_001944422.1</td><td>XP_001944457.1</td><td>2842 (3e-88)</td></tr><tr><td>signaling</td><td>imd pathway</td><td>key</td><td>kenny</td><td>IKKgamma</td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>not found</td></tr><tr><td>signaling</td><td>imd pathway</td><td>lap2</td><td>Inhibitor of apoptosis 2</td><td></td><td>ACYPI000445</td><td>100159034</td><td>XM_001942899.1</td><td>XP_001942934.1</td><td>60268 (2e-27)</td></tr><tr><td>signaling</td><td>imd pathway</td><td>ird5</td><td>immune response deficiency 5</td><td>IKK</td><td>ACYPI004933</td><td>100163880</td><td>XM_001952347.1</td><td>XP_001952382.1</td><td>7578 (3e-90)</td></tr><tr><td>signaling</td><td>jnk pathway</td><td>hep</td><td>hemipterous</td><td></td><td>ACYPI005993</td><td>100165019</td><td>XM_001944492.1</td><td>XP_001944527.1</td><td>8122 (8e-46)</td></tr><tr><td>signaling</td><td>jnk pathway</td><td>bsk</td><td>basket</td><td>MAPK, JNK</td><td>ACYPI004372</td><td>100163276</td><td>XM_001945425.1</td><td>XP_001945460.1</td><td>1929 (1e-88)</td></tr><tr><td>signaling</td><td>jnk pathway</td><td>Jra</td><td>Jun-related antigen</td><td></td><td>ACYPI002386</td><td>100161138</td><td>XM_001947521.1</td><td>XP_001947556.1</td><td>6809 (1e-89)</td></tr><tr><td>signaling</td><td>jnk pathway</td><td>kay</td><td>kayak</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>signaling</td><td>JNK pathway</td><td>egr</td><td>eiger</td><td></td><td>ACYPI001133</td><td>100159786</td><td>XM_001952555.1</td><td>XP_001952590.1</td><td>1287(2e-131)</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Abaecin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Alloferon</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Andropin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Apisimin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr></tbody></table>
<table><thead><tr><th>Class</th><th>Role in Insect Immunity</th><th>Gene Symbol</th><th>Gene Name</th><th>Other Gene Names</th><th>ACYPI identifier</th><th>NCBI gene ID</th><th>NCBI transcript ID</th><th>NCBI protein ID</th><th>Myzus ID (e-value)</th></tr></thead><tbody><tr><td>response</td><td>antimicrobial peptide</td><td>Att</td><td>Attacin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td>Cec</td><td>Cecropin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Coleoptericin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td>Def</td><td>Defensin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Diptericin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Drosocin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Drosomycin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Formicin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Gambicin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Gomesin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Heliocin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Holotricin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Lebocin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Megourin</td><td></td><td>not found, though see text</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Metchnikowin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Moricin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Penaeidin</td><td></td><td>not found, though see text</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Polyphemusin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Spingerin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Tachyplesin</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td></td><td>Virescein</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td>Thm1</td><td>Thaumatin1</td><td>Tha, Thn</td><td>ACYPI005841</td><td>100164856</td><td>XM_001942683.1</td><td>XP_001942718.1</td><td>81361 (1e-8)</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td>Thm2</td><td>Thaumatin2</td><td>Tha, Thn</td><td>ACYPI001394</td><td>100160062</td><td>XM_001942537.1</td><td>XP_001942572.1</td><td>81361 (2e-8)</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td>Thm3</td><td>Thaumatin3</td><td>Tha, Thn</td><td>ACYPI009605</td><td>100168942</td><td>XM_001942744.1</td><td>XP_001942779.1</td><td>81361 (2e-9)</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td>Thm4</td><td>Thaumatin4</td><td>Tha, Thn</td><td>ACYPI003287</td><td>100162111</td><td>XM_001942495.1</td><td>XP_001942530.1</td><td>81361 (2e-9)</td></tr><tr><td>response</td><td>antimicrobial peptide</td><td>Thm5</td><td>Thaumatin5</td><td>Tha, Thn</td><td>ACYPI007568</td><td>100166717</td><td>XM_001951871.1</td><td>XP_001951906.1</td><td>9364 (5e-22)</td></tr></tbody></table>
<table><thead><tr><th>Class</th><th>Role in Insect Immunity</th><th>Gene Symbol</th><th>Gene Name</th><th>Other Gene Names</th><th>ACYPI identifier</th><th>NCBI gene ID</th><th>NCBI transcript ID</th><th>NCBI protein ID</th><th>Myzus ID (e-value)</th></tr></thead><tbody><tr><td>response</td><td>antimicrobial peptide</td><td>Thm6</td><td>Thaumatin6</td><td>Tha, Thn</td><td>ACYPI005301</td><td>100164271</td><td>XM_001942753.1</td><td>XP_001942788.1</td><td>81361 (3e-15)</td></tr><tr><td>response</td><td>microbial degradation</td><td>Lys1</td><td>Lysozyme, i-type</td><td></td><td>ACYPI002175</td><td>100160909</td><td>XM_001949053.1</td><td>XP_001949088.1</td><td>3094 (8e-63)</td></tr><tr><td>response</td><td>microbial degradation</td><td>Lys2</td><td>Lysozyme, i-type</td><td></td><td>ACYPI009125</td><td>100168424</td><td>XM_001949177.1</td><td>XP_001949212.1</td><td>584 (3e-80)</td></tr><tr><td>response</td><td>microbial degradation</td><td>Lys3</td><td>Lysozyme, i-type</td><td></td><td>ACYPI008509</td><td>100167742</td><td>XM_001949283.1</td><td>XP_001949318.1</td><td>74639 (7e-27)</td></tr><tr><td>response</td><td>fungal degradation</td><td>Cht1</td><td>Chitinase-like protein 1</td><td></td><td>ACYPI001365</td><td>100160032</td><td>XM_001943565.1</td><td>XP_001943600.1</td><td>3640 (0)</td></tr><tr><td>response</td><td>fungal degradation</td><td>Cht2</td><td>Chitinase-like protein 2</td><td>Drosophila chitinase 3</td><td>ACYPI010095</td><td>100169480</td><td>XM_001943003.1</td><td>XP_001943038.1</td><td>5202 (1e-52)</td></tr><tr><td>response</td><td>fungal degradation</td><td>Cht3</td><td>Chitinase-like protein 3</td><td></td><td>ACYPI001396</td><td>100160065</td><td>XM_001942561.1</td><td>XP_001942596.1</td><td>5202 (6e-73)</td></tr><tr><td>response</td><td>fungal degradation</td><td>Cht4</td><td>Chitinase-like protein 4</td><td></td><td>ACYPI006403</td><td>100165452</td><td>XM_001950345.1</td><td>XP_001950380.1</td><td>5202 (2e-168)</td></tr><tr><td>response</td><td>fungal degradation</td><td>Cht5</td><td>Chitinase-like protein 5</td><td></td><td>ACYPI009964</td><td>100169337</td><td>XM_001947381.1</td><td>XP_001947416.1</td><td>5202 (2e-52)</td></tr><tr><td>response</td><td>fungal degradation</td><td>Cht6</td><td>Chitinase-like protein 6</td><td></td><td>ACYPI009878</td><td>100169240</td><td>XM_001952683.1</td><td>XP_001952718.1</td><td>5202 (3e-50)</td></tr><tr><td>response</td><td>fungal degradation</td><td>Cht7</td><td>Chitinase-like protein 7</td><td></td><td>ACYPI005756</td><td>100164767</td><td>XM_001947852.1</td><td>XP_001947887.1</td><td>1327 (4e-162)</td></tr><tr><td>response</td><td>general stress response</td><td>TotA</td><td>Turandot A</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>general stress response</td><td>TotB</td><td>Turandot B</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>general stress response</td><td>TotC</td><td>Turandot C</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>general stress response</td><td>TotF</td><td>Turandot F</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>general stress response</td><td>TotM</td><td>Turandot M</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>general stress response</td><td>TotZ</td><td>Turandot Z</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>mark pathogens for phagocytosis</td><td>Tepl</td><td>Thiolester containing protein I</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>mark pathogens for phagocytosis</td><td>TeplI</td><td>Thiolester containing protein II</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>mark pathogens for phagocytosis</td><td>TeplII-1</td><td>Thiolester containing protein III - 1, partial</td><td></td><td>ACYPI005292</td><td>100164261</td><td>XM_001944313.1</td><td>XP_001944348.1</td><td>no hit</td></tr><tr><td>response</td><td>mark pathogens for phagocytosis</td><td>TeplII-2</td><td>Thiolester containing protein III - 2</td><td></td><td>ACYPI000145</td><td>100158705</td><td>XM_001945685.1</td><td>XP_001945720.1</td><td>no hit</td></tr><tr><td>response</td><td>mark pathogens for phagocytosis</td><td>TeplIV</td><td>Thiolester containing protein IV</td><td></td><td>not found</td><td>not found</td><td>not found</td><td>not found</td><td>n/a</td></tr><tr><td>response</td><td>prophenoloxidase response</td><td>ProPO1</td><td>Prophenoloxidase 1</td><td>Diphenol oxidase A3</td><td>ACYPI001367</td><td>100160034</td><td>XM_001949272.1</td><td>XP_001949307.1</td><td>5431 (5e-147)</td></tr><tr><td>response</td><td>prophenoloxidase response</td><td>ProPO2</td><td>Prophenoloxidase 2</td><td>Diphenol oxidase A3</td><td>ACYPI004484</td><td>100163393</td><td>XM_001951102.1</td><td>XP_001951137.1</td><td>5721 (e3-133)</td></tr><tr><td>response</td><td>production of nitric oxide, a toxic gas</td><td>Nos</td><td>Nitric oxide synthase</td><td></td><td>ACYPI001689</td><td>100160390</td><td>XM_001946174.1</td><td>XP_001946209.1</td><td>462 (1e-35)</td></tr></tbody></table>
<table><thead><tr><th>Class</th><th>Role in Insect Immunity</th><th>Gene Symbol</th><th>Gene Name</th><th>Other Gene Names</th><th>ACYPI identifier</th><th>NCBI gene ID</th><th>NCBI transcript ID</th><th>NCBI protein ID</th><th>Myzus ID (e-value)</th></tr></thead><tbody><tr><td>response</td><td>general stress response</td><td>Hsc5</td><td>Heat shock cognate 5</td><td></td><td>ACYPI004693</td><td>100163620</td><td>XM_001950464.1</td><td>XP_001950499.1</td><td></td></tr><tr><td>response</td><td>general stress response</td><td>Hsc70</td><td>Heat shock cognate 70</td><td></td><td>ACYPI007166</td><td>100166283</td><td>XM_001948031.1</td><td>XP_001948066.1</td><td>1624 (0)</td></tr><tr><td>response</td><td>general stress response</td><td>Hsc70-1</td><td>Heat shock cognate 70 - 1</td><td></td><td>ACYPI000474</td><td>100159065</td><td>XM_001951198.1</td><td>XP_001951233.1</td><td>182 (0)</td></tr><tr><td>response</td><td>general stress response</td><td>Hsc70-2</td><td>Heat shock cognate 70 - 2</td><td></td><td>ACYPI004809</td><td>100163748</td><td>XM_001951351.1</td><td>XP_001951386.1</td><td>182 (0)</td></tr><tr><td>response</td><td>general stress response</td><td>Hsc70Cb</td><td>Heat shock cognate 70 - Cb</td><td></td><td>ACYPI004544</td><td>100163455</td><td>XM_001951757.1</td><td>XP_001951792.1</td><td>2297 (0)</td></tr><tr><td>response</td><td>general stress response</td><td>Hsp14</td><td>Heat shock protein 14</td><td></td><td>ACYPI002719</td><td>100161502</td><td>XM_001945733.1</td><td>XP_001945768.1</td><td>3375 (0)</td></tr><tr><td>response</td><td>general stress response</td><td>Hsp21.4</td><td>Heat shock protein 21.4</td><td></td><td>ACYPI003907</td><td>100162777</td><td>XM_001949367.1</td><td>XP_001949402.1</td><td>6174 (2e-108)</td></tr><tr><td>response</td><td>general stress response</td><td>Hsp60</td><td>Heat shock protein 60</td><td></td><td>ACYPI009253</td><td>100168563</td><td>XM_001951338.1</td><td>XP_001951373.1</td><td>317 (6e-155)</td></tr><tr><td>response</td><td>general stress response</td><td>Hsp70Aa</td><td>Heat shock protein 70Aa</td><td></td><td>ACYPI009117</td><td>100168413</td><td>XM_001951880.1</td><td>XP_001951915.1</td><td>182 (0)</td></tr><tr><td>response</td><td>general stress response</td><td>Hsp70Ab</td><td>Heat shock protein 70Ab</td><td></td><td>ACYPI007961</td><td>100167145</td><td>XM_001949626.1</td><td>XP_001949661.1</td><td>182 (0)</td></tr><tr><td>response</td><td>general stress response</td><td>Hsp70Ba</td><td>Heat shock protein 70Ba</td><td></td><td>ACYPI008763</td><td>100168026</td><td>XM_001949802.1</td><td>XP_001949837.1</td><td>182 (0)</td></tr><tr><td>response</td><td>general stress response</td><td>Hsp83</td><td>Heat shock protein 83</td><td></td><td>ACYPI009380</td><td>100168702</td><td>XM_001944726.1</td><td>XP_001944761.1</td><td>1268 (0)</td></tr><tr><td>response</td><td>general stress response</td><td>Hsp83</td><td>Heat shock protein 83</td><td></td><td>ACYPI002010</td><td>100160736</td><td>XM_001943137.1</td><td>XP_001943172.1</td><td>1268 (0)</td></tr><tr><td>response</td><td>general stress response</td><td>Hsp90</td><td>Heat shock protein 90</td><td></td><td>ACYPI002398</td><td>100161155</td><td>XM_001951175.1</td><td>XP_001951210.1</td><td>5927 (1e-149)</td></tr><tr><td>response</td><td>general stress response</td><td>Hsp90</td><td>Heat shock protein 90</td><td></td><td>ACYPI009915</td><td>100169283</td><td>XM_001948902.1</td><td>XP_001948937.1</td><td>3803 (0)</td></tr><tr><td>response</td><td>detoxification</td><td>Gst</td><td>Glutathione S-transferase-like, microsomal</td><td></td><td>ACYPI004835</td><td>100163775</td><td>XM_001951200.1</td><td>XP_001951235.1</td><td>3849 (8e-76)</td></tr><tr><td>response</td><td>detoxification</td><td>Gst</td><td>Glutathione S-transferase-like, microsomal</td><td></td><td>ACYPI006691</td><td>100165764</td><td>XM_001946296.1</td><td>XP_001946331.1</td><td>3849 (5e-37)</td></tr><tr><td>response</td><td>detoxification</td><td>Gst</td><td>Glutathione S-transferase-like, sigma class</td><td></td><td>ACYPI000794</td><td>100159421</td><td>XM_001952064.1</td><td>XP_001952099.1</td><td>3513 (3e-99)</td></tr><tr><td>response</td><td>detoxification</td><td>Gst</td><td>Glutathione S-transferase-like, sigma class</td><td></td><td>ACYPI002127</td><td>100160859</td><td>XM_001952005.1</td><td>XP_001952040.1</td><td>3896 (1e-105)</td></tr><tr><td>response</td><td>detoxification</td><td>Gst</td><td>Glutathione S-transferase-like, sigma class</td><td></td><td>ACYPI002679</td><td>100161459</td><td>XM_001952021.1</td><td>XP_001952056.1</td><td>3896 (1e-100)</td></tr><tr><td>response</td><td>detoxification</td><td>Gst</td><td>Glutathione S-transferase-like, sigma class</td><td></td><td>ACYPI009326</td><td>100168645</td><td>XM_001952392.1</td><td>XP_001952427.1</td><td>1197 (6e-82)</td></tr><tr><td>response</td><td>detoxification</td><td>Gst</td><td>Glutathione S-transferase-like, sigma class</td><td></td><td>ACYPI009519</td><td>100168850</td><td>XM_001946569.1</td><td>XP_001946604.1</td><td>1197 (2e-102)</td></tr><tr><td>response</td><td>detoxification</td><td>Gst</td><td>Glutathione S-transferase-like, sigma class</td><td></td><td>ACYPI009520</td><td>100168850</td><td>XM_001946504.1</td><td>XP_001946539.1</td><td>1197 (2e-102)</td></tr><tr><td>response</td><td>detoxification</td><td>GstD</td><td>Glutathione S-transferase, delta class</td><td></td><td>ACYPI006899</td><td>100165990</td><td>XM_001951401.1</td><td>XP_001951436.1</td><td>2376 (4e-56)</td></tr><tr><td>response</td><td>detoxification</td><td>GstD</td><td>Glutathione S-transferase, delta class</td><td></td><td>ACYPI009586</td><td>100168923</td><td>XM_001952561.1</td><td>XP_001952596.1</td><td>2376 (5e-38)</td></tr></tbody></table>
<table><thead><tr><td>Class</td><td>Role in Insect Immunity</td><td>Gene Symbol</td><td>Gene Name</td><td>Other Gene Names</td><td>ACYPI identifier</td><td>NCBI gene ID</td><td>NCBI transcript ID</td><td>NCBI protein ID</td><td>Myzus ID (e-value)</td></tr></thead><tbody><tr><td>response</td><td>detoxification</td><td>GstD10</td><td>Glutathione S-transferase, delta class</td><td></td><td>ACYPI008042</td><td>100167231</td><td>XM_001948159.1</td><td>XP_001948194.1</td><td>2376 (7e-73)</td></tr><tr><td>response</td><td>detoxification</td><td>GstD4</td><td>Glutathione S-transferase, delta class</td><td></td><td>ACYPI001068</td><td>100159718</td><td>XM_001942679.1</td><td>XP_001942714.1</td><td>2376 (1e-84)</td></tr><tr><td>response</td><td>detoxification</td><td>GstD6</td><td>Glutathione S-transferase, delta class</td><td></td><td>ACYPI006598</td><td>100165666</td><td>XM_001952338.1</td><td>XP_001952373.1</td><td>2376 (4e-53)</td></tr><tr><td>response</td><td>detoxification</td><td>GstD6</td><td>Glutathione S-transferase, delta class, partial</td><td></td><td>ACYPI008550</td><td>100167788</td><td>XM_001952381.1</td><td>XP_001952416.1</td><td>2376 (3e-52)</td></tr><tr><td>response</td><td>detoxification</td><td>GstD8</td><td>Glutathione S-transferase, delta class</td><td></td><td>ACYPI008657</td><td>100167906</td><td>XM_001942576.1</td><td>XP_001942611.1</td><td>2376 (9e-112)</td></tr><tr><td>response</td><td>detoxification</td><td>GstD9</td><td>Glutathione S-transferase, delta class</td><td></td><td>ACYPI005620</td><td>100164626</td><td>XM_001950500.1</td><td>XP_001950535.1</td><td>569 (1e-123)</td></tr><tr><td>response</td><td>detoxification</td><td>Gst</td><td>Glutathione S-transferase, theta class</td><td></td><td>ACYPI007233</td><td>100166353</td><td>XM_001949321.1</td><td>XP_001949356.1</td><td>6981 (2e-79)</td></tr><tr><td>response</td><td>detoxification</td><td>Gst</td><td>Glutathione S-transferase, theta class</td><td></td><td>ACYPI009122</td><td>100168419</td><td>XM_001949359.1</td><td>XP_001949394.1</td><td>9615 (3e-73)</td></tr><tr><td>response</td><td>alarm pheromone production</td><td>IPPS</td><td>Isoprenyl diphosphate synthase</td><td></td><td>ACYPI000050</td><td>100144905</td><td>NM_001126161.3</td><td>NP_001119633.3</td><td>912 (0)</td></tr><tr><td>response</td><td>alarm pheromone production</td><td>FPPS</td><td>similar to Farnesyl diphosphate synthase 2</td><td></td><td>ACYPI007080</td><td>100166187</td><td>XM_001950388.1</td><td>XP_001950423.1</td><td>912 (0)</td></tr></tbody></table>
<table><caption>Table S2. Samples for qPCR expression study.</caption><thead><tr><th>Sample Name</th><th>Sample Handling Notes</th></tr></thead><tbody><tr><td colspan="2"><strong>Bacterial Stabbing Experiment</strong></td></tr><tr><td>No stab control</td><td>Frozen 8hrs after exposure of treatment aphids</td></tr><tr><td>Sterile stab</td><td>Frozen 8hrs after exposure</td></tr><tr><td><em>E.coli</em> stab</td><td>Frozen 8hrs after exposure</td></tr><tr><td>Gram- pathogen stab</td><td>Frozen 8hrs after exposure</td></tr><tr><td>Gram+ pathogen stab</td><td>Frozen 8hrs after exposure</td></tr><tr><td colspan="2"><strong>Bacterial Feeding Experiment</strong></td></tr><tr><td>Feed control</td><td>Frozen 12hrs after exposure to diet</td></tr><tr><td><em>E. coli</em> feed</td><td>Frozen 12hrs after exposure to diet</td></tr><tr><td>Gram- pathogen feed</td><td>Frozen 12hrs after exposure to diet</td></tr><tr><td colspan="2"><strong>Fungal Shower Experiment</strong></td></tr><tr><td>Fungus control</td><td>Frozen 24hrs after exposure</td></tr><tr><td>Fungus infected</td><td>Frozen 24hrs after exposure</td></tr><tr><td colspan="2"><strong>Viral Stabbing Experiment</strong></td></tr><tr><td>Virus control (no stab)</td><td>Frozen 16hrs after exposure</td></tr><tr><td>Virus infected</td><td>Frozen 16hrs after exposure</td></tr><tr><td colspan="2"><strong>Stress Experiment</strong></td></tr><tr><td>No stress Control</td><td>Frozen at beginning of stress experiment</td></tr><tr><td>Alarm pheromone</td><td>Frozen minutes after exposure to EBF</td></tr><tr><td>Post alarm pheromone</td><td>Frozen 12hrs after exposure to EBF</td></tr><tr><td>Heat stress</td><td>Frozen immediately after 36°C heat shock</td></tr><tr><td>Post heat stress</td><td>Frozen 8hrs after being returned to 20°C</td></tr><tr><td>Starvation</td><td>Frozen 12hrs after being removed from plant</td></tr></tbody></table>
<table><caption>Table S3. Primers for qPCR expression study.</caption><thead><tr><td>Gene symbol</td><td>Gene name</td><td>Putative function</td><td>ACYPI ID</td><td>Primer Pair (5' to 3')</td></tr></thead><tbody><tr><td>GNBP2</td><td>gram-negative binding protein 2</td><td>recognition</td><td>ACYPI006143</td><td>gnbp2_1f: AATTTCCGTGATGGGTGTTTAAAGT<br>gnbp2_1r: TTTGTTTTCATCCATGTTGATGAC</td></tr><tr><td>Gale1</td><td>galectin 1</td><td>recognition and response</td><td>ACYPI001371</td><td>gale_1f: GCTCCAATACTCAATCCGACTCTT<br>gale_1r: CATCGTCCTTGTTTCAAACC</td></tr><tr><td>TI</td><td>toll</td><td>toll signaling pathway</td><td>ACYPI000177</td><td>tl1_1f: GAGCTCACCGTTTAAACTTTGTCA<br>tl1_1r: CATCAACTGAACGAGCAATTTGA</td></tr><tr><td>Cact</td><td>cactus</td><td>toll signaling pathway</td><td>ACYPI006820</td><td>cact_1f: GATGGCCAAAGTGCTCTTCATT<br>cact_1r: GAGCTTTCATTAGGTGTTTCACAATTT</td></tr><tr><td>DI</td><td>dorsal 1b</td><td>toll signaling pathway</td><td>ACYPI003588</td><td>dl_1f: CAAGAGAATAGAAAACCACATCGTCTA<br>dl_1r: AAACATCAGTGTTATGCGGCTAAG</td></tr><tr><td>DIB</td><td>dorsal 1b</td><td>toll signaling pathway</td><td>ACYPI005133</td><td>dlb_1f: CTCTCAGAGTACGAGAAGAAATAAGAGTAGAT<br>dlb_1r: AAACATCAGTGTTATGCGGCTAAG</td></tr><tr><td>Myd88</td><td>myeloid differentiation primary<br>response gene</td><td>toll signaling pathway</td><td>ACYPI001638</td><td>myd88_1f: TGCATGTTAAATGCCACGAAA<br>myd88_1r: TCCTCTCCAATCCCTGGGTAA</td></tr><tr><td>IRD5</td><td>immune response deficient 5</td><td>imd/jnk pathway</td><td>ACYPI004933</td><td>IRD5_1f: TGCTTATCTTGCACCGGAAGT<br>IRD5_1r: ACTATGACTCCAACACTCCACATATCTAA</td></tr><tr><td>Bsk</td><td>basket (JNK)</td><td>jnk pathway</td><td>ACYPI004372</td><td>Basket_1f: TTTGATCGATTATTCCTGATGTACT<br>Basket_1r: GTGCTTGGCTTGCTTTTAGTTTATT</td></tr><tr><td>JRA1</td><td>Jun-related antigen</td><td>imd/jnk pathway</td><td>ACYPI002386</td><td>Jra_1f: AAATCAAACTCGAAAGGAAAAGACA<br>Jra_1r: TTCGGCGGCATTTGGA</td></tr><tr><td>IAP2</td><td>inhibitor of apoptosis 2</td><td>imd/jnk pathway</td><td>ACYPI000445</td><td>lap2_10f: TCGATGAACACAAACGTCACAA<br>lap2_10r: GTTCACCAGTTTCCTTATGATTTTCAA</td></tr><tr><td>Stat92E-2</td><td>Signal-transducer and activator of<br>transcription 2</td><td>Jak/Stat pathway</td><td>ACYPI005642</td><td>Stat2_37f: TCATTAGTTCAGTGGAAACGTCAAC<br>Stat2_37r: AATCACACAATTTCTCACACCAAGTT</td></tr><tr><td>Lys1</td><td>lysozyme, i-type</td><td>response to bacteria</td><td>ACYPI002175</td><td>lysoz1_1f: CGCACAGGACTGCAACCA<br>lysoz1_1r: GGATGGCCGCGTAATCAG</td></tr><tr><td>Lys2</td><td>lysozyme, i-type</td><td>response to bacteria</td><td>ACYPI009125</td><td>Lys2_10f: GCGTCAGAGACCCGTATTGC<br>Lys2_10r: GCAATCCTTTGCGTATCTCTGTATG</td></tr><tr><td>Lys3</td><td>lysozyme, i-type</td><td>response to bacteria</td><td>ACYPI008509</td><td>lysoz3_1f: CCGGTCAGTAGCAGAGGAAAGT<br>lysoz3_1r: ATGAGCTCTCGCGTAGTTTGG</td></tr><tr><td>Thm2</td><td>thaumatin 2</td><td>response (antimicrobial peptide)</td><td>ACYPI001394</td><td>Tha2_1r1f: CAACAGTAAAGGAAAATGCGAAAC<br>Tha2_1r1r: TGGCACGCCCATGATACC</td></tr><tr><td>Thm3</td><td>thaumatin 3</td><td>response (antimicrobial peptide)</td><td>ACYPI009605</td><td>Tha3_1f: GGGCAGGCAGGATTTGG<br>Tha3_1r: TTGGATCTTGTTCCCGCAAT</td></tr><tr><td>Thm4</td><td>thaumatin 4</td><td>response (antimicrobial peptide)</td><td>ACYPI003287</td><td>Tha4_1f: GGCGGGCAGGATTTGG<br>Tha4_1r: GTGGATCTTGTTCCCGCAAT</td></tr><tr><td>Thm6</td><td>thaumatin 6</td><td>response (antimicrobial peptide)</td><td>ACYPI005301</td><td>Tha6_1f: AAAATGCAGCGCTCAAGGA<br>Tha6_1r: CACTTGATCTTGTTCCCGCAAT</td></tr><tr><td>HSP60</td><td>heat shock protein 60</td><td>response to stress</td><td>ACYPI009253</td><td>hsp60_1f: GATGCAATGAACGACGAATATGTTA<br>hsp60_1r: CTGACAACTTTGGTTGGATCGA</td></tr><tr><td>HSC70</td><td>heat shock cognate 70</td><td>response to stress</td><td>ACYPI007166</td><td>HP3f: TTGGGTGGAGAAGATTTTGA<br>HP3r: ATGACTGGCAGAAAGACCAC</td></tr><tr><td>HSP83</td><td>heat shock protein 83</td><td>response to stress</td><td>ACYPI002010</td><td>HP6f: CCGTACTGATCCTGGTGAAC<br>HP6r: GCCAATGAATTGAGAGTGCT</td></tr></tbody></table>
<table><caption>**Table S4. Expression of recognition and signaling genes.** Values indicate the relative expression of a gene in a sample relative to expression in the appropriate control sample, +/- one standard deviation. For the bacterial stabbing experiment, we compared a sterile stab sample to a no stab sample only for genes that showed greater than 2-fold upregulation in one of the bacteria-stabbed samples. Relative expression values should be interpreted with caution as they are based on only a single experimental replicate pooling five aphids.</caption><thead><tr><th></th><th>GNBP2</th><th>GALE1</th><th>TOLL</th><th>CACTUS</th><th>DOR1</th><th>DOR1B</th><th>MYD88</th><th>IRD5</th><th>JNK</th><th>JRA1</th><th>IAP2</th><th>JAK</th><th>STAT92E2</th></tr></thead><tbody><tr><td>No stab control</td><td>1.00+/-0.05</td><td>1.00+/-0.08</td><td>1.00+/-0.03</td><td>1.00+/-0.06</td><td>1.00+/-0.16</td><td>1.00+/-0.05</td><td>1.00+/-0.12</td><td>1.00+/-0.07</td><td>1.00+/-0.06</td><td>1.00+/-0.02</td><td>1.00+/-0.04</td><td>1.00+/-0.12</td><td>1.00+/-0.11</td></tr><tr><td>Sterile stab</td><td></td><td></td><td>3.01+/-0.07</td><td>0.66+/-0.02</td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.48+/-0.03</td><td></td><td></td></tr><tr><td><i>E. coli stab</i></td><td>0.49+/-0.05</td><td>0.66+/-0.02</td><td>5.53+/-0.48</td><td>2.18+/-0.13</td><td>1.66+/-0.07</td><td>0.72+/-0.03</td><td>0.82+/-0.06</td><td>0.45+/-0.04</td><td>0.87+/-0.05</td><td>1.19+/-0.03</td><td>1.64+/-0.08</td><td>0.84+/-0.78</td><td>0.46+/-0.04</td></tr><tr><td>Gram-pathogen stab</td><td>0.59+/-0.06</td><td>0.78+/-0.12</td><td>2.52+/-0.21</td><td>1.78+/-0.33</td><td>0.84+/-0.06</td><td>0.65+/-0.02</td><td>1.26+/-0.08</td><td>0.43+/-0.06</td><td>0.76+/-0.02</td><td>0.85+/-0.06</td><td>2.00+/-0.06</td><td>0.77+/-0.06</td><td>0.59+/-0.04</td></tr><tr><td>Gram+pathogen stab</td><td>0.82+/-0.04</td><td>0.69+/-0.02</td><td>2.28+/-0.22</td><td>1.42+/-0.06</td><td>0.89+/-0.05</td><td>0.51+/-0.05</td><td>0.99+/-0.05</td><td>0.51+/-0.05</td><td>0.69+/-0.04</td><td>0.91+/-0.05</td><td>2.43+/-0.16</td><td>0.73+/-0.04</td><td>0.42+/-0.02</td></tr><tr><td>Feed control</td><td>1.00+/-0.05</td><td>1.00+/-0.07</td><td>1.00+/-0.10</td><td>1.00+/-0.07</td><td>1.00+/-0.05</td><td>1.00+/-0.08</td><td>1.00+/-0.07</td><td>1.00+/-0.03</td><td>1.00+/-0.10</td><td>1.00+/-0.07</td><td>1.00+/-0.06</td><td>1.00+/-0.08</td><td>1.00+/-0.08</td></tr><tr><td><i>E. coli</i> feed</td><td>1.01+/-0.05</td><td>0.77+/-0.05</td><td>1.45+/-0.19</td><td>2.06+/-0.12</td><td>0.88+/-0.07</td><td>0.80+/-0.05</td><td>0.91+/-0.03</td><td>1.00+/-0.03</td><td>0.72+/-0.05</td><td>1.04+/-0.07</td><td>1.15+/-0.06</td><td>0.77+/-0.02</td><td>0.74+/-0.10</td></tr><tr><td>Gram-pathogen feed</td><td>0.82+/-0.06</td><td>0.63+/-0.05</td><td>1.41+/-0.15</td><td>1.35+/-0.05</td><td>0.84+/-0.08</td><td>0.70+/-0.03</td><td>0.63+/-0.03</td><td>1.03+/-0.04</td><td>0.70+/-0.05</td><td>0.95+/-0.03</td><td>1.46+/-0.10</td><td>0.64+/-0.02</td><td>0.69+/-0.07</td></tr><tr><td>Fungus control</td><td>1.00+/-0.07</td><td>1.00+/-0.03</td><td>1.00+/-0.08</td><td>1.00+/-0.02</td><td>1.00+/-0.07</td><td>1.00+/-0.04</td><td>1.00+/-0.09</td><td>1.00+/-0.09</td><td>1.00+/-0.06</td><td>1.00+/-0.08</td><td>1.00+/-0.04</td><td>1.00+/-0.07</td><td>1.00+/-0.04</td></tr><tr><td>Fungus infected</td><td>0.91+/-0.03</td><td>1.20+/-0.18</td><td>1.16+/-0.10</td><td>0.87+/-0.11</td><td>1.06+/-0.02</td><td>1.00+/-0.07</td><td>1.00+/-0.05</td><td>0.62+/-0.07</td><td>1.30+/-0.10</td><td>1.00+/-0.03</td><td>0.88+/-0.03</td><td>1.32+/-0.07</td><td>1.32+/-0.09</td></tr><tr><td>Virus control</td><td>1.00+/-0.02</td><td>1.00+/-0.04</td><td>1.00+/-0.03</td><td>1.00+/-0.03</td><td>1.00+/-0.08</td><td>1.00+/-0.06</td><td>1.00+/-0.02</td><td>1.00+/-0.04</td><td>1.00+/-0.06</td><td>1.00+/-0.07</td><td>1.00+/-0.10</td><td>1.00+/-0.03</td><td>1.00+/-0.12</td></tr><tr><td>Virus infected</td><td>0.88+/-0.03</td><td>1.01+/-0.04</td><td>1.23+/-0.14</td><td>0.83+/-0.02</td><td>1.59+/-0.05</td><td>0.97+/-0.04</td><td>0.87+/-0.05</td><td>0.92+/-0.06</td><td>0.93+/-0.03</td><td>1.16+/-0.06</td><td>0.98+/-0.07</td><td>1.40+/-0.11</td><td>2.42+/-0.27</td></tr><tr><td>No stress control</td><td>1.00+/-0.10</td><td>1.00+/-0.14</td><td>1.00+/-0.03</td><td>1.00+/-0.02</td><td>1.00+/-0.07</td><td>1.00+/-0.07</td><td>1.00+/-0.04</td><td>1.00+/-0.06</td><td>1.00+/-0.04</td><td>1.00+/-0.04</td><td>1.00+/-0.08</td><td>1.00+/-0.06</td><td>1.00+/-0.06</td></tr><tr><td>Alarm pheromone</td><td>0.79+/-0.05</td><td>0.80+/-0.07</td><td>0.94+/-0.03</td><td>2.41+/-0.24</td><td>0.96+/-0.05</td><td>0.87+/-0.04</td><td>0.74+/-0.02</td><td>0.95+/-0.13</td><td>0.97+/-0.06</td><td>0.96+/-0.03</td><td>1.37+/-0.09</td><td>1.15+/-0.07</td><td>0.98+/-0.05</td></tr><tr><td>Post alarm pheromone</td><td>0.54+/-0.03</td><td>0.94+/-0.12</td><td>0.34+/-0.05</td><td>2.46+/-0.10</td><td>0.78+/-0.15</td><td>0.98+/-0.07</td><td>0.96+/-0.07</td><td>1.03+/-0.14</td><td>0.99+/-0.06</td><td>0.64+/-0.13</td><td>2.11+/-0.14</td><td>1.08+/-0.08</td><td>1.00+/-0.07</td></tr><tr><td>Heat stress</td><td>1.35+/-0.07</td><td>1.14+/-0.05</td><td>0.74+/-0.08</td><td>1.48+/-0.06</td><td>2.59+/-0.17</td><td>1.71+/-0.06</td><td>1.01+/-0.03</td><td>0.94+/-0.07</td><td>1.14+/-0.07</td><td>1.17+/-0.16</td><td>0.68+/-0.02</td><td>1.27+/-0.22</td><td>1.97+/-0.12</td></tr><tr><td>Post heat stress</td><td>0.76+/-0.04</td><td>0.69+/-0.09</td><td>0.48+/-0.02</td><td>1.68+/-0.10</td><td>0.75+/-0.11</td><td>0.63+/-0.08</td><td>0.58+/-0.03</td><td>0.67+/-0.04</td><td>0.77+/-0.01</td><td>0.69+/-0.02</td><td>2.11+/-0.12</td><td>0.76+/-0.02</td><td>0.58+/-0.07</td></tr><tr><td>Starvation</td><td>2.14+/-0.42</td><td>1.16+/-0.03</td><td>0.64+/-0.05</td><td>3.29+/-0.27</td><td>1.79+/-0.12</td><td>1.36+/-0.06</td><td>0.76+/-0.06</td><td>0.86+/-0.11</td><td>1.20+/-0.06</td><td>0.44+/-0.08</td><td>1.13+/-0.14</td><td>1.37+/-0.07</td><td>1.03+/-0.12</td></tr></tbody></table>
<table><caption>**Table S5. Expression of response genes.** Values indicate the relative expression of a gene in a sample relative to expression in the appropriate control sample, +/- S.D. For the bacterial stabbing experiment, we compared a sterile stab sample to a no stab sample only for genes that showed greater than 2-fold upregulation in one of the bacteria-stabbed samples. Relative expression values should be interpreted with caution as they are based on only a single experimental replicate pooling five aphids.</caption><thead><tr><th></th><th>LYS1</th><th>LYS2</th><th>LYS3</th><th>THM2</th><th>THM3</th><th>THM4</th><th>THM6</th><th>HSP60</th><th>HSC70</th><th>HSP83</th></tr></thead><tbody><tr><td>No stab control</td><td>1.00+/-0.05</td><td>1.00+/-0.07</td><td>1.00+/-0.03</td><td>1.00+/-0.04</td><td>1.00+/-0.09</td><td>1.00+/-0.06</td><td>1.00+/-0.06</td><td>1.00+/-0.05</td><td>1.00+/-0.05</td><td>1.00+/-0.05</td></tr><tr><td>Sterile stab</td><td></td><td></td><td></td><td>0.74+/-0.07</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><i>E. coli</i> stab</td><td>1.00+/-0.03</td><td>1.35+/-0.08</td><td>1.76+/-0.15</td><td>2.37+/-0.07</td><td>0.34+/-0.05</td><td>0.29+/-0.07</td><td>0.39+/-0.03</td><td>0.81+/-0.04</td><td>0.58+/-0.04</td><td>0.80+/-0.06</td></tr><tr><td>Gram-pathogen stab</td><td>1.12+/-0.07</td><td>1.41+/-0.05</td><td>1.57+/-0.07</td><td>1.80+/-0.03</td><td>1.06+/-0.04</td><td>0.86+/-0.08</td><td>1.60+/-0.06</td><td>0.96+/-0.05</td><td>0.69+/-0.05</td><td>1.26+/-0.07</td></tr><tr><td>Gram+pathogen stab</td><td>1.12+/-0.04</td><td>1.59+/-0.17</td><td>1.62+/-0.06</td><td>0.56+/-0.05</td><td>0.58+/-0.04</td><td>0.51+/-0.04</td><td>1.01+/-0.07</td><td>0.84+/-0.04</td><td>0.66+/-0.02</td><td>0.97+/-0.04</td></tr><tr><td>Feed control</td><td>1.00+/-0.02</td><td>1.00+/-0.12</td><td>1.00+/-0.04</td><td>1.00+/-0.04</td><td>1.00+/-0.05</td><td>1.00+/-0.05</td><td>1.00+/-0.06</td><td>1.00+/-0.09</td><td>1.00+/-0.04</td><td>1.00+/-0.07</td></tr><tr><td><i>E. coli</i> feed</td><td>2.16+/-0.13</td><td>1.17+/-0.05</td><td>1.18+/-0.12</td><td>0.77+/-0.06</td><td>0.67+/-0.05</td><td>0.69+/-0.04</td><td>0.59+/-0.02</td><td>1.17+/-0.14</td><td>0.80+/-0.05</td><td>0.77+/-0.03</td></tr><tr><td>Gram-pathogen feed</td><td>2.66+/-0.22</td><td>0.91+/-0.05</td><td>1.09+/-0.02</td><td>0.30+/-0.08</td><td>0.39+/-0.02</td><td>0.41+/-0.02</td><td>0.67+/-0.03</td><td>0.88+/-0.05</td><td>0.76+/-0.03</td><td>0.64+/-0.03</td></tr><tr><td>Fungus control</td><td>1.00+/-0.01</td><td>1.00+/-0.10</td><td>1.00+/-0.05</td><td>1.00+/-0.11</td><td>1.00+/-0.06</td><td>1.00+/-0.07</td><td>1.00+/-0.03</td><td>1.00+/-0.05</td><td>1.00+/-0.04</td><td>1.00+/-0.01</td></tr><tr><td>Fungus infected</td><td>0.87+/-0.15</td><td>0.78+/-0.03</td><td>0.85+/-0.05</td><td>1.10+/-0.30</td><td>1.07+/-0.06</td><td>1.30+/-0.06</td><td>1.43+/-0.06</td><td>0.92+/-0.19</td><td>0.97+/-0.09</td><td>2.31+/-0.06</td></tr><tr><td>Virus control</td><td>1.00+/-0.04</td><td>1.00+/-0.02</td><td>1.00+/-0.02</td><td>1.00+/-0.14</td><td>1.00+/-0.16</td><td>1.00+/-0.12</td><td>1.00+/-0.10</td><td>1.00+/-0.06</td><td>1.00+/-0.02</td><td>1.00+/-0.02</td></tr><tr><td>Virus infected</td><td>0.92+/-0.04</td><td>0.67+/-0.03</td><td>0.64+/-0.02</td><td>0.64+/-0.06</td><td>0.95+/-0.14</td><td>0.96+/-0.11</td><td>0.93+/-0.13</td><td>0.74+/-0.02</td><td>0.77+/-0.07</td><td>1.20+/-0.06</td></tr><tr><td>No stress control</td><td>1.00+/-0.02</td><td>1.00+/-0.05</td><td>1.00+/-0.02</td><td>1.00+/-0.06</td><td>1.00+/-0.11</td><td>1.00+/-0.04</td><td>1.00+/-0.05</td><td>1.00+/-0.08</td><td>1.00+/-0.07</td><td>1.00+/-0.05</td></tr><tr><td>Alarm pheromone</td><td>0.87+/-0.13</td><td>0.92+/-0.03</td><td>0.86+/-0.03</td><td>0.30+/-0.04</td><td>0.49+/-0.01</td><td>1.05+/-0.04</td><td>1.37+/-0.05</td><td>0.77+/-0.02</td><td>1.14+/-0.02</td><td>1.21+/-0.05</td></tr><tr><td>Post alarm pheromone</td><td>0.89+/-0.04</td><td>0.76+/-0.04</td><td>0.93+/-0.01</td><td>0.34+/-0.07</td><td>0.64+/-0.03</td><td>0.65+/-0.05</td><td>0.22+/-0.05</td><td>1.44+/-0.15</td><td>1.20+/-0.05</td><td>1.28+/-0.03</td></tr><tr><td>Heat stress</td><td>1.48+/-0.04</td><td>0.98+/-0.05</td><td>0.66+/-0.02</td><td>1.70+/-0.10</td><td>0.95+/-0.05</td><td>0.68+/-0.02</td><td>0.35+/-0.06</td><td>1.55+/-0.09</td><td>2.07+/-0.06</td><td>5.54+/-0.27</td></tr><tr><td>Post heat stress</td><td>0.78+/-0.13</td><td>0.61+/-0.02</td><td>0.67+/-0.04</td><td>0.31+/-0.07</td><td>0.85+/-0.04</td><td>0.74+/-0.06</td><td>0.64+/-0.05</td><td>0.92+/-0.02</td><td>1.01+/-0.07</td><td>0.26+/-0.01</td></tr><tr><td>Starvation</td><td>1.30+/-0.11</td><td>2.96+/-0.23</td><td>1.19+/-0.06</td><td>0.75+/-0.02</td><td>0.90+/-0.05</td><td>0.93+/-0.04</td><td>0.53+/-0.04</td><td>1.09+/-0.08</td><td>1.02+/-0.10</td><td>0.64+/-0.04</td></tr></tbody></table>
<table><caption>Table S6. Gut EST library statistics.</caption><thead><tr><th>Library ID</th><th>TD2a library</th><th>TD2b library</th></tr><tr><th>Tissue treatment</th><th>ID0AFF</th><th>ID0AAG</th></tr><tr><th></th><th>Control<br>digestive tract</th><th>Gram-challenged<br>digestive tract</th></tr></thead><tbody><tr><td>Number of clones sequenced</td><td>5283</td><td>4043</td></tr><tr><td>Mean clone length</td><td>490 ± 160</td><td>602 ± 219</td></tr><tr><td>Median clone length</td><td>548</td><td>694</td></tr><tr><td>N seq. &lt; 100 bp</td><td>210</td><td>197</td></tr><tr><td>Clones with no blastX hit (E≥10)<sup>a</sup></td><td>737</td><td>633</td></tr><tr><td>% without hit</td><td>14,0%</td><td>15,7%</td></tr><tr><td>Clones with blastX hit (E&lt;10)</td><td>4546 (4065*)</td><td>3410 (2921*)</td></tr><tr><td>Clones with non-sign. hits (10<sup>-3</sup> – 10)</td><td>1408</td><td>936</td></tr><tr><td>Clones with sign. hits (E ≤ 10<sup>-3</sup>)</td><td>3138</td><td>2474</td></tr><tr><td>% with sign. hit</td><td>59,4%</td><td>61,2%</td></tr><tr><td>Uniprot hits from blastX (total)</td><td>2192</td><td>806</td></tr><tr><td>Uniprot hits from blastX (significant)</td><td>1486</td><td>578</td></tr><tr><td>N hits with more than 10 clones</td><td>56</td><td>50</td></tr><tr><td>N hits with more than 1% expression</td><td>6</td><td>12</td></tr><tr><td>N hits with more than 2% expression</td><td>1</td><td>9</td></tr><tr><td>Hits with GO annotation (%)<sup>b</sup></td><td>79 %</td><td>80 %</td></tr><tr><td>Hits without GO annotation (total Fatigo)</td><td>312 (1495)</td><td>117 (579)</td></tr><tr><td>N Contigs (Ap v5 clustering)</td><td>2128</td><td>875</td></tr><tr><td>redundancy index (% contigs vs clones)</td><td>40.3 %</td><td>21.6 %</td></tr><tr><td>Nb specific contigs (control vs challenged)</td><td>1724</td><td>471</td></tr><tr><td>specificity rate (% specific vs total)</td><td>81%</td><td>54%</td></tr><tr><td>Fatigo analysis, total genes analysed</td><td>1495</td><td>579</td></tr><tr><td>Go class (4th level)<sup>c</sup></td><td></td><td></td></tr><tr><td>biopolymer metabolism (0.005 – 0.088)</td><td>21.0 %</td><td>11.3 %</td></tr><tr><td>transport (0.029 – 0.45)</td><td>19.1 %</td><td>27.1 %</td></tr><tr><td>establishment of localization (0.038 – 0.64)</td><td>19.2 %</td><td>27.0 %</td></tr><tr><td>cellular metabolism</td><td>78.4 %</td><td>71.7 %</td></tr><tr><td>cell organization and biogenesis</td><td>7.4 %</td><td>3.8 %</td></tr><tr><td>primary metabolism</td><td>67.7 %</td><td>62.3 %</td></tr><tr><td>macromolecule metabolism</td><td>46.9 %</td><td>44.0 %</td></tr><tr><td>biosynthesis</td><td>24.3 %</td><td>26.4 %</td></tr><tr><td>catabolism</td><td>14.3 %</td><td>12.6 %</td></tr></tbody></table>

a : blastX performed through the blast parsing script runblastncbi (courtesy Laurent Duret), on Uniprot DB (May 2005, release UniprotKB 4.0, pbil server pbil.univ-lyon1.fr)

b : FATIGO links to GO ontology (June 2005, UniprotKB release 4.0, www.fatigo.org)

c : beneath % of class are given the *unadjusted* and adjusted (step-down min p) p values for library comparison, as computed by Fatigo (Fisher exact test). For non-discriminant classes (no p given, p≥10%), only main classes are listed (>10% representation)

* : corrected for hit redundancy in blast results (low complexity or modular proteins)
<table><caption>**Table S7. List of selected ESTs from the subtracted library.** Results include the highest homologous match for each EST against the *A. pisum* NCBI refseq set (blastX), the publicly available *A. Pisum* ESTs (blastN), and the SwissProt databases (blastX). ESTs have been selected according to either apparent differential gene expression or to their similarities to putative immune-related genes. Differential expression status of ESTs was analyzed using a dot-blot experiment as described in the materials and methods. The change in gene expression after *E. coli* infection relative to the untreated aphid is given as qualitative data (+: overexpression; -: underexpression; =: no differential gene expression). Descriptions for aphid matches are based on *A. pisum* EST cluster descriptions, available at the *Acyrthosiphon Pisum* EST Database [11], and/or on refseq descriptions.</caption><thead><tr><th rowspan="2">GenBank<br>Acc. Num.</th><th colspan="2">EST</th><th colspan="4">Aphid Hits</th><th colspan="3">Swiss Prot Hits</th></tr><tr><th>Length</th><th>Relative<br>expression</th><th>A. pisum gene<br>(evalue)</th><th>A. pisum EST<br>(evalue)</th><th>A. pisum<br>EST cluster</th><th>Description<br>based on gene or EST</th><th>Swiss Prot ID<br>(evalue)</th><th>Organism</th><th>Description</th></tr></thead><tbody><tr><td>GD186025</td><td>237</td><td>-</td><td>no hit</td><td>EX619631<br>(e-104)</td><td>APG20589</td><td></td><td>no hit</td><td></td><td></td></tr><tr><td>GD186052</td><td>629</td><td>-</td><td>ACYPI006141<br>(5e-31)</td><td>FF333609<br>(0.0)</td><td>APG03947</td><td>similar to eukaryotic translation<br>initiation factor 5</td><td>Q9VXK6<br>(9e-26)</td><td>Drosophila<br>melanogaster</td><td>eukaryotic translation initiation factor 5</td></tr><tr><td>GD186090</td><td>299</td><td>-</td><td>ACYPI004024<br>(3e-11)</td><td>FF338507<br>(e-106)</td><td>APG01676</td><td>transport, small GTPase<br>mediated signal transduction</td><td>Q9VZ23<br>(2e-07)</td><td>Drosophila<br>melanogaster</td><td>GTP-binding nuclear protein Ran</td></tr><tr><td>GD186102</td><td>425</td><td>-</td><td>no hit</td><td>DY224167</td><td>APD07979</td><td></td><td>no hit</td><td></td><td></td></tr><tr><td>GD185918</td><td>445</td><td>+</td><td>no hit</td><td>FF317856</td><td>APG05151</td><td></td><td>no hit</td><td></td><td></td></tr><tr><td>GD185990</td><td>539</td><td>+</td><td>ACYPI005949<br>(5e-99)</td><td>FF334362<br>(0.0)</td><td>APG08004</td><td>Rps3 ribosomal protein S3</td><td>P48153<br>(2e-81)</td><td>Manduca<br>sexta</td><td>40S ribosomal protein S3</td></tr><tr><td>GD186053</td><td>320</td><td>+</td><td>ACYPI006749<br>(8e-34)</td><td>EX620733<br>(e-169)</td><td>APG08261</td><td>methionine biosynthesis</td><td>no hit</td><td></td><td></td></tr><tr><td>GD186057</td><td>748</td><td>+</td><td>ACYPI006004<br>(2e-189)</td><td>FF324610<br>(0.0)</td><td>APG11294</td><td>similar to latent nuclear antigen</td><td>Q8IMP6<br>(7e-23)</td><td>Drosophila<br>melanogaster</td><td>protein SPT2 homolog</td></tr><tr><td>GD186146</td><td>540</td><td>+</td><td>ACYPI003334<br>(6e-62)</td><td>EX607065<br>(0.0)</td><td>APG09767</td><td></td><td>Q6GMF8<br>(5e-46)</td><td>Danio rerio</td><td>rhomboid family member 1</td></tr><tr><td>GD186208</td><td>397</td><td>+</td><td>no hit</td><td>FF317879<br>(0.0)</td><td>APG01972</td><td>nuclear mRNA splicing</td><td>no hit</td><td></td><td></td></tr><tr><td>GD186213</td><td>399</td><td>+</td><td>no hit</td><td>EX653226<br>(e-113)</td><td>APG17996</td><td></td><td>no hit</td><td></td><td></td></tr><tr><td>GD186220</td><td>314</td><td>+</td><td>ACYPI000343<br>(8e-59)</td><td>CN762809<br>(e-155)</td><td>APD04091</td><td>similar to ubiquitin specific<br>protease 7</td><td>Q9VYQ8<br>(5e-17)</td><td>Drosophila<br>melanogaster</td><td>ubiquitin carboxyl-terminal hydrolase 7</td></tr><tr><td>GD186223</td><td>571</td><td>+</td><td>ACYPI007035<br>(4e-85)</td><td>FF317872<br>(0.0)</td><td>APG06079</td><td>similar to coiled-coil domain-<br>containing protein 132</td><td>Q8CI71<br>(1e-19)</td><td>Mus<br>musculus</td><td>coiled-coil domain-containing protein 132</td></tr><tr><td>GD186225</td><td>333</td><td>+</td><td>no hit</td><td>no hit</td><td></td><td></td><td>no hit</td><td></td><td></td></tr><tr><td>GD186231</td><td>379</td><td>+</td><td>ACYPI003578<br>(2e-58)</td><td>FF314239<br>(0.0)</td><td>APG03794</td><td>similar to signal recognition<br>particle 72 kDa protein</td><td>P33731<br>(7e-15)</td><td>Canis lupus<br>familiaris</td><td>signal recognition particle 72 kDa protein</td></tr><tr><td>GD186337</td><td>674</td><td>+</td><td>no hit</td><td>FF333448<br>(e-115)</td><td>APG13156</td><td>amino acid transport</td><td>Q54S12<br>(0.12)</td><td>Dictyostelium<br>discoideum</td><td>transmembrane protein 104 homolog</td></tr><tr><td>GD186362</td><td>397</td><td>+</td><td>no hit</td><td>DY223529<br>(e-103)</td><td>APD03776</td><td></td><td>no hit</td><td></td><td></td></tr><tr><td>GD186383</td><td>375</td><td>+</td><td>no hit</td><td>FF305066<br>(0.0)</td><td>APG03552</td><td>protein amino acid<br>phosphorylation</td><td>Q16513<br>(0.003)</td><td>Homo<br>sapiens</td><td>serine/threonine-protein kinase N2</td></tr><tr><td>GD186386</td><td>361</td><td>+</td><td>no hit</td><td>FF330011</td><td>APG09318</td><td></td><td>no hit</td><td></td><td></td></tr></tbody></table>
<table><thead><tr><th rowspan="2">GenBank<br>Acc. Num.</th><th colspan="2">EST</th><th colspan="3">Aphid Hits</th><th colspan="3">Swiss Prot Hits</th></tr><tr><th>Length</th><th>Relative<br>expression</th><th>A. pisum gene<br>(evalue)</th><th>A. pisum EST<br>(evalue)<br>(e-115)</th><th>A. pisum<br>EST cluster</th><th>Description<br>based on gene or EST</th><th>Swiss Prot ID<br>(evalue)</th><th>Organism</th><th>Description</th></tr></thead><tbody><tr><td>GD185976</td><td>283</td><td>=</td><td>ACYPI002027<br>(2e-58)</td><td>CV838512<br>(e-154)</td><td>APD02867</td><td>thrombospondin-like</td><td>Q9C0I4<br>(1e-16)</td><td>Homo<br>sapiens</td><td>thrombospondin type-1 domain-<br>containing protein 7B</td></tr><tr><td>GD186047</td><td>347</td><td>=</td><td>ACYPI000111<br>(1e-68)</td><td>no hit</td><td></td><td>similar to adam</td><td>Q9VAC5<br>(2e-53)</td><td>Drosophila<br>melanogaster</td><td>Adam 17-like protease</td></tr><tr><td>GD186063</td><td>1130</td><td>=</td><td>ACYPI000719<br>(3e-136)</td><td>FF336689<br>(0.0)</td><td>APG09006</td><td></td><td>P13284<br>(7e-13)</td><td>Homo<br>sapiens</td><td>gamma-interferon-inducible lysosomal<br>thiol reductase precursor</td></tr><tr><td>GD186100</td><td>284</td><td>=</td><td>ACYPI008118<br>(4e-102)</td><td>FF315088<br>(0.0)</td><td>APG16211</td><td>similar to tyrosine protein<br>kinase</td><td>Q24592<br>(2e-08)</td><td>Drosophila<br>melanogaster</td><td>tyrosine-protein kinase hopscotch (Jak)</td></tr><tr><td>GD186119</td><td>325</td><td>=</td><td>ACYPI005016<br>(2e-51)</td><td>FF320908<br>(e-176)</td><td>APG08844</td><td>serine protease inhibitor<br>(serpin 1)</td><td>P48594<br>(2e-17)</td><td>Homo<br>sapiens</td><td>serpin B4</td></tr><tr><td>GD186120</td><td>353</td><td>=</td><td>ACYPI005540<br>(6e-63)</td><td>CN759023<br>(0.0)</td><td>APD04504</td><td>MAPKKK cascade</td><td>Q5E9X2<br>(3e-45)</td><td>Bos taurus</td><td>dual specificity mitogen-activated<br>protein kinase 6</td></tr><tr><td>GD186131</td><td>517</td><td>=</td><td>ACYPI005016<br>(5e-86)</td><td>FF336182<br>(0.0)</td><td>APG07692</td><td>serine-protease inhibitor<br>(serpin 1)</td><td>P48594<br>(2e-17)</td><td>Homo<br>sapiens</td><td>serpin B4</td></tr><tr><td>GD186142</td><td>328</td><td>=</td><td>no hit</td><td>EX639251<br>(e-110)</td><td>APG19100</td><td>receptor activity</td><td>Q5XIN3<br>(1e-07)</td><td>Rattus<br>norvegicus</td><td>TRAF3-interacting protein 1</td></tr><tr><td>GD186162</td><td>330</td><td>=</td><td>ACYPI003236<br>(3e-59)</td><td>no hit</td><td></td><td>similar to sunday driver</td><td>Q9ESN9<br>(9e-34)</td><td>Mus<br>musculus</td><td>JNK-interacting protein 3</td></tr><tr><td>GD186173</td><td>245</td><td>=</td><td>no hit</td><td>CN761251<br>(e-119)</td><td>APD04293</td><td>programs cell death</td><td>Q3UU35<br>(0.8)</td><td>Mus<br>musculus</td><td>Ovostatin homolog</td></tr><tr><td>GD186257</td><td>345</td><td>=</td><td>ACYPI006651<br>(7e-32)</td><td>CN764436<br>(e-163)</td><td>APD05533</td><td>similar to membrane-<br>associated LPS-inducible TNF<br>alpha factor</td><td>Q8QGW7<br>(7e-10)</td><td>Gallus gallus</td><td>LPS-induced TNF-alpha factor<br>homolog</td></tr><tr><td>GD186266</td><td>346</td><td>=</td><td>ACYPI002465<br>(3e-28)</td><td>CN756373<br>(e-173)</td><td>APD05909</td><td>similar to macrophage<br>migration inhibitory factor</td><td>P91850<br>(3e-08)</td><td>Brugia malayi</td><td>Macrophage migration inhibitory factor<br>homolog</td></tr><tr><td>GD186363</td><td>268</td><td>=</td><td>no hit</td><td>EX650498<br>(e-126)</td><td>APG05258</td><td>sugar binding</td><td>no hit</td><td></td><td></td></tr></tbody></table>
<table><caption>**Table S7. List of selected ESTs from the subtracted library.** Results include the highest homologous match for each EST against the *A. pisum* NCBI refseq set (blastX), the publicly available *A. Pisum* ESTs (blastN), and the SwissProt databases (blastX). ESTs have been selected according to either apparent differential gene expression or to their similarities to putative immune-related genes. Differential expression status of ESTs was analyzed using a dot-blot experiment as described in the materials and methods. The change in gene expression after *E. coli* infection relative to the untreated aphid is given as qualitative data (+: overexpression; -: underexpression; =: no differential gene expression). Descriptions for aphid matches are based on *A. pisum* EST cluster descriptions, available at the *Acyrthosiphon Pisum* EST Database [11], and/or on refseq descriptions.</caption><thead><tr><th colspan="3">EST</th><th colspan="3">Aphid Hits</th><th colspan="3">Swiss Prot Hits</th></tr><tr><th>GenBank<br>Acc. Num.</th><th>Length</th><th>Relative<br>expression</th><th>A. pisum gene<br>(evalue)</th><th>A. pisum EST<br>(evalue)</th><th>A. pisum<br>EST cluster</th><th>Description<br>based on gene or EST</th><th>Swiss Prot ID<br>(evalue)</th><th>Organism</th><th>Description</th></tr></thead><tbody><tr><td>GD186025</td><td>237</td><td>-</td><td>no hit</td><td>EX619631<br>(e-104)</td><td>APG20589</td><td></td><td>no hit</td><td></td><td></td></tr><tr><td>GD186052</td><td>629</td><td>-</td><td>ACYPI006141<br>(5e-31)</td><td>FF333609<br>(0.0)</td><td>APG03947</td><td>similar to eukaryotic translation<br>initiation factor 5</td><td>Q9VXK6<br>(9e-26)</td><td>Drosophila<br>melanogaster</td><td>eukaryotic translation initiation factor 5</td></tr><tr><td>GD186090</td><td>299</td><td>-</td><td>ACYPI004024<br>(3e-11)</td><td>FF338507<br>(e-106)</td><td>APG01676</td><td>transport, small GTPase<br>mediated signal transduction</td><td>Q9VZ23<br>(2e-07)</td><td>Drosophila<br>melanogaster</td><td>GTP-binding nuclear protein Ran</td></tr><tr><td>GD186102</td><td>425</td><td>-</td><td>no hit</td><td>DY224167</td><td>APD07979</td><td></td><td>no hit</td><td></td><td></td></tr><tr><td>GD185918</td><td>445</td><td>+</td><td>no hit</td><td>FF317856</td><td>APG05151</td><td></td><td>no hit</td><td></td><td></td></tr><tr><td>GD185990</td><td>539</td><td>+</td><td>ACYPI005949<br>(5e-99)</td><td>FF334362<br>(0.0)</td><td>APG08004</td><td>Rps3 ribosomal protein S3</td><td>P48153<br>(2e-81)</td><td>Manduca<br>sexta</td><td>40S ribosomal protein S3</td></tr><tr><td>GD186053</td><td>320</td><td>+</td><td>ACYPI006749<br>(8e-34)</td><td>EX620733<br>(e-169)</td><td>APG08261</td><td>methionine biosynthesis</td><td>no hit</td><td></td><td></td></tr><tr><td>GD186057</td><td>748</td><td>+</td><td>ACYPI006004<br>(2e-189)</td><td>FF324610<br>(0.0)</td><td>APG11294</td><td>similar to latent nuclear antigen</td><td>Q8IMP6<br>(7e-23)</td><td>Drosophila<br>melanogaster</td><td>protein SPT2 homolog</td></tr><tr><td>GD186146</td><td>540</td><td>+</td><td>ACYPI003334<br>(6e-62)</td><td>EX607065<br>(0.0)</td><td>APG09767</td><td></td><td>Q6GMF8<br>(5e-46)</td><td>Danio rerio</td><td>rhomboid family member 1</td></tr><tr><td>GD186208</td><td>397</td><td>+</td><td>no hit</td><td>FF317879<br>(0.0)</td><td>APG01972</td><td>nuclear mRNA splicing</td><td>no hit</td><td></td><td></td></tr><tr><td>GD186213</td><td>399</td><td>+</td><td>no hit</td><td>EX653226<br>(e-113)</td><td>APG17996</td><td></td><td>no hit</td><td></td><td></td></tr><tr><td>GD186220</td><td>314</td><td>+</td><td>ACYPI000343<br>(8e-59)</td><td>CN762809<br>(e-155)</td><td>APD04091</td><td>similar to ubiquitin specific<br>protease 7</td><td>Q9VYQ8<br>(5e-17)</td><td>Drosophila<br>melanogaster</td><td>ubiquitin carboxyl-terminal hydrolase 7</td></tr><tr><td>GD186223</td><td>571</td><td>+</td><td>ACYPI007035<br>(4e-85)</td><td>FF317872<br>(0.0)</td><td>APG06079</td><td>similar to coiled-coil domain-<br>containing protein 132</td><td>Q8CI71<br>(1e-19)</td><td>Mus<br>musculus</td><td>coiled-coil domain-containing protein 132</td></tr><tr><td>GD186225</td><td>333</td><td>+</td><td>no hit</td><td>no hit</td><td></td><td></td><td>no hit</td><td></td><td></td></tr><tr><td>GD186231</td><td>379</td><td>+</td><td>ACYPI003578<br>(2e-58)</td><td>FF314239<br>(0.0)</td><td>APG03794</td><td>similar to signal recognition<br>particle 72 kDa protein</td><td>P33731<br>(7e-15)</td><td>Canis lupus<br>familiaris</td><td>signal recognition particle 72 kDa protein</td></tr><tr><td>GD186337</td><td>674</td><td>+</td><td>no hit</td><td>FF333448<br>(e-115)</td><td>APG13156</td><td>amino acid transport</td><td>Q54S12<br>(0.12)</td><td>Dictyostelium<br>discoideum</td><td>transmembrane protein 104 homolog</td></tr><tr><td>GD186362</td><td>397</td><td>+</td><td>no hit</td><td>DY223529<br>(e-103)</td><td>APD03776</td><td></td><td>no hit</td><td></td><td></td></tr><tr><td>GD186383</td><td>375</td><td>+</td><td>no hit</td><td>FF305066<br>(0.0)</td><td>APG03552</td><td>protein amino acid<br>phosphorylation</td><td>Q16513<br>(0.003)</td><td>Homo<br>sapiens</td><td>serine/threonine-protein kinase N2</td></tr><tr><td>GD186386</td><td>361</td><td>+</td><td>no hit</td><td>FF330011</td><td>APG09318</td><td></td><td>no hit</td><td></td><td></td></tr></tbody></table>
<table><thead><tr><th rowspan="2">GenBank<br>Acc. Num.</th><th colspan="2">EST</th><th colspan="3">Aphid Hits</th><th colspan="3">Swiss Prot Hits</th></tr><tr><th>Length</th><th>Relative<br>expression</th><th>A. pisum gene<br>(evalue)</th><th>A. pisum EST<br>(evalue)</th><th>A. pisum<br>EST cluster</th><th>Description<br>based on gene or EST</th><th>Swiss Prot ID<br>(evalue)</th><th>Organism</th><th>Description</th></tr></thead><tbody><tr><td>GD185976</td><td>283</td><td>=</td><td>ACYPI002027<br>(2e-58)</td><td>CV838512<br>(e-154)</td><td>APD02867</td><td>thrombospondin-like</td><td>Q9C0I4<br>(1e-16)</td><td>Homo<br>sapiens</td><td>thrombospondin type-1 domain-<br>containing protein 7B</td></tr><tr><td>GD186047</td><td>347</td><td>=</td><td>ACYPI000111<br>(1e-68)</td><td>no hit</td><td></td><td>similar to adam</td><td>Q9VAC5<br>(2e-53)</td><td>Drosophila<br>melanogaster</td><td>Adam 17-like protease</td></tr><tr><td>GD186063</td><td>1130</td><td>=</td><td>ACYPI000719<br>(3e-136)</td><td>FF336689<br>(0.0)</td><td>APG09006</td><td></td><td>P13284<br>(7e-13)</td><td>Homo<br>sapiens</td><td>gamma-interferon-inducible lysosomal<br>thiol reductase precursor</td></tr><tr><td>GD186100</td><td>284</td><td>=</td><td>ACYPI008118<br>(4e-102)</td><td>FF315088<br>(0.0)</td><td>APG16211</td><td>similar to tyrosine protein<br>kinase</td><td>Q24592<br>(2e-08)</td><td>Drosophila<br>melanogaster</td><td>tyrosine-protein kinase hopscotch (Jak)</td></tr><tr><td>GD186119</td><td>325</td><td>=</td><td>ACYPI005016<br>(2e-51)</td><td>FF320908<br>(e-176)</td><td>APG08844</td><td>serine protease inhibitor<br>(serpin 1)</td><td>P48594<br>(2e-17)</td><td>Homo<br>sapiens</td><td>serpin B4</td></tr><tr><td>GD186120</td><td>353</td><td>=</td><td>ACYPI005540<br>(6e-63)</td><td>CN759023<br>(0.0)</td><td>APD04504</td><td>MAPKKK cascade</td><td>Q5E9X2<br>(3e-45)</td><td>Bos taurus</td><td>dual specificity mitogen-activated<br>protein kinase 6</td></tr><tr><td>GD186131</td><td>517</td><td>=</td><td>ACYPI005016<br>(5e-86)</td><td>FF336182<br>(0.0)</td><td>APG07692</td><td>serine-protease inhibitor<br>(serpin 1)</td><td>P48594<br>(2e-17)</td><td>Homo<br>sapiens</td><td>serpin B4</td></tr><tr><td>GD186142</td><td>328</td><td>=</td><td>no hit</td><td>EX639251<br>(e-110)</td><td>APG19100</td><td>receptor activity</td><td>Q5XIN3<br>(1e-07)</td><td>Rattus<br>norvegicus</td><td>TRAF3-interacting protein 1</td></tr><tr><td>GD186162</td><td>330</td><td>=</td><td>ACYPI003236<br>(3e-59)</td><td>no hit</td><td></td><td>similar to sunday driver</td><td>Q9ESN9<br>(9e-34)</td><td>Mus<br>musculus</td><td>JNK-interacting protein 3</td></tr><tr><td>GD186173</td><td>245</td><td>=</td><td>no hit</td><td>CN761251<br>(e-119)</td><td>APD04293</td><td>programs cell death</td><td>Q3UU35<br>(0.8)</td><td>Mus<br>musculus</td><td>Ovostatin homolog</td></tr><tr><td>GD186257</td><td>345</td><td>=</td><td>ACYPI006651<br>(7e-32)</td><td>CN764436<br>(e-163)</td><td>APD05533</td><td>similar to membrane-<br>associated LPS-inducible TNF<br>alpha factor</td><td>Q8QGW7<br>(7e-10)</td><td>Gallus gallus</td><td>LPS-induced TNF-alpha factor<br>homolog</td></tr><tr><td>GD186266</td><td>346</td><td>=</td><td>ACYPI002465<br>(3e-28)</td><td>CN756373<br>(e-173)</td><td>APD05909</td><td>similar to macrophage<br>migration inhibitory factor</td><td>P91850<br>(3e-08)</td><td>Brugia malayi</td><td>Macrophage migration inhibitory factor<br>homolog</td></tr><tr><td>GD186363</td><td>268</td><td>=</td><td>no hit</td><td>EX650498<br>(e-126)</td><td>APG05258</td><td>sugar binding</td><td>no hit</td><td></td><td></td></tr></tbody></table>
<caption>**Figure S1.** Maximum likelihood phylogenies of selected immune gene families. (a) Gram-negative binding proteins; (b) Spätzles; (c) Tolls, note that there is no support for *A. pisum* tolls clading with *D. melanogaster* Tehao; (d) Lysozymes; (e) Teps; (f) Prophenoloxidases; and, (g) low molecular weight heat shock proteins (Hsp83, Hsp90 not included). * represents approximate likelihood ratio test (aLRT) support > 80. (Aga: *Anopheles gambiae*; Ame: *Apis Mellifera*; Api: *Acythrosiphon pisum*; Bom: *Bombyx mori*; Cel: *Caenorhabditis elegans*; Cin: *Ciona intestinalis*; Dme = *Drosophila melanogaster*; Dpu = *Daphnia pulex*; Nvi: *Nasonia vitripennis*; Phu: *Pediculus humanus*; Tca: *Tribolium castaneum*.)</caption>

<figure><img src="image_21.png" /><figcaption>(a)</figcaption></figure>
<figure><img src="image_22.png" /><figcaption>(b)</figcaption></figure>
(c)

<figure><img src="image_23.png" /></figure>
<figure><img src="image_24.png" /><figcaption>(d)</figcaption></figure>
(e)

<figure><img src="image_25.png" /></figure>
<figure><img src="image_26.png" /><figcaption>(f)</figcaption></figure>
(g)

<figure><img src="image_27.png" /></figure>
<figure><img src="image_28.png" /><figcaption>**Figure S2.** Alignments of putative antimicrobial peptides megourin and penaeidin. (a) Putative pea aphid megourin (pea_aphid) aligned with 3 megourins of the aphid *Megoura viciae* (MEGVI). (b) Putative pea aphid penaeidin (ACYPI37769-PA) aligned with penaeidins from the shrimp *Penaeus vannamei* (PENVA) and *Penaeus (Litopenaeus) setiferus* (LITS).</figcaption></figure>
**Figure S3.** Survival curves for experimental infections associated with the qPCR study. (a) In the bacterial feeding experiment, 30 aphids per condition were fed for 20hrs on artificial diet containing bacteria or on control diet and then transferred to plants and monitored for survival. Though survival data for the bacterial stabbing experiment, in which aphids were stabbed with bacteria-contaminated needles, was not collected daily, after two days, overall survival was similar to the feeding trials, with 100% survival of control aphids, 80% survival of *E. coli*-stabbed aphids, 30% survival of Gram-positive pathogen-stabbed aphids, and 0% survival of Gram-negative pathogen-stabbed aphids (n = 10 per condition). (b) In the fungal shower experiment, 22 aphids were exposed to a shower of fungal spores for two hours and then transferred to plants. Control aphids were not exposed. (c) In the viral stabbing experiment, 20 aphids were stabbed with a virus-contaminated needle and then transferred to plants. Control aphids were not stabbed.

<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>
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