TMP 7913

Research Article
Innate
Journal of J Innate Immun Received: March 15, 2013
Immunity DOI: 10.1159/000353734 Accepted after revision: June 13, 2013
Published online: August 27, 2013
Genome-Wide Transcriptional Analysis of

Drosophila Larvae Infected by Entomopathogenic
Nematodes Shows Involvement of Complement,
Recognition and Extracellular Matrix Proteins
Badrul Arefin a Lucie Kucerova b Pavel Dobes c Robert Markus a
Hynek Strnad d Zhi Wang a Pavel Hyrsl c Michal Zurovec b Ulrich Theopold a
a
Department of Molecular Biosciences, Wenner-Gren Institute, Stockholm University, Stockholm, Sweden;
b
Biology Center, Academy of Sciences of the Czech Republic, Institute of Entomology, Ceske Budejovice,
c
Department of Animal Physiology and Immunology, Institute of Experimental Biology, Masaryk University,
Brno, and d Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Key Words ute to controlling nematobacterial infections encode: a

Basement membrane · Coagulation · Hemocyte · homolog of thioester-containing complement protein 3, a
Insect immunity · Nematodes · Recognition molecule · basement membrane component (glutactin), a recognition
Thioester-containing proteins protein (GNBP-like 3) and possibly several small peptides. Of
note is that several of these genes have not previously been
implicated in immune responses. © 2013 S. Karger AG, Basel
Abstract
Heterorhabditis bacteriophora is an entomopathogenic nem-
atode (EPN) which infects its host by accessing the hemo-
lymph where it releases endosymbiotic bacteria of the spe- Introduction
cies Photorhabdus luminescens. We performed a genome-
wide transcriptional analysis of the Drosophila response to Entomopathogenic nematodes (EPNs) are natural
EPN infection at the time point at which the nematodes pathogens used to control insect pests. In most cases, they
reached the hemolymph either via the cuticle or the gut and associate with symbiotic bacteria, which they release once
the bacteria had started to multiply. Many of the most inside their insect host [1]. Both nematode-derived [2] and
strongly induced genes have been implicated in immune re- bacterial factors [3] interfere to varying extent with host
sponses in other infection models. Mapping of the complete immune reactions often resulting in the ultimate death of
set of differentially regulated genes showed the hallmarks of the insect [1]. During recent years, EPNs have increasingly
a wound response, but also identified a large fraction of EPN- been used to probe the insect immune system and identify
specific transcripts. Several genes identified by transcrip-
tome profiling or their homologues play protective roles
during nematode infections. Genes that positively contrib- Badrul Arefin and Lucie Kucerova share first authorship.
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Stockholm University Library
© 2013 S. Karger AG, Basel Dr. Ulrich Theopold

1662–811X/13/0000–0000$38.00/0 Department of Molecular Biosciences, Wenner-Gren Institute, Stockholm University
Svante Arrheniusväg 20c
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E-Mail karger@karger.com This is an Open Access article licensed under the terms of the
SE–10691 Stockholm (Sweden)
www.karger.com/jin Creative Commons Attribution-NonCommercial 3.0 Un-
E-Mail uli.theopold @ su.se
ported license (CC BY-NC) (www.karger.com/OA-license),
applicable to the online version of the article only. Distribu-
tion permitted for non-commercial purposes only.
genes and factors that have the potential to protect against Materials and Methods
EPN infections [4]. Some of this work was performed on
Nematode Culture
target pest species, but also on well-established models for EPNs H. bacteriophora (strain H222 isolated from Pouzdrany,
insect immunity [4–8]. Surprisingly, when Drosophila me- Czech Republic) were cultured in vivo on larvae of the greater wax
lanogaster larvae were infected with the combination of moth Galleria mellonella at room temperature. Released infective
the EPN Heterorhabditis bacteriophora and the bacteri- juveniles (IJs) were collected and stored at room temperature in tap
um Photorhabdus luminescens, the canonical Toll and imd water with 0.075% formaldehyde. Small pieces of sponge were placed
to the storage dishes to provide a solid medium for nematodes.
pathways, which are protective against bacterial and fungal
infections, had little effect [5]. Instead, the Drosophila co- Nematode Infections
agulation system reduced nematode infectivity and further Nematode infections were performed according to the protocol
work established that this involves a close collaboration described previously [14]. Here we further modified the microtiter
between humoral- and cell-derived factors, which together plate infection assay to provide more consistent data and enable
easy comparison of larger groups of genes and their knockdown in
act against EPN infections [6, 9]. In accordance with previ- different tissues. For the infection assays, Drosophila eggs were col-
ous work on other hosts [10], eicosanoids also appear to lected 6 h after transferring flies to new food with addition of yeast;
contribute to this response in fly larvae [6]. 60 h later, larvae were collected, rinsed briefly in 25 ° C tap water

In addition to the interest in EPNs as pest control and placed individually in the wells of a microtiter plate. EPNs
agents and as tools to study insect innate immunity, the were used for infection 1 or 2 months after their release from G.
mellonella cadavers to ensure optimal pathogenicity and were di-
study of infectious nematodes has also implications for luted to 25 IJs/larva. Ten microliters of the EPN suspension were
medical research. Filarial nematodes use insects as vec- added to each well; the plate was covered with Parafilm® and kept
tors to infect vertebrates, such as cattle and humans, at 25 ° C at a 12-hour light/dark cycle. In all infection experiments,

sometimes causing debilitating and fatal diseases such as larval mortality caused by EPNs was scored 48 h after infection
river blindness, and heart and lymphatic filariasis [4]. [14]. The mortality results of the screens were normalized to the
respective control and the fold increase in mortality is shown. For
This has stimulated several systematic studies on the microarray analysis and quantitative PCR verification, third instar
transcriptome changes that occur in vector insects after larvae (88 h after egg laying) were infected with H. bacteriophora
infection with filarial nematodes [11–13]. For example, harboring green fluorescent protein (GFP)-expressing P. lumi-
the innate immune responses against the causative agents nescens in microtiter plates (100 IJs/larva). After 2 h of infection,
of lymphatic filariasis (caused by Brugia pahangi) were Drosophila larvae were rinsed briefly in water and transferred to
vials with fly food. They were scored for a GFP signal after addi-
characterized in their insect host (Armigeres subalbatus), tional 6 h (confirmation of Photorhabdus septicemia) and used for
and several putative immune-related molecules were RNA isolation. To improve visualization of EPN invasion into
found to be induced [12]. A closely related species (B. ma- their hosts and the hemocyte response to the infection (fig. 1), we
layi) to which Armigeres is resistant showed a distinctly performed nematode infections in plastic bags [14]. Larvae of D.
different pattern of induction [12]. Ultimately, the hope melanogaster expressing GFP under the control of DDC (dopa de-
carboxylase) promoter were infected with EPNs harboring the
behind these studies is to increase our understanding of wild-type strain of P. luminescens. To simultaneously monitor the
these responses and identify protective factors [4]. This infection process and hemocyte recruitment, larvae expressing red
may permit blocking or at least delaying transmission of fluorescent protein (RFP) in hemocytes were infected with H. bac-
the nematodes. teriophora harboring GFP-expressing P. luminescens. In both cas-
One obstacle for the functional studies in vector hosts es, the infection was performed using a high dose of EPNs (300 IJs/
larva). The larvae were washed 2 h after infection and transferred
is the more restricted repertoire of molecular techniques to the new plastic bag without EPNs. Eight hours after infection,
available for their genetic manipulation. The goal of the larvae were checked for the signs of infection (melanized wounds:
work presented here is that the extended knowledge of fig. 1).
Drosophila immunity will advance our general under-
standing of the responses against both EPNs and filarial Fly Strains
Fly strains were kept under standard conditions. All RNAi lines
nematodes. To this end, we performed a genome-wide came from the Vienna Drosophila RNAi Center [15] and NIG-Fly
analysis of the Drosophila transcriptome response after Stock Center (see online suppl. table 1B for further details; see
EPN infection. In addition, we tested a panel of mutant www.karger.com/doi/10.1159/000353734 for all online suppl. ma-
Drosophila lines in representative genes detected in our terial). Two GD and one KK RNAi lines from the Vienna Dro-
screen together with some other candidates and identi- sophila RNAi Center were used for a single candidate gene primar-
ily upon availability. Glutactin mutant GltEY22126, which contains
fied several promising gene products which slow down insertion of P(EPgy2) transposable element within the second cod-
EPN infections in Drosophila larvae. ing exon, was obtained from the Bloomington Stock Center. Bruno
Lemaitre kindly provided the mutants GNBP-3hades [16], PGRP-
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2 J Innate Immun Arefin /Kucerova /Dobes /Markus /Strnad /

DOI: 10.1159/000353734 Wang /Hyrsl /Zurovec /Theopold

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a
b c
d e
f g
h i jj k
k
Fig. 1. Infection routes of nematodes and hemocyte recruitment. (g, the punctate signal) and the DDC-GFP signal (more diffuse
a Infections can occur via the mouth, hindgut or by penetrat- signal in the gut epithelium, surrounding the wound site). h, i Ses-
ing the cuticle of Drosophila larvae. Arrows indicate entry sites. sile hemocytes (red) in hml-GAL4/UAS-RFP larva; noninfected
b, c Noninfected DDC-GFP-expressing larva [visualized by stereo- larva shows hemocyte clusters (h) and after infection the clusters
microscopy (b) and fluorescence channel (c)]. d, e DDC-GFP lar- disperse (j). The punctate signal corresponds to hemocytes. i Non-
va infected by nematodes. Note that melanin spots (two of which infected larva seen in the fluorescence channel. To trace the infec-
are indicated by arrows and also shown at larger magnification) tion the larva was infected with nematodes harboring GFP-ex-
are visible accompanied by local DDC activation [stereomicros- pressing bacteria (green). Arrows indicate the wound (j, k) and
copy (d) and fluorescence channel (e)]. f, g Upon entry via the arrowheads show bacteria (k). Inset shows bacteria at a higher
mouth or hindgut, nematodes create wounds (arrow) in the gut; magnification (all infected samples were analyzed 16 h after infec-
phase-contrast picture (f), fixed dissected gut stained with DAPI tion with nematodes). Inf./Non-inf. = Infected/noninfected.
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Nematode Infection of Drosophila J Innate Immun 3

DOI: 10.1159/000353734
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SAseml [17], PGRP-LCE12 [18], PGRP-LE112 [19] and PGRP-LF200 Quantitative PCR
[20] (see online suppl. table 1B for further details). Infection of Total RNA from infected and noninfected Drosophila larvae
Drosophila larvae was compared with either wild-type w1118 or was isolated using the RiboZol RNA extraction reagent (Amresco).
crosses between Gal4 drivers and w1118 (compare figure legends The RNA was further purified by NucleoSpin RNA II kit (Mache-
4–6 for details). Gal4-expressing driver lines with specificity for rey-Nagel) including an on-column digestion step with rDNase I.
either the fat body (ppl-Gal4) or hemocytes (he-Gal4 and hml- Total RNA (1,000 ng) was applied for reverse transcription using
Gal4) were used. The DDC expression pattern or hemocyte local- PrimeScript Reverse Transcriptase (Takara) and oligo(dT)(17-
ization was assessed in DDC-GFP and hml-GAL4 UAS-RFP flies, mer). Quantitative PCR was performed using the HOT FIREPol
respectively (see online suppl. table 1B for details). The vkg-GFP EvaGreen qPCR Mix Plus (Solis BioDyne, Tartu, Estonia). The
(G00454) strain was obtained from the Flytrap collection. W; PCR reaction volume was 20 μl, containing 5 μl of diluted cDNA
Idgf3/+; UAS-Idgf3/+ served as our wild-type stock for the micro- and 250 nM primers. The amplification was carried out in an Eco
array analysis. The data presented here are part of a larger study, real-time PCR system (Illumina) for 45 cycles (95 ° C for 15 s; an-

which includes addressing the influence of Idgf3 on nematode in- nealing temperature dependent on primer pair for 30 s; 72 ° C for
fection. We have performed the controls for the heterozygous lar- 20 s) following an initial denaturation/Pol activation step (95 ° C
vae and found no significant difference (see online suppl. fig. 4). for 15 min). Each sample was analyzed in triplicate. Primers (on-
In line with our previously published results [21], we observe that line suppl. table 1) were designed with Lasergene PrimerSelect
mortality rates are consistent among the wild-type strains used in Software (DNASTAR) to assure that each amplicon was specific.
this study, only Oregon R (not used otherwise in this study) shows Melting analysis confirmed a single product for each primer pair
slightly but not significantly (p = 0.102) increased mortality. reaction. The product size was verified by gel electrophoresis. Data
were analyzed and quantified with the Illumina EcoStudy software.
RNA Isolation Relative mRNA levels were normalized to Rack1 and rp49 expres-
Total RNA from nematode-infected and naive third instar Dro- sion and standardized to the noninfected sample. The results are
sophila larvae was extracted using RiboZol RNA extraction reagent presented as the mean log2-transformed fold changes (infected/
(Amresco) according to the manufacturer’s protocol and subse- noninfected larvae) in transcript levels ±SEM of 2 independent
quently cleaned with NucleoSpin RNA II kit (Macherey-Nagel). biological replicates.
Quality and concentration of the RNA were measured with a
NanoDrop 2000 spectrophotometer (Thermo Scientific). RNA in- Microscopy
tegrity was analyzed in an Agilent 2100 Bioanalyzer. We included Leica MZ FLIII fluorescence stereomicroscope coupled to a
only samples with an intact RNA profile. Panasonic DMC-G2 camera was used to visualize wounds, DDC-
GFP localization and hemocyte recruitment. Images of dissected
Expression Profiling guts and tissues stained with DAPI were taken with a Hamamatsu
The Affymetrix GeneChip® Drosophila Genome 2.0 Array ORCA-ER camera (C4742-95) attached to a Zeiss Axioplan 2 mi-
System was used for microarray analysis following the standard croscope. Confocal images were taken in a Zeiss LSM 510 Meta
protocol [100 ng RNA was amplified with GeneChip 3′ IVT Ex- microscope.
press Kit (Affymetrix) and 10 μg of labeled cRNA was hybridized
to the chip according to the manufacturer’s instructions]. Statistical Evaluation of Infection Experiments
All experiments were run at least in triplicate using 48 Dro-
Statistical Analysis of Array Data sophila larvae per replicate. Each experiment was repeated inde-
Analysis was performed in three replicates. Data were prepro- pendently at least twice. The results are expressed as the mean ±
cessed in Partek Genomic Suit (Partek). In short, the transcription SD. Mortality rates were compared to the respective control cross
profiles were background corrected using the GCRMA method, between driver lines and w1118, which was always included to ac-
quantile normalized and variance stabilized using base-2 logarith- count for differences between different sets of experiments. The
mic transformation. Analysis of variance yielded transcripts dif- level of significance for the individual comparisons was deter-
ferentially expressed between analyzed samples (within LIMMA mined using Student’s t test (unpaired, two-sided).
[22]); Storey’s q values [23] were used to select significantly differ-
entially transcribed genes, q < 0.05. The transcription data are
MIAME compliant and deposited in the ArrayExpress database
(accession E-MTAB-1542). Results
All statistical analyses were performed in R (http://www.R-
project.org) and within Bioconductor [24]. Differentially ex- EPN Infections in Drosophila Occur via Two Entry Sites
pressed genes were selected for gene set enrichment analysis
(GSEA). We performed GSEA on genes that mapped to KEGG To follow the infection of Drosophila larvae after incu-
pathways [25] and have defined gene ontology (GO) terms [26] bation with EPNs, we used a combination of H. bacte-
using the Fisher test and approach of Tian et al. [27]. For GSEA, riophora and its associated bacterium P. luminescens ex-
pathways with q < 0.05 and |log FC| ≥0.4 (where FC = fold change) pressing GFP [5]. To assess the infection status of the lar-
after hypergeometric testing were considered differentially ex- vae, we relied on a recently developed method that allows
pressed (online suppl. table 4) but pathways at q < 0.05 and |log
FC| ≥0.4 are also shown (online suppl. table 4, right-most column continuous monitoring based on the detection of GFP-
part). expressing Photorhabdus [5, 14]. The entry of the nema-
todes into the hemocoel of Drosophila larvae causes
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wounds, which melanize (fig. 1d–e) and the release of The transcripts that are shared with the array results from
bacterial symbionts subsequently leads to bacteremia earlier studies include the majority of the transcripts in
(fig. 1k). Similar to other EPNs, Heterorhabditis was the highly regulated fraction (fig. 2; online suppl. table 3).
found to enter larvae both via the cuticle and the underly- The majority of EPN-regulated transcripts (485) are not
ing epidermis and via the gut epithelium (fig. 1; online differentially regulated under the other conditions [fig. 2c,
suppl. fig. 1A, B). When using a GFP-expressing Dro- category D; online suppl. table 3 (complete survey of in-
sophila strain under the control of the DDC promoter, duced genes)].
induction of the reporter in cells in the wider area sur- GO analysis using AmiGO (http://amigo.geneontol-
rounding the wound was observed in both locations ogy.org) of the top 100 list, which included the most
(fig. 1e, g; online suppl. fig. 1C for comparison). Similar strongly positively and negatively regulated genes, shows
to other infection models that involve cellular immunity a highly significant enrichment for immune-related
[28], hemocytes are recruited from their sessile compart- genes: antimicrobial peptides, immune-induced genes
ments into the circulation (fig. 1h–k). (IMs and Edin), a thioester-containing protein (TEP) 2
and Relish, the key transcriptional activator of the imd
Transcriptome Changes upon EPN Infection pathway (fig. 2b; online suppl. table 2). This is particu-
To obtain a complete picture of the larval immune re- larly true when GO analysis was performed on those
sponse against EPNs, we performed a genome-wide tran- genes that had previously been found to be induced in
scriptional analysis comparing infected and noninfected bacterial infection models (table 1, categories A, B, F).
animals at the same stage, feeding third instar larvae (ap- Nematode- and wasp-specific genes mapped to addition-
prox. 96 h after egg laying). Since the duration of the in- al pathways, most of which are related to developmental
fection and the multiple attempts of nematodes to infect processes. When all differentially regulated transcripts
larvae (fig. 1d, e) precluded an exact determination of the were mapped to the KEGG database, several additional
time point of infection, the infected samples were chosen developmental pathways were identified. Among those,
such that the GFP signal due to the release of bacteria the fly equivalent of vertebrate oocyte maturation, Wnt
from the nematode intestine had just passed the detection signaling, and ubiquitin-mediated proteolysis were the
threshold 6 h after exposure to EPNs. In this way, we en- most significantly enriched (online suppl. table 4). Other
sured that the samples had reached the same stage of the pathways were found enriched with lower significance,
infection and the sampled transcriptome represents the although some of these are connected to the Wnt path-
early stage of a developing bacteremia. way (online suppl. fig. 2).
Samples were collected in triplicate and RNA was hy- To confirm the data obtained by microarrays, we used
bridized to Affymetrix GeneChip Drosophila Genome real-time RT-PCR on genes selected from representatives
2.0 arrays. After statistical analysis and setting the statisti- of all categories in figure 2c covering different induction
cal threshold to a value of q < 0.05 and the threshold of levels including repression. This analysis largely confirms
induction at 2, a total of 642 transcripts were found dif- the results from the array study, however one of the genes
ferentially regulated upon nematode infection. A com- (PGRP-LF) displayed a lower fold change |log FC <1|
plete heatmap of the 100 most strongly regulated tran- (fig. 3, online suppl. table 6).
scripts shows that the replicates from the infected and In summary, the genome-wide transcriptional analysis
noninfected samples are well separated and that the ma- identifies both immune-related genes as well as members
jority of regulated genes are induced (fig. 2a). Explorato- of other pathways. In general, immune genes appear most
ry analysis using principal component analysis confirms strongly regulated. The identification of the additional
the similarity between the triplicates of induced and non- pathways requires further analysis, but a likely explana-
induced samples (fig. 2a and data not shown). The list of tion for their induction is a requirement during closure
all transcripts induced upon infection with Heterorhabdi- and healing of the wounds afflicted by the nematodes.
tis/Photorhabdus was compared to earlier studies, which
included bacterial (both nonpathogenic and pathogenic)- Functional Tests of Candidate Genes in Response to
and wasp-infected larvae and revealed a core set of 17 Nematodes
genes induced under all four conditions (fig. 2c, category To test their contribution to the response to nema-
A; online suppl. table 3). Additional transcripts were todes, we used RNAi lines and mutants for candidate
shared between EPN-infected larvae and the three other genes identified in the arrays and examined the viability
conditions (fig. 2a, c, additional categories B, C, E–H). of such larvae upon EPN infection. As a criterion used for
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a
2
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selecting candidate defense genes from the list of induced organ. Having observed that upon EPN infection, hemo-
transcripts, we focused on those for which a function in cytes are recruited into the circulation (fig. 1j, k), we sup-
immunity had been established in other infection models plemented this approach using a second, hemocyte-spe-
[29] as well as on their paralogs, which had not yet been cific driver (fig. 4a). Using a selection of significantly in-
implicated in immune reactions. duced immune and developmental genes identified in
our array, we failed to find any effects when the genes
Functional Tests Using RNAi Lines were knocked down in hemocytes. In contrast, when us-
Since the transcriptional response against nematodes ing gene silencing in the fat body, significant effects were
and their bacteria shows a strong immune signature, we observed for Gram-negative binding protein-like protein
decided to score mortality rates after knocking down can- (GNBP-like 3), attacin A (one line), and a third one for
didate genes in the fat body, the key Drosophila immune an immune-induced peptide (IM18). For the GNPB-like
Table 1. Gene ontology analysis of genes regulated by nematobacterial infection
Region Gene Category Biological process

name No.
A 17 all GO:0019731 antibacterial humoral response

GO:0006952 defense response
GO:0042742 defense response to bacterium
B 15 nematode + wasps + G+ and G– bacteria GO:0006952 defense response
GO:0050829 defense response to Gram– bacterium
GO:0042742 defense response to bacterium
C 71 nematodes + wasps GO:0048731 system development
GO:0007275 multicellular organismal development
GO:0009653 anatomical structure morphogenesis
D 485 nematode GO:0030154 cell differentiation
GO:0048699 generation of neurons
GO:0048869 cellular developmental process
E 15 nematodes + G+ and G– bacteria no significant hit
F 11 nematodes + G+ and G– bacteria + pathogenic G– bacteria GO:0019731 antibacterial humoral response
GO:0019731 antibacterial humoral response
G 22 nematodes + pathogenic G– bacteria no significant hit
H 6 nematodes + wasps + pathogenic G– bacteria no significant hit
Significantly enriched pathways after GO analysis using AmiGO. The top list of enriched GO terms is shown for all the nematode-
regulated categories shown in the Venn diagram in figure 2c. G+/G– = Gram-positive/Gram-negative.
Fig. 2. The significantly regulated transcripts after nematobacte- of the categories compare the Venn diagram in c. b GO classifica-
rial infection are enriched for immune genes. a A heatmap repre- tion of the 100 most strongly up-regulated genes. Immune re-
senting the 100 most strongly regulated transcripts from the mi- sponse molecules occupy a fourth of the top 100 genes (see also
croarray (columns c1–c3 = Control: noninfected; i1–i3 = infected online suppl. table 2). c Venn diagram showing differentially regu-
larvae). Each column represents an independent sample. Color key lated transcripts after infection with common Gram-negative (G–)
and density plot represent the level of regulation. Dark intensities and -positive (G+) bacteria [32, 34], pathogenic G– bacterial wasps
indicate the most up- and down-regulated genes, respectively. The [33, 35, 49] and nematodes (this work) in Drosophila larvae. 381
one-letter code to the right of the heatmap indicates whether the and 104 transcripts are specifically up- or down-regulated after
gene was previously detected in other genome-wide analysis of nematobacterial infection (comprising altogether 485 differential-
Drosophila larval immune response (category A–C, G, H) or is spe- ly regulated transcripts in category D).
cifically regulated upon nematode infection (D); for a description
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12
10
Fold change (log2)

8
6
4
2
Fig. 3. Validation of microarray data using
quantitative RT-PCR. Selected genes from 0
the different categories of genes regulat-
–2
PGRP-SB1
Drosomycin
Tep1
Tep2
Spds
PGRP-LF
ed by nematobacterial infection were ana-
IM3
GNBP-like
CG7607
Attacin A
Diptericin B
Glutactin
lyzed by quantitative PCR using RNA sam-
ples from naive and infected larvae. The
relative expression level (ratio infected/
noninfected larvae) is shown as log2 mean
from 2 independent experiments ± SEM.
Knockdown in fat body Knockdown in hemocytes

6 6
Normalized mortality (fold)
5 5

**
4 4
**
3 * 3 **
2 2
1 1
0 0
larval translucida 18977 GD
Wrinkled 8269 GD
Diptericin B 28736 GD
Drosomycin 2703 GD
GNBP-like 3 7545 GD
IM2 43372 GD
IM3 29214 GD
IM18 49651 GD
starry night 51382 GD
Argonaute 2 49473 GD
Argonaute 2 51521 GD
Akap200 5647 GD
starry night 1665 GD
Attacin-A 50320 GD Non-inf.

Attacin-A 9287 GD
Attacin-A 50319 GD
Attacin-A 50320 GD
PGRP-SB1 101298 KK
P PLX w1118
He x w1118
Argonaute 2 100356 KK
Akap200 102374 KK
Akap200 109996 KK
starry night 107993 KK

Diptericin B 102607 KK
Metchnikowin 109740 KK
GNBP-like 3 107358 KK
SPH93 104307 KK
PPL X w1118
13422R-3 (GNBP-like 3)
a Immune molecules Development Neg. contro l b NIG RNAi
Fig. 4. Increased Drosophila mortality upon nematobacterial infec- crosses, which were set to 1. Attacin A-noninfected (Non-inf.) is a
tion after knocking down the expression of genes selected from the parallel handling and developmental control. b The increase in
array data. a Selected RNAi lines (see online suppl. table 1B for mortality observed for GNBP-like 3 was confirmed using an inde-
further details) were crossed with a fat body (ppl-GAL4) or hemo- pendent RNAi line from the NIG-Fly collection. Data presented
cyte-specific driver (he-GAL4) and infected with H. bacteriophora are means ± SD; t test: * p < 0.05; ** p < 0.01, only for lines with
at a dose of 25 IJs/larva at 25 ° C. Mortality was scored after 48 h
significantly increased mortality.
and is presented after normalization to the respective control
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5
** **

4
** *
3
Fig. 5. Candidate screen for Drosophila 2

genes involved in defense against nemato-
1
bacterial infection using available mutants.
Mutants for recognition molecules (see on- 0
line suppl. table 1B for further details) and
TEP4
TEP3
PPL X w1118
Glutactin
TEP2
w-A5001
w1118
PGRP-LC
TEP23
PGRP-LE
GNBP-3
PGRP-LF
PGRP-SA
the basement membrane component glu-
tactin were infected with H. bacteriophora
at a dose of 25 IJs/larva at 29 ° C. Mortality

was scored as in figure 4. * p < 0.05; ** p <

0.01.
3 gene, a similar trend was found for a second line while Glutactin Is Required in Hemocytes
two additional attacin A lines failed to reproduce the phe- Since hemocytes are known to contribute to the for-
notype. Increased mortality was also seen for an indepen- mation of the basement membrane [31], we speculated
dent GNBP-like 3 line from a different collection, provid- that interfering with glutactin production specifically in
ing additional confirmation of our results (fig. 4b). For these cells might also have an impact on EPN infection.
IM18, only one line was available. These results indicate This idea is further supported by the observation that glu-
that the candidate genes obtained by transcriptional pro- tactin shows enriched expression in hemocytes compared
filing, and in particular GNBP-like 3, likely play a role in to whole larvae [32]. As controls, we also knocked down
the immunity against EPNs, although the data from RNAi expression of several other genes with enriched expres-
studies require further verification. sion in hemocytes, including two C-type lectins, SPARC
and two phagocytic receptors (see online suppl. table 5 for
Functional Tests of Mutants a list of the genes and the ratio of hemocyte enrichment).
Based on the microarray results, we extended our assay Indeed, while none of the control knockdowns led to in-
to an additional set of key immune molecules, for which creased susceptibility, two out of three glutactin RNAi
mutant fly strains were available (fig. 5), including mem- lines crossed with the first hemocyte-specific driver
bers of well-characterized classes of pattern recognition (Hemese) showed an increase in mortality after EPN in-
receptors, such as GNBPs and peptidoglycan recognition fection, while the third available line failed to do so. When
proteins (PGRPs), as well as some members of Drosophi- this line was crossed with a different hemocyte GAL4 line
la TEPs, three of which had also been found changed after (Hemolectin), it turned out that it presented higher sus-
EPN infection (see online suppl. table 3) and the base- ceptibility, too (fig. 6b). This means that the expression of
ment membrane protein glutactin. Amongst the recogni- glutactin in hemocytes contributes to protection against
tion proteins, a significant influence on EPN infections EPNs. To assess the damage afflicted to the basement
was observed for PGRP-LF and for TEP3. The most sig- membrane during nematode infection, we infected larvae
nificant difference was obtained with TEP3 mutants ei- that express a GFP-tagged version of collagen IV (in Dro-
ther alone or in combination with TEP2, both of which sophila also called Viking), one of the major components
had previously been tested in in vivo infections of Dro- of the basement membrane. While Viking normally
sophila without any detectable effects [30]. Finally, we ob- forms a fibrous network surrounding the gut, transmi-
served increased mortality of glutactin mutants, support- gration of nematodes creates wounds that melanize as
ing a protective function of the basement membrane. visible in figure 1 and lack Viking in an area that extends
This result is consistent with the idea that upon entry of beyond melanization (online suppl. fig. 3). This under-
EPNs into Drosophila larvae, the basement membrane lines the need for creating a replacement for the basement
has a protective function since it is encountered by nem- membrane.
atodes crossing either gut or cuticular epidermis.
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DOI: 10.1159/000353734
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a b
Fig. 6. Screen for genes involved in the defense against nematobac- bacteriophora at a dose of 25 IJs/ larva at 29 ° C. The effects for glu-

terial infection using candidate genes with hemocyte-enriched tactin knockdown were confirmed with an early hemocyte driver
transcription. a RNAi lines for selected hemocyte-enriched genes (hml-Gal4, b). Data presented are means ± SD; t test: * p < 0.05;
(see online suppl. table 1B and text for further details) were crossed ** p < 0.01. Mortality was scored as in figure 4.
with a hemocyte-specific driver (he-Gal4) and infected with H.
Discussion maturation pathway, Wnt signaling and ubiquitin-me-

diated proteolysis. Additional pathways are identified
Here we provide a genome-wide characterization of at lower significance levels. It is worth noting that some
the transcriptional changes that occur upon infection of of these pathways are connected, such as the ubiquitin
Drosophila larvae with the EPN H. bacteriophora and its and mitogen-activated protein kinase pathways, both of
associated bacteria P. luminescens. Our initial character- which are linked to Wnt signaling (online suppl. fig. 2).
ization of the infection process shows that the gut epithe- In addition, upstream of mitogen-activated protein ki-
lium, the cuticle and the underlying epidermis are tar- nases, the receptor tyrosine kinase stitcher has been
geted, leading to activation of a wound DDC-GFP report- shown to be involved in wound healing in the embryo
er construct (fig. 1). Our microarray analysis therefore [36] and may play a similar role in larvae.
covers all major events that occur as a consequence of the One possible explanation for the activation of Dro-
entry of the nematode and the influence of both the nem- sophila equivalent to the mammalian oocyte maturation
atode and its bacteria on the host immune system. pathway might be that this pathway includes mainly a
A highly significant enrichment for immune genes ob- number of general cell cycle regulators and such proteins
tained in the set of most strongly induced genes (fig. 2) are also induced in a proliferative response of various so-
supports the notion that an immune response against matic tissues upon nematode wounding (online suppl.
Photorhabdus has been initiated at the time when the GFP fig. 2). These include Polo-like kinases, which are key
signal indicating the presence of bacteria appeared in the regulators of cell division, the mitotic cyclin B and pro-
hemolymph. In addition to immune genes shared with tein kinase A. An effect of wounding is also supported by
earlier reports on septic injury due to bacteria or wasp the fact that genes in Wnt and hedgehog signaling, as well
infestation [32–35], some genes with proposed immune as components of the extracellular matrix, are enriched
functions are specifically induced in the presence of EPNs in our gene set. All three pathways have been shown to
(fig. 2c). When genes with weaker levels of induction are be important during wound healing acting at different
included and mapped to known pathways, three path- stages in vertebrates and may play comparable roles in
ways turn up as most significantly enriched: an oocyte flies [37, 38]. For example, the Wnt pathway may support
130.237.94.172 - 11/23/2013 12:01:37 PM


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the establishment of planar cell polarity during epider- for this discrepancy may be a JNK-dependent induction
mal wound healing similar to its role during develop- and/or a developmentally regulated constitutive expres-
ment and the extracellular matrix may play a role similar sion of this gene. Recent data regarding infections of adult
to the granulation tissue that forms after wounding in flies with EPNs suggest that AMPs can be induced by bac-
vertebrates [39]. Hemocytes may be crucial during this teria-free (axenic) nematodes, most likely also as a part
process by contributing collagen IV [40] and glutactin of wounding defense [8]. Notably, the mammalian AMP
(this work) in a localized fashion. Ultimately, wound LL-37 contributes to wound healing [45] as part of its
healing converges on the activation of small ρ GTPases, pleiotropic effects [46].
which have a well-defined role during wound healing in Finally, consistent with our notion that one of the
Drosophila [41, 42]. However, this occurs mostly at the pathways we identified in the GSEA was the synthesis of
posttranslational level and is less amenable to transcrip- extracellular matrix, we found that the ECM protein glu-
tome studies. tactin protects against EPN infections. This extends our
For a preliminary functional analysis of the genes in- previous findings that clot components reduce EPN mor-
volved in the nematobacterial response, we focused on tality. We propose that the basement membrane compo-
several of the induced genes from the array including the nents cooperate with the clot matrix to seal the wounds
basement membrane component glutactin and a set of afflicted by nematodes. This potentially occurs by linking
known recognition molecules (fig. 5). The data confirm the clot to the wound edges and/or sealing it off on the
earlier observations from others and our group that the hemolymph side, such a process is akin to the formation
imd and Toll pathways that are required for induction of of the granulation tissue during wound healing in verte-
immune genes are dispensable for the response against brates [39]. Similarly, the recent observation that the
EPNs, since mutants in the receptors required for their complement system interacts with the clotting cascade is
activation do not show increased mortality [5, 21]. We consistent with our observation that both the clotting and
attribute the effects we observed with PGRP-LF, which is the C3 homolog TEP3 act against nematobacterial infec-
a negative regulator of the imd pathway, to the previous- tions [47]. Taken together, both the list of genes we found
ly observed developmental defects [20]. Alternatively, to be regulated by EPN infection and the genes we have
PGRP-LF, which acts as a negative regulator of imd sig- tested functionally show specific signatures, which are
naling [20, 43], may aid to combat nematodes indirectly not shared with other infection models. Both sets of genes
by positively regulating other effector pathways. Most in- provide a rich source for further testing in different set-
terestingly, mutants in one of the TEP3 survive EPN in- tings such as septic and aseptic wounding and infection
fections significantly less than controls and other TEP with bacteria that are pathogenic to Drosophila such as
mutants. Of note, the same mutants had previously failed Pseudomonas entomophila and Serratia marcescens [34,
to show any developmental or immune phenotype [30]. 48].
A trend to increased susceptibility was also found for the
recognition molecule GNBP-like 3.
We also tried to interfere with the induction of some Acknowledgments
of the highly regulated immune genes identified in the ar-
The authors’ work is supported by the Swedish Research Coun-
ray by their specific knockdown in both hemocytes and
cil (U.T.), STINT (Swedish Foundation for International Coop-
the fat body. Since robust knockdowns are difficult to eration in Research and Higher Education), the Knut and Alice
achieve in this setting, it is not too surprising that these Wallenberg Foundation (U.T.), the Swedish Cancer Foundation
data are more variable than our previous findings. Nev- (U.T.), the European Social Fund and the state budget of the Czech
ertheless, two RNAi lines with specificity for small im- Republic (P.H. and P.D.) and the European Union Seventh Frame-
work Program (FP7/2007-2013) under grant agreement No.
mune peptides (attacin A and IM18) did also show an
316304 (M.Z.). We thank Dominique Ferrandon for providing the
effect. Although these data require verification, for ex- TEP mutant lines, Zdenek Mracek for the wild-type strain of H.
ample due to possible off-target effects (online suppl. ta- bacteriophora and Todd Ciche for GFP-expressing P. luminescens.
ble 1B), they seem to contradict the concept that the imd Authors are grateful to the service laboratory at the Institute of
pathway is not required during the anti-EPN response. Molecular Genetics and especially to Martina Chmelikova for
technical assistance.
However, they are consistent with the possible attacin
function implicated from its differential induction in a
different worm infection model involving a tapeworm
and the flour beetle as a host [44]. Possible explanations
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DOI: 10.1159/000353734
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References
1 Eleftherianos I, ffrench-Constant RH, Clarke 14 Dobes P, Wang Z, Markus R, Theopold U, 26 Ashburner M, Ball CA, Blake JA, Botstein D,
DJ, Dowling AJ, Reynolds SE: Dissecting the Hyrsl P: An improved method for nematode Butler H, Cherry JM, Davis AP, Dolinski K,
immune response to the entomopathogen infection assays in Drosophila larvae. Fly Dwight SS, Eppig JT, Harris MA, Hill DP, Is-
Photorhabdus. Trends Microbiol 2010; 18: (Austin) 2012;6:75–79. sel-Tarver L, Kasarskis A, Lewis S, Matese JC,
552–560. 15 Dietzl G, Chen D, Schnorrer F, Su KC, Bari- Richardson JE, Ringwald M, Rubin GM, Sher-
2 Balasubramanian N, Nascimento G, Ferreira nova Y, Fellner M, Gasser B, Kinsey K, Oppel lock G: Gene ontology: tool for the unification
R, Martinez M, Simoes N: Pepsin-like aspar- S, Scheiblauer S, Couto A, Marra V, Keleman of biology. The gene ontology consortium.
tic protease (Sc-ASP155) cloning, molecular K, Dickson BJ: A genome-wide transgenic Nat Genet 2000;25:25–29.
characterization and gene expression analy- RNAi library for conditional gene inactiva- 27 Tian L, Greenberg SA, Kong SW, Altschuler
sis in developmental stages of nematode tion in Drosophila. Nature 2007;448:151–156. J, Kohane IS, Park PJ: Discovering statistically
Steinernema carpocapsae. Gene 2012; 500: 16 Gottar M, Gobert V, Matskevich AA, Reich- significant pathways in expression profiling
164–171. hart JM, Wang C, Butt TM, Belvin M, Hoff- studies. Proc Natl Acad Sci USA 2005; 102:
3 Lang AE, Schmidt G, Schlosser A, Hey TD, mann JA, Ferrandon D: Dual detection of 13544–13549.
Larrinua IM, Sheets JJ, Mannherz HG, Ak- fungal infections in Drosophila via recogni- 28 Zettervall CJ, Anderl I, Williams MJ, Palmer
tories K: Photorhabdus luminescens toxins tion of glucans and sensing of virulence fac- R, Kurucz E, Ando I, Hultmark D: A directed
ADP-ribosylate actin and RhoA to force actin tors. Cell 2006;127:1425–1437. screen for genes involved in Drosophila blood
clustering. Science 2010;327:1139–1142. 17 Michel T, Reichhart JM, Hoffmann JA, Royet cell activation. Proc Natl Acad Sci USA 2004;
4 Castillo JC, Reynolds SE, Eleftherianos I: In- J: Drosophila Toll is activated by Gram-posi- 101:14192–14197.
sect immune responses to nematode para- tive bacteria through a circulating peptido- 29 Lemaitre B, Hoffmann J: The host defense of
sites. Trends Parasitol 2011;27:537–547. glycan recognition protein. Nature 2001;414: Drosophila melanogaster. Annu Rev Immu-
5 Hallem EA, Rengarajan M, Ciche TA, Stern- 756–759. nol 2007;25:697–743.
berg PW: Nematodes, bacteria, and flies: a tri- 18 Gottar M, Gobert V, Michel T, Belvin M, 30 Bou Aoun R, Hetru C, Troxler L, Doucet D,
partite model for nematode parasitism. Curr Duyk G, Hoffmann JA, Ferrandon D, Royet J: Ferrandon D, Matt N: Analysis of thioester-
Biol 2007;17:898–904. The Drosophila immune response against containing proteins during the innate im-
6 Hyrsl P, Dobes P, Wang Z, Hauling T, Wil- Gram-negative bacteria is mediated by a pep- mune response of Drosophila melanogaster. J
helmsson C, Theopold U: Clotting factors and tidoglycan recognition protein. Nature 2002; Innate Immun 2011;3:52–64.
eicosanoids protect against nematode infec- 416:640–644. 31 Lavine MD, Strand MR: Insect hemocytes and
tions. J Innate Immun 2011;3:65–70. 19 Takehana A, Yano T, Mita S, Kotani A, Oshi- their role in immunity. Insect Biochem Mol
7 Castillo JC, Shokal U, Eleftherianos I: A novel ma Y, Kurata S: Peptidoglycan recognition Biol 2002;32:1295–1309.
method for infecting Drosophila adult flies protein (PRGP)-LE and PGRP-LC act syner- 32 Irving P, Ubeda JM, Doucet D, Troxler L, La-
with insect pathogenic nematodes. Virulence gistically in Drosophila immunity. EMBO J gueux M, Zachary D, Hoffmann JA, Hetru C,
2012;3:339–347. 2004;23:4690–4700. Meister M: New insights into Drosophila lar-
8 Castillo JC, Shokal U, Eleftherianos I: Im- 20 Maillet F, Bischoff V, Vignal C, Hoffmann J, val haemocyte functions through genome-
mune gene transcription in Drosophila adult Royet J: The Drosophila peptidoglycan recog- wide analysis. Cell Microbiol 2005;7:335–350.
flies infected by entomopathogenic nema- nition protein PGRP-LF blocks PFRP-LC and 33 Wertheim B, Kraaijeveld AR, Schuster E,
todes and their mutualistic bacteria. J Insect IMD/JNK pathway activation. Cell Host Mi- Blanc E, Hopkins M, Pletcher SD, Strand MR,
Physiol 2013;59:179–185. crobe 2008;3:293–303. Partridge L, Godfray HC: Genome-wide gene
9 Wang R, Liang Z, Hal M, Söderhäll K: A trans- 21 Wang Z, Wilhelmsson C, Hyrsl P, Loof TG, expression in response to parasitoid attack in
glutaminase involved in the coagulation sys- Dobes P, Klupp M, Loseva O, Mörgelin M, Drosophila. Genome Biol 2005;6:R94.
tem of the freshwater crayfish, Pacifastacus Ikle J, Cripps RM, Herwald H, Theopold U: 34 Vodovar N, Vinals M, Liehl P, Basset A, De-
leniusculus. Tissue localisation and cDNA Pathogen entrapment by transglutaminase – grouard J, Spellman P, Boccard F, Lemaitre B:
cloning. Fish Shellfish Immunol 2001; 11: a conserved early innate immune mechanism. Drosophila host defense after oral infection by
623–637. PLoS Pathog 2010;6:e1000763. an entomopathogenic Pseudomonas species.
10 Kim Y, Ji D, Cho S, Park Y: Two groups of 22 Smyth GK: Linear models and empirical Proc Natl Acad Sci USA 2005; 102: 11414–
entomopathogenic bacteria, Photorhabdus bayes methods for assessing differential ex- 11419.
and Xenorhabdus, share an inhibitory action pression in microarray experiments. Stat 35 Schlenke TA, Morales J, Govind S, Clark AG:
against phospholipase A2 to induce host im- Appl Genet Mol Biol 2004;3:article3. Contrasting infection strategies in generalist
munodepression. J Invertebr Pathol 2005; 89: 23 Storey JD, Tibshirani R: Statistical signifi- and specialist wasp parasitoids of Drosophila
258–264. cance for genomewide studies. Proc Natl melanogaster. PLoS Pathog 2007;3:1486–1501.
11 Aliota MT, Fuchs JF, Mayhew GF, Chen CC, Acad Sci USA 2003;100:9440–9445. 36 Wang S, Tsarouhas V, Xylourgidis N, Sabri N,
Christensen BM: Mosquito transcriptome 24 Gentleman RC, Carey VJ, Bates DM, Bolstad Tiklova K, Nautiyal N, Gallio M, Samakovlis
changes and filarial worm resistance in Ar- B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge C: The tyrosine kinase stitcher activates
migeres subalbatus. BMC Genomics 2007; 8: Y, Gentry J, Hornik K, Hothorn T, Huber W, grainy head and epidermal wound healing in
463. Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Drosophila. Nat Cell Biol 2009;11:890–895.
12 Aliota MT, Fuchs JF, Rocheleau TA, Clark Rossini AJ, Sawitzki G, Smith C, Smyth G, 37 Razzell W, Wood W, Martin P: Swatting flies:
AK, Hillyer JF, Chen CC, Christensen BM: Tierney L, Yang JY, Zhang J: Bioconductor: modelling wound healing and inflammation
Mosquito transcriptome profiles and filarial open software development for computation- in Drosophila. Dis Model Mech 2011; 4: 569–
worm susceptibility in Armigeres subalbatus. al biology and bioinformatics. Genome Biol 574.
PLoS Negl Trop Dis 2010;4:e666. 2004;5:R80. 38 Shaw TJ, Martin P: Wound repair at a glance.
13 Erickson SM, Xi Z, Mayhew GF, Ramirez JL, 25 Kanehisa M, Goto S: Kegg: Kyoto encyclope- J Cell Sci 2009;122:3209–3213.
Aliota MT, Christensen BM, Dimopoulos G: dia of genes and genomes. Nucleic Acids Res 39 Sonnemann KJ, Bement WM: Wound repair:
Mosquito infection responses to developing 2000;28:27–30. toward understanding and integration of sin-
filarial worms. PLoS Negl Trop Dis 2009; gle-cell and multicellular wound responses.
3:e529. Annu Rev Cell Dev Biol 2011;27:237–263.
130.237.94.172 - 11/23/2013 12:01:37 PM


Downloaded by:
40 Adachi T, Tomita M, Yoshizato K: Synthesis 43 Basbous N, Coste F, Leone P, Vincentelli R, 47 Nikolajsen CL, Scavenius C, Enghild JJ: Hu-
of prolyl 4-hydroxylase alpha subunit and Royet J, Kellenberger C, Roussel A: The Dro- man complement C3 is a substrate for trans-
type IV collagen in hemocytic granular cells sophila peptidoglycan-recognition protein LF glutaminases. A functional link between non-
of silkworm, Bombyx mori: Involvement of interacts with peptidoglycan-recognition protease-based members of the coagulation
type IV collagen in self-defense reaction and protein LC to downregulate the Imd pathway. and complement cascades. Biochemistry
metamorphosis. Matrix Biol 2005; 24: 136– EMBO Rep 2011;12:327–333. 2012;51:4735–4742.
154. 44 Zhong D, Wang MH, Pai A, Yan G: Tran- 48 Cronin SJ, Nehme NT, Limmer S, Liegeois S,
41 Stramer B, Wood W, Galko MJ, Redd MJ, Ja- scription profiling of immune genes during Pospisilik JA, Schramek D, Leibbrandt A, Si-
cinto A, Parkhurst SM, Martin P: Live imag- parasite infection in susceptible and resistant moes Rde M, Gruber S, Puc U, Ebersberger
ing of wound inflammation in Drosophila strains of the flour beetles (Tribolium casta- I, Zoranovic T, Neely GG, von Haeseler A,
embryos reveals key roles for small GTPases neum). Exp Parasitol 2013;134:61–67. Ferrandon D, Penninger JM: Genome-wide
during in vivo cell migration. J Cell Biol 2005; 45 Ramos R, Silva JP, Rodrigues AC, Costa R, RNAi screen identifies genes involved in in-
168:567–573. Guardao L, Schmitt F, Soares R, Vilanova M, testinal pathogenic bacterial infection. Sci-
42 Lesch C, Jo J, Wu Y, Fish GS, Galko MJ: A Domingues L, Gama M: Wound healing ence 2009;325:340–343.
targeted UAS-RNAi screen in Drosophila lar- activity of the human antimicrobial peptide 49 Lee MJ, Mondal A, Small C, Paddibhatla I,
vae identifies wound closure genes regulating LL37. Peptides 2011;32:1469–1476. Kawaguchi A, Govind S: A database for the
distinct cellular processes. Genetics 2010;186: 46 Vandamme D, Landuyt B, Luyten W, Schoofs analysis of immunity genes in Drosophila:
943–957. L: A comprehensive summary of LL-37, the Padma database. Fly (Austin) 2011; 5: 155–
factotum human cathelicidin peptide. Cell 161.
Immunol 2012;280:22–35.
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A
Arefin, Fig.S1
Arefin, Fig.S2
Non‐inf. PC Non‐inf. Vkg‐GFP Non‐inf. Merge
Inf. PC Inf. Vkg‐GFP Inf. Merge
Arefin, Fig.S3
E3CF<=GH?-"F3CA<=GA2"I/3=-J"
!"
#"
$"
%"
&"
'"
Arefin, Fig.S4
(###)""
(###)"*"(+",-./%01234
567+"89:4,-./%"
77;45<=&"*"(###)"
>?45<=&"*"(###)"
1<@A3@":"
BC?.3@"D"
Fig. S1. Internal gut wounds caused by the nematode are visible through the
cuticle (A and B, note the uniform activity of the DDC-GFP reporter, [1]). C: An
artificial wound made by a needle (125 micrometer in diameter) is shown as control
(reproducing the local activation of the DDC-GFP reporter like in Fig. 1D).
Fig. S2. KEGG map for the Drosophila Wnt signaling pathway.
Fig. S3. Damage of the basement membrane after nematode infection of
Drosophila larvae. Gut preparations from nematode-infected and control larvae (non-
infected) that express a Collagen IV (viking)-GFP fusion protein [2] were analyzed
using confocal microscopy. Note the loss of Viking(GFP) signal after nematode entry,
which extends beyond the melanized area (White line highlights the melanized area
boundary in green channel (Vkg-GFP), PC: phase contrast, ).
Fig. S4. No significant changes in mortality are observed among Drosophila
control lines, and the heterozygote strain, which was used in microarray. All the
indicated controls and the heterozygote (w; Idgf3/+; UAS-Idgf3/+) were subjected to
infection with Heterorhabditis bacteriophora at a dose 25 IJs per larva at 25° C.
Mortality was scored 48h after the infection. No significant changes in mortality were
detected among the lines. Data presented are means ±SD (see Material and Methods
for further details).

Arefin, Table S1
Gene Primer name Sequence Annealing

temperature
Rack1 Rack1 fw CCC GTG ACA AGA CCC TGA T 50 °C
Rack1 rev TAG TTG CCA TCG GAG GAG AG
rp49 rp49 fw CTT CAT CCG CCA CCA GTC 50 °C
rp49 rev GGC GAC GCA CTC TGT TGT
IM3 IM3 fw TTG GGT CTG CTG GCT CTG 52 °C
IM3 rev TTC AAC TGG CAT CCT TCA TTC
Diptericin B DiptB fw CTA TTC ATT GGA CTG GCT TGT G 54 °C
DiptB rev GTC CAT TGG GGC TCT GC
Drosomycin Drs fw CCC TCT TCG CTG TCC TGA 52 °C
Drs rev TTA GCA TCC TTC GCA CCA G
Attacin A AttA fw TGG TCA TGG TGC CTC TTT G 54 °C
AttA rev GAT TGT GTC TGC CAT TGT TGA
PGRP-SB1 SB1 fw GGG TGC GGT GTC TGC TC 52 °C
SB1 rev TCA CCG GCC ACG ATA AAG
GNBP-like CG13422 fw CCA AGG CCA CCG TCA AG 52 °C
CG13422 rev TCG CTC AGA TCG TCC ATT T
Tep1 Tep2 fw GTC CTG CTC GCC CTT CTC 52 °C
Tep2 rev TCA AAT GCC AAA ACT CTA TGT CA
Tep2 Tep1 fw CGT TCT GCT GGC TTT CTT C 52 °C
Tep1 rev ATA CTG GTC GTC CGT CTT GTC
Spds Spds fw GCG ATG GCC TGT GGT TT 52 °C
Spds rev CAG CGC GTG TTT CAT TAG C
Glutactin Glt fw TAT CCG GTA CAA GAG CCA CAG 54 °C
Glt rev TTT CGG GGA GAT TTT CGT T
CG7607 CG7607 fw TAC ATA GCC GCC CAA AGA A 52 °C
CG7607 rev AGC CAT GCA AAA GAA AGT GAT
PGRP-LF LF fw CGC CAT CAT GTT TCA CTC AA 52 °C
LF rev GAC CAA TAA AGG CCA CGA CTA
Arefin, Table S1B
VDRC lines
Array genes (fig.4)
y)
ra
on
r
oa
si
e
es
om
r
ic
pr
(m
os
ex
ID
l
ve
dy
ro
t
an
le
bo
h
e
C
m
am
n
at
ge
ed
or
io
ty
f
n
ct
r
sf
ili
rt
al
o
ta
e
pe
du
n
se
b
rv
en
ia
ff
o
Ty
La
In
In
Tr
O
N
V
C
1 Attacin A CG10146 9287 GD 1 viable 3 yes Strongly induced

4 DiptericinB CG10794 102607 KK 0 viable 2 yes Strongly induced
5 DiptericinB CG10794 28736 GD 1 viable 3 yes Strongly induced
6 Drosomycin CG10810 2703 GD 5 viable 2 yes Strongly induced
7 GNBP like 3 CG13422 107358 KK 0 viable 2 yes Strongly induced
8 GNBP like 3 CG13422 7545 GD 0 viable 3 yes Strongly induced
9 IM2 CG18106 43372 GD 3 viable 2 yes Strongly induced
10 IM3 CG16844 29214 GD 2 viable 1 yes Strongly induced
11 IM18 CG10332 49651 GD 0 viable 3 yes Moderate
12 Metchnikowin CG8175 109740 KK 1 viable 2 yes Strongly induced
13 PGRP-SB1 CG9681 101298 KK 0 viable 2 yes Strongly induced
14 SPH93 CG6639 104307 KK 0 viable 2 yes Moderate
15 Akap200 CG13388 102374 KK 0 viable 2 yes 2 fold (LogFC)
16 Akap200 CG13388 109996 KK 0 viable 2 yes 2 fold (LogFC)
17 Akap200 CG13388 5647 GD 0 viable 2 yes 2 fold (LogFC)
18 Starry night CG11895 107993 KK 0 viable 2 Not available Moderate
19 Starry night CG11895 1665 GD 0 viable 3 Not available Moderate
20 Starry night CG11895 51382 GD 0 viable 2 Not available Moderate
21 Argonaute 2 CG7439 100356 KK 0 viable 2 yes no
22 Argonaute 2 CG7439 49473 GD 0 viable 2 yes no
23 Argonaute 2 CG7439 51521 GD 0 viable 2 yes no
24 Larval translucida
CG32372 18977 GD 2 viable 3 yes no
25 Wrinkled CG5123 8269 GD 0 viable 3 no no
Hemocytes enriched genes (fig. 6)

26 Lectin24DB CG2958 105118 KK 0 viable 2
27 Lectin24DB CG2958 45294 GD 2 viable 3
28 Lectin28C CG7106 104201 KK 0 viable 2
29 SPARC CG6378 100566 KK 1 viable 2
30 SPARC CG6378 16677 GD 1 viable 2
31 Eater CG6124 4301 GD 0 viable 3
32 dSR-C1 CG4099 49964 GD 0 viable 2
33 Glutactin CG9280 101918 KK 1 viable 2
34 Glutactin CG9280 15428 GD 1 viable 3
35 Glutactin CG9280 15429 GD 1 viable 2
36 CG4250 CG4250 101949 KK 0 viable 2
NIG lines (Fig.4)
37 GNBP like 3 CG13422 13422R-3 viable 3
Mutant lines (Fig.5)
Bloomington linesNo
(Fig.5)Gene name CG no Genotype Stock no. Type Mutant type Viability
38 Glutactin CG9280 22539 Mutant P-element insertion
viable
From Bruno Lemaitre lab (Fig.5)

39 GNBP-3 CG5008 GNBP-3/TM6c Mutant lethal
40 PGRP-SA CG11709 y, w, DD, Seml (PGRP-SA)Mutant viable
41 PGRP-LC CG4432 PGRP-LCÊ12D3 Mutant viable
42 PGRP-LE CG8995 y1 w67c23 PGRP-LE112 Mutant viable
43 PGRP-LF CG4437 PGRP-LF200 Mutant lethal
From Dominique Ferrandom lab (Fig.5)

44 Tep2 CG7052 Tep2 (d11521) Mutant viable
45 Tep3 CG7068 Tep3 (d03976) Mutant viable
46 Tep4 CG10363 Tep4(EY04656) Mutant viable
47 Tep2,3 Double mutant
FRT medited deletion
viable
Reporter lines
48 Ddc-GFP - 1.4 kb Ddc-GFP trnasgenic line was obtained from William McGinnis lab
49 hml-GAL4;UAS-RFP w1118 iso;+; hml-Gal4 UAS-RFP line was generated by recombining hml-Gal4 and UAS-RFP on 3rd chromosome
50 vkg-GFP CG16858 G00454 protein-trap viable obtained from Cooley lab (Flytrap)
Arefin, Table S3
Injection of G+ and G- bacteria (FC)
Pathogenic G- Bacteria (FC)

Nematodes (log2 FC)
Wasps (Code/FC)
Gene Name Abbreviation Probe Set class
Immune induced molecule 3 IM3 1622893_at 8.99 7.858111968 12.05 4.1 A - all infections
elevated during infection edin 1634366_at 8.76 13.28267974 C – shared EPN+wasps
Immune induced molecule 4 IM4 1626345_at 8.23 7.265109147 2.62 B - missing in pathogenic bacteria
Attacin-C AttC 1641419_at 7.94 10.45256917 28.08 11.655 A - all infections
Immune induced molecule 1 IM1 1633053_at 7.87 2.870099541 26.73 2.685 A - all infections
IM2-like CG15065 1631660_at 7.81 14 9.4 2.365 A - all infections
Defensin Def 1634271_at 7.81 2.86 E - shared EPN+bac. injection
Diptericin B DptB 1638235_at 7.29 4.04834199 9.270157347 9.95 A - all infections
GNBP-like CG13422 1627271_at 7.1 2 9.6 B - missing in pathogenic bacteria
Dmel_CG15067 CG15067 1625174_at 6.95 2 10.46 B - missing in pathogenic bacteria
Dmel_CG33470 CG33470 1639019_s_at 6.93 D - EPN specific
Immune induced molecule 2 IM2 1640360_at 6.84 2.091029944 7.62 B - missing in pathogenic bacteria
Immune induced molecule 23 IM23 1629530_at 6.75 2 13.12 B - missing in pathogenic bacteria
Drosomycin Drs 1635189_at 6.02 3.318574591 11.7879722 5.48 A - all infections
Immune induced molecule 10 IM10 1626319_a_at 5.95 14 23.24764177 2.62 A - all infections
Metchnikowin Mtk 1627613_at 5.5 9.252935299 37.81 13.865 A - all infections
Attacin-A AttA 1627551_s_at 5.46 18.76399116 2.319343217 B - missing in pathogenic bacteria
Attacin-A AttA 1625124_at 5.44 13.85899334 52.02091148 18.65 A - all infections
Dmel_CG30026 CG30026 1631370_at 5.29 D - EPN specific
Peptidoglycan recognition protein SB1 PGRP-SB1 1636490_at 5.27 10.20658452 3.54 5.44 A - all infections
Dmel_CG15068 CG15068 1635060_at 5.15 10.81605612 C – shared EPN+wasps
Cecropin C CecC 1632719_at 5 1.69 1.98 F - missing in wasp infection
Cecropin B CecB 1626530_at 4.45 4.357954545 1.83 B - missing in pathogenic bacteria
Attacin-D AttD 1631475_at 3.84 2.03 3.805 F - missing in wasp infection
Dmel_CG16775 CG16775 1623884_at 3.57 5.935 G – shared EPN+pathogenic G- bacteria
Dmel_CG18067 CG18067 1640144_at 3.28 13 7.103805806 2.255 A - all infections
Dmel_CG18107 CG18107 1638772_at 2.95 3.338274045 2.63 B - missing in pathogenic bacteria
gp150-like CG14762 1632809_at 2.95 D - EPN specific
Dmel_CG4716 CG4716 1629046_a_at 2.94 8 C – shared EPN+wasps
Dmel_CG30029 CR30029 1636492_at 2.92 1.68 G – shared EPN+pathogenic G- bacteria
accord accord 1634633_s_at 2.92 D - EPN specific
Fibrinogen-like CG5550 1629201_at 2.81 2.364948282 2.32 13.7 A - all infections
Dmel_CG4716 CG4716 1628963_at 2.7 2.09 2.7 F - missing in wasp infection
Thioester-containing protein 1 Tep1 1637734_at 2.66 12 C – shared EPN+wasps
Zn finger homeodomain 1 zfh1 1628262_a_at 2.54 D - EPN specific
Dmel_CG6639 CG6639 1625698_at 2.47 12 4.81 2.16 A - all infections
Chronologically inappropriate morphogenesis chinmo 1629484_s_at 2.45 D - EPN specific
Mucin 26B Muc26B 1632017_at 2.44 1.638986293 2.075 F - missing in wasp infection
Mucin 96D Muc96D 1636058_at 2.44 D - EPN specific
polychaetoid pyd 1637322_at 2.4 0.653075935 C – shared EPN+wasps
Mucin 96D Muc96D 1635733_x_at 2.4 D - EPN specific
Insulin-like receptor InR 1629141_at 2.35 D - EPN specific
starry night stan 1626087_at 2.32 D - EPN specific
Dmel_CG8160 CG8160 1638648_at 2.31 7.75063567 7.6 2.98 A - all infections
zye CG5847 1623710_at 2.31 1.162055336 C – shared EPN+wasps
Beta amyloid protein precursor-like Appl 1624033_at 2.31 0.797154497 C – shared EPN+wasps
Transposon.63 1632295_s_at 2.31 D - EPN specific
Dmel_CG14695 CG14695 1639704_at 2.29 1.394326241 1.9 H - missing in bac. Injection
NK7.1 CG8524 1631303_s_at 2.22 D - EPN specific
anachronism ana 1630502_at 2.21 D - EPN specific
no receptor potential A norpA 1636576_s_at 2.21 D - EPN specific
Chronologically inappropriate morphogenesis chinmo 1628005_at 2.16 D - EPN specific
moody CG4322 1633112_at 2.14 D - EPN specific
grappa gpp 1638708_s_at 2.12 D - EPN specific
DISCO Interacting Protein 1 DIP1 1624215_s_at 2.09 D - EPN specific
anon-fast-evolving-1H7 CR42862 1625672_s_at 2.08 D - EPN specific
klumpfuss klu 1629347_at 2.08 D - EPN specific
Dmel_CG6279 1638969_at 2.07 D - EPN specific
Dmel_CG13461 CG13461 1635959_at 2.06 2.274807593 E - shared EPN+bac. injection
anon-fast-evolving-1H7 CG32477 1640472_at 2.05 1.68 1.885 F - missing in wasp infection
wech CG42396 1639785_s_at 2.04 0.8495051 C – shared EPN+wasps
Neurofibromin 1 Nf1 1625048_at 2.03 D - EPN specific
Mucin 68Ca Muc68Ca 1638341_s_at 2.03 D - EPN specific
Muscle-specific protein 300 Msp-300 1632945_at 2.02 D - EPN specific
Neurofibromin 1 Nf1 1634083_at 2.02 D - EPN specific
Odorant-binding protein 49a Obp49a 1624932_at 2.01 D - EPN specific
polyA-binding protein interacting protein 2 Paip2 1628632_at 2.01 D - EPN specific
Dmel_CG8388 CG8388 1627323_at 2 D - EPN specific
Thioester-containing protein 2 Tep2 1630067_a_at 1.99 7.195665353 4.089814116 B - missing in pathogenic bacteria
SRPK SRPK 1632130_s_at 1.98 D - EPN specific
IGF-II mRNA-binding protein Imp 1623455_s_at 1.97 D - EPN specific
headcase hdc 1629307_s_at 1.96 0.594499033 C – shared EPN+wasps
twin of eyeless toy 1633512_at 1.96 D - EPN specific
Dmel_CG12071 CG12071 1624143_a_at 1.95 D - EPN specific
Fasciclin 1 Fas1 1624183_a_at 1.95 D - EPN specific
Cenp-C Cenp-C 1629217_at 1.94 D - EPN specific
Bruce CG6303 1634454_at 1.93 0.954526709 C – shared EPN+wasps
Transposon.17 1624819_s_at 1.93 D - EPN specific
cable Cbl 1635580_at 1.93 D - EPN specific
grappa gpp 1627796_s_at 1.89 D - EPN specific
Mucin 68Ca Muc68Ca 1631700_at 1.89 D - EPN specific
pericentrin-like cp309 1634009_at 1.88 D - EPN specific
Kinesin heavy chain 73 Khc-73 1627136_at 1.87 D - EPN specific
Elongase 68α Elo68α 1635229_at 1.87 D - EPN specific
LD07388P/CAPRICIOUS LD07388 1634920_at 1.86 D - EPN specific
Dmel_CG16711 CG16711 1624565_a_at 1.85 2.467849224 C – shared EPN+wasps
derailed drl 1624297_at 1.85 D - EPN specific
Muscle-specific protein 300 Msp-300 1627250_at 1.85 D - EPN specific
Tie-like receptor tyrosine kinase Tie 1629129_at 1.85 D - EPN specific
Dmel_CG10332/Immune induced molecule 18 CG10332/IM18 1622952_at 1.83 D - EPN specific
Relish Rel 1627376_at 1.82 4 3.60014622 3.415 A - all infections
leak lea 1636905_at 1.82 0.943275096 C – shared EPN+wasps
Peptidoglycan recognition protein LF PGRP-LF 1633145_at 1.8 D - EPN specific
KP78b KP78b 1639894_at 1.8 D - EPN specific
lethal (3) neo38 l(3)neo38 1640598_s_at 1.78 D - EPN specific
armitage armi 1623907_at 1.76 D - EPN specific
dumpy dp 1634075_at 1.76 D - EPN specific
Cyclin T CycT 1637897_at 1.75 0.716867073 C – shared EPN+wasps
Darkener of apricot Doa 1640892_a_at 1.73 D - EPN specific
Distal-less Dll 1636088_at 1.71 D - EPN specific
Cht6 Cht6 1633303_at 1.69 D - EPN specific
anastral spindle 1 ana1 1637397_a_at 1.69 D - EPN specific
tartan trn 1639235_at 1.69 D - EPN specific
Thor CG8846 1635900_at 1.67 1.71 3.44 F - missing in wasp infection
mutagen-sensitive 210 mus210 1632827_a_at 1.67 D - EPN specific
nemo nmo 1624300_s_at 1.66 0.870678928 C – shared EPN+wasps
schnurri shn 1625445_s_at 1.66 D - EPN specific
trio trio 1635047_s_at 1.66 D - EPN specific
Myosin binding subunit Mbs 1630456_at 1.65 D - EPN specific
Connectin Con 1639913_at 1.63 0.912106008 C – shared EPN+wasps
telomere fusion tefu 1630729_at 1.63 D - EPN specific
unc-13 unc-13 1635684_a_at 1.63 D - EPN specific
Syncrip Syp 1640760_at 1.62 0.27532097 2.416948355 B - missing in pathogenic bacteria
scabrous sca 1636998_at 1.62 0.876908394 C – shared EPN+wasps
Prosap Prosap 1625236_s_at 1.62 D - EPN specific
stumps stumps 1634063_a_at 1.61 D - EPN specific
staufen stau 1633016_a_at 1.6 D - EPN specific
dim γ-tubulin 5 dgt5 1641436_at 1.6 D - EPN specific
enabled ena 1627191_a_at 1.59 D - EPN specific
CHKov1 CHKov1 1625997_s_at 1.58 D - EPN specific
eukaryotic translation initiation factor 4G2 eIF4G2 1637987_at 1.55 0.862151836 C – shared EPN+wasps
Cullin-3 Cul-3 1624970_s_at 1.55 D - EPN specific
golden goal gogo 1625852_at 1.55 D - EPN specific
mRNA-like ncRNA in embryogenesis 2 MRE2 1629325_at 1.55 D - EPN specific
abrupt ab 1629702_a_at 1.55 D - EPN specific
naked cuticle nkd 1630361_at 1.55 D - EPN specific
Myocardin-related transcription factor Mrtf 1636679_at 1.55 D - EPN specific
anastral spindle 3 ana3 1640805_at 1.55 D - EPN specific
serrano sano 1627971_s_at 1.54 D - EPN specific
Thd1 Thd1 1636521_at 1.54 D - EPN specific
Nutrient Amino Acid Transporter 1 NAAT1 1639528_at 1.54 D - EPN specific
Jumonji, AT rich interactive domain 2 Jarid2 1634092_at 1.52 0.858938891 C – shared EPN+wasps
enhanced adult sensory threshold east 1624378_at 1.52 D - EPN specific
couch potato cpo 1632644_s_at 1.52 D - EPN specific
Dmel_CG42356 CG42356 1632958_a_at 1.5 2.33 G – shared EPN+pathogenic G- bacteria
mustard mtd 1626842_a_at 1.5 D - EPN specific
nervous fingers 1 nerfin-1 1634039_at 1.5 D - EPN specific
longitudinals lacking lola 1635096_at 1.5 D - EPN specific
onecut onecut 1630376_at 1.49 D - EPN specific
bangles and beads bnb 1632734_s_at 1.49 D - EPN specific
Cirl Cirl 1640640_at 1.49 D - EPN specific
Dmel_CG31780 CG31780 1624619_s_at 1.48 7.268118978 C – shared EPN+wasps
Checkpoint suppressor homologue CHES-1-like 1629295_at 1.48 D - EPN specific
Not1 Not1 1638560_a_at 1.47 D - EPN specific
Phosphodiesterase 11 Pde11 1639582_at 1.47 D - EPN specific
APC-like Apc 1628548_at 1.46 1.37 G – shared EPN+pathogenic G- bacteria
polyhomeotic proximal ph-p 1623441_at 1.46 D - EPN specific
Myocyte enhancer factor 2 Mef2 1626392_s_at 1.46 D - EPN specific
decapentaplegic dpp 1630026_s_at 1.46 D - EPN specific
Protein C kinase 98E Pkc98E 1631059_at 1.46 D - EPN specific
Alhambra Alh 1630415_at 1.45 1.02075416 C – shared EPN+wasps
kon-tiki kon 1625687_at 1.45 D - EPN specific
Calmodulin-binding transcription activator Camta 1626232_at 1.45 D - EPN specific
polo polo 1636189_at 1.45 D - EPN specific
karst kst 1637710_at 1.45 D - EPN specific
fat ft 1624125_at 1.44 D - EPN specific
mushroom-body expressed mub 1625921_at 1.44 D - EPN specific
skuld skd 1631516_s_at 1.44 D - EPN specific
asense ase 1635124_at 1.44 D - EPN specific
anterior open aop 1627394_s_at 1.43 1.00640539 C – shared EPN+wasps
Stromalin SA 1626710_at 1.42 D - EPN specific
Meiotic central spindle Meics 1629709_at 1.42 D - EPN specific
Suppressor of zeste 2 Su(z)2 1633866_at 1.42 D - EPN specific
longitudinals lacking lola 1640945_at 1.41 2.573891626 C – shared EPN+wasps
trithorax trx 1624533_s_at 1.41 0.936943992 C – shared EPN+wasps
ftz transcription factor 1 ftz-f1 1624520_a_at 1.41 D - EPN specific
Liprin-γ Liprin-γ 1625011_at 1.41 D - EPN specific
Tis11 homolog Tis11 1623863_a_at 1.4 D - EPN specific
spalt major salm 1627881_at 1.4 D - EPN specific
SoxNeuro SoxN 1631408_at 1.4 D - EPN specific
Dp110 Pi3K92E 1631594_s_at 1.4 D - EPN specific
dally-like dlp 1636974_at 1.4 D - EPN specific
Dmel_CG12868 CG12868 1635030_at 1.39 3.062181652 8 F - missing in wasp infection
purity of essence poe 1634654_at 1.39 0.854332193 C – shared EPN+wasps
B52 B52 1633821_at 1.39 D - EPN specific
rad50 rad50 1638887_a_at 1.39 D - EPN specific
roundabout robo 1626774_s_at 1.38 D - EPN specific
gliolectin glec 1628743_at 1.38 D - EPN specific
midline fasciclin mfas 1632298_s_at 1.38 D - EPN specific
split ends spen 1641518_a_at 1.38 D - EPN specific
hibris hbs 1637539_a_at 1.37 0.74710544 C – shared EPN+wasps
myoblast city mbc 1631013_at 1.37 2.135 G – shared EPN+pathogenic G- bacteria
wings apart-like wapl 1636092_a_at 1.37 D - EPN specific
Sox21b CG32139 1637750_at 1.36 0.459252157 C – shared EPN+wasps
CAP-D2 condensin subunit CAP-D2 1625447_at 1.36 D - EPN specific
ariadne 2 ari-2 1625556_at 1.36 D - EPN specific
Smad on X Smox 1629290_at 1.35 D - EPN specific
disconnected disco 1639940_at 1.35 D - EPN specific
Synaptotagmin 4 Syt4 1641475_at 1.35 D - EPN specific
par-1 par-1 1628849_at 1.34 D - EPN specific
Tollo Tollo 1637481_at 1.34 D - EPN specific
hopscotch hop 1639072_at 1.33 2 C – shared EPN+wasps
insensitive insv 1624375_at 1.33 D - EPN specific
scribbler sbb 1626352_at 1.33 D - EPN specific
pou domain motif 3 pdm3 1631222_at 1.32 3.545454545 C – shared EPN+wasps
gluon glu 1635123_at 1.32 3.052313883 C – shared EPN+wasps
mutagen-sensitive 312 mus312 1629113_a_at 1.32 D - EPN specific
pointed pnt 1630010_a_at 1.32 D - EPN specific
chiffon chif 1630995_at 1.32 D - EPN specific
Interacts with the C terminus of ELL 1 Ice1 1631109_at 1.32 D - EPN specific
Centrosomal protein 190kD Cp190 1631940_s_at 1.32 D - EPN specific
Ankyrin 2 Ank2 1633313_at 1.32 D - EPN specific
vielfaltig vfl 1638370_s_at 1.32 D - EPN specific
pavarotti pav 1623405_at 1.31 D - EPN specific
Sin3A Sin3A 1630165_s_at 1.31 D - EPN specific
longitudinals lacking lola 1634495_s_at 1.3 0.837751405 C – shared EPN+wasps
optic ganglion reduced ogre 1628323_s_at 1.3 2.21608288 E - shared EPN+bac. injection
Promyelocytic leukemia zinc finger ortholog Plzf 1639534_at 1.3 D - EPN specific
Leukocyte-antigen-related-like Lar 1637537_at 1.29 0.92228936 C – shared EPN+wasps
multiple ankyrin repeats single KH domain mask 1641226_a_at 1.29 0.875724028 C – shared EPN+wasps
found in neurons fne 1633852_at 1.29 0.717516202 C – shared EPN+wasps
Glutactin Glt 1630515_s_at 1.29 1.06 G – shared EPN+pathogenic G- bacteria
squeeze sqz 1630772_at 1.29 D - EPN specific
Megalin mgl 1641272_at 1.29 D - EPN specific
prospero pros 1635500_a_at 1.28 D - EPN specific
female sterile (1) homeotic fs(1)h 1625127_at 1.27 0.680376656 C – shared EPN+wasps
nejire nej 1622925_at 1.27 D - EPN specific
roughest rst 1625366_at 1.27 D - EPN specific
Dmel_CG6181 Ge-1 1632251_s_at 1.27 D - EPN specific
monkey king mkg 1622892_s_at 1.26 1.21 G – shared EPN+pathogenic G- bacteria
longitudinals lacking lola 1625768_s_at 1.26 D - EPN specific
Dicer-1 Dcr-1 1627580_at 1.26 D - EPN specific
BRWD3 BRWD3 1640098_at 1.26 D - EPN specific
Dmel_CG13624 CG13624 1636321_s_at 1.25 1.48 G – shared EPN+pathogenic G- bacteria
debra dbr 1628243_at 1.25 D - EPN specific
Spc105-related Spc105R 1628318_at 1.25 D - EPN specific
Dmel_CG42319 CG42319 1624969_s_at 1.24 1.0203125 C – shared EPN+wasps
Serpin 28Dc Spn28Dc 1636145_at 1.24 1.579484809 E - shared EPN+bac. injection
growth arrest and DNA damage-inducible gene 45 Gadd45 1625139_at 1.24 1.16 G – shared EPN+pathogenic G- bacteria
Brahma associated protein 170kD Bap170 1628423_at 1.24 D - EPN specific
homeodomain interacting protein kinase hipk 1639306_s_at 1.23 0.900560193 C – shared EPN+wasps
Polycomblike Pcl 1623525_at 1.23 0.87116529 C – shared EPN+wasps
slender lobes sle 1624252_s_at 1.23 2.137019888 E - shared EPN+bac. injection
asterless asl 1625999_at 1.23 D - EPN specific
Bub1-related kinase BubR1 1641015_at 1.23 D - EPN specific
Dmel_CG15784 CG15784 1640884_at 1.22 2.122161816 3.943964525 2.285 A - all infections
necrotic nec 1636653_at 1.22 2.349722029 C – shared EPN+wasps
Daxx-like protein DLP 1630023_at 1.21 D - EPN specific
Myosin heavy chain-like Mhcl 1632231_a_at 1.21 D - EPN specific
mutagen-sensitive 201 mus201 1637542_s_at 1.21 D - EPN specific
Fasciclin 2 Fas2 1638956_at 1.21 D - EPN specific
Programmed cell death 4 ortholog Pdcd4 1640020_at 1.21 D - EPN specific
Hybrid male rescue Hmr 1641024_at 1.21 D - EPN specific
A kinase anchor protein 200 Akap200 1640838_s_at 1.2 2.384756328 2.015 F - missing in wasp infection
dachsous ds 1640627_at 1.2 1.049431232 C – shared EPN+wasps
Fancd2 Fancd2 1640096_at 1.2 D - EPN specific
toutatis tou 1640433_a_at 1.2 D - EPN specific
Cadherin 96Ca Cad96Ca 1623418_at 1.19 0.399106685 E - shared EPN+bac. injection
Tenascin accessory Ten-a 1623755_at 1.19 D - EPN specific
pr-set7 pr-set7 1624463_s_at 1.19 D - EPN specific
Breast cancer 2, early onset homolog Brca2 1624941_at 1.19 D - EPN specific
sister-of-Sex-lethal ssx 1627150_at 1.19 D - EPN specific
Ceramidase CDase 1639396_s_at 1.19 D - EPN specific
mirror mirr 1639798_at 1.19 D - EPN specific
Toll Tl 1639321_s_at 1.18 3 2.472136618 1.56 A - all infections
Imaginal disc growth factor 1 Idgf1 1633237_at 1.18 2.404311432 C – shared EPN+wasps
toucan toc 1641048_a_at 1.18 1.033009352 C – shared EPN+wasps
Transferrin 1 Tsf1 1632430_at 1.18 2.29 G – shared EPN+pathogenic G- bacteria
Na,K-ATPase Interacting NKAIN 1625992_s_at 1.18 D - EPN specific
odd paired opa 1628865_at 1.18 D - EPN specific
DNA ligase I DNA-ligI 1630390_at 1.18 D - EPN specific
WNK kinase Wnk 1635378_at 1.18 D - EPN specific
lethal (1) G0148 l(1)G0148 1639411_at 1.18 D - EPN specific
male-specific lethal 1 msl-1 1639438_at 1.18 D - EPN specific
Ankyrin 2 Ank2 1640337_a_at 1.18 D - EPN specific
Cadherin 87A Cad87A 1641575_at 1.18 D - EPN specific
Regulatory particle non-ATPase 5 Rpn5 1639091_at 1.17 2.037034174 E - shared EPN+bac. injection
cactus cact 1629899_at 1.17 D - EPN specific
retinal degeneration C rdgC 1634061_a_at 1.17 D - EPN specific
grappa gpp 1637016_at 1.17 D - EPN specific
Elongation factor 4a CG9932 1638653_a_at 1.17 D - EPN specific
Down syndrome cell adhesion molecule Dscam 1637619_s_at 1.16 0.818395743 C – shared EPN+wasps
Smc5 Smc5 1623590_s_at 1.16 D - EPN specific
mini spindles msps 1628172_at 1.16 D - EPN specific
abnormal oocyte abo 1628418_at 1.16 D - EPN specific
ATAC complex component 2 Atac2 1632308_at 1.16 D - EPN specific
Sulfated Sulf1 1635007_at 1.16 D - EPN specific
masquerade mas 1639190_at 1.16 D - EPN specific
knockout ko 1641310_at 1.16 D - EPN specific
SD27140 SD27140 1641365_s_at 1.16 D - EPN specific
Suppressor of cytokine signaling at 36E Socs36E 1637703_a_at 1.15 2.219902805 1.61 H - missing in bac. Injection
expanded ex 1625970_at 1.15 1.166760884 C – shared EPN+wasps
terribly reduced optic lobes trol 1640223_a_at 1.15 0.582856592 C – shared EPN+wasps
Inducer of meiosis 4 Ime4 1624263_at 1.15 D - EPN specific
windei wde 1626478_at 1.15 D - EPN specific
MAN1 MAN1 1631696_s_at 1.15 D - EPN specific
tay bridge tay 1633390_at 1.15 D - EPN specific
centrosomin cnn 1635619_a_at 1.15 D - EPN specific
Cht6 Cht6 1639149_at 1.15 D - EPN specific
Dmel_CG9449 CG9449 1628150_a_at 1.14 1.87 G – shared EPN+pathogenic G- bacteria
lethal (3) L1231 l(3)L1231 1629496_at 1.14 D - EPN specific
alan shepard shep 1637552_s_at 1.14 D - EPN specific
enoki mushroom enok 1639061_at 1.14 D - EPN specific
unkept unk 1633020_at 1.13 0.92385834 C – shared EPN+wasps
Ubiquitin-specific protease 64E Ubp64E 1624727_s_at 1.13 D - EPN specific
convoluted conv 1629638_at 1.13 D - EPN specific
Na,K-ATPase Interacting NKAIN 1633752_at 1.13 D - EPN specific
little imaginal discs lid 1633855_s_at 1.13 D - EPN specific
Cadherin 74A Cad74A 1635742_s_at 1.13 D - EPN specific
Integrator 1 IntS1 1636297_at 1.13 D - EPN specific
Lap1 Lap1 1639189_at 1.13 D - EPN specific
lemming A lmgA 1639494_at 1.13 D - EPN specific
RNA polymerase II 215kD subunit RpII215 1640764_at 1.13 D - EPN specific
LIM-kinase1 LIMK1 1641452_a_at 1.13 D - EPN specific
glass gl 1623923_a_at 1.12 D - EPN specific
Kinesin-like protein at 61F Klp61F 1624620_at 1.12 D - EPN specific
pickled eggs pigs 1626090_at 1.12 D - EPN specific
pyramus pyr 1631552_at 1.12 D - EPN specific
SMC2 SMC2 1634149_at 1.12 D - EPN specific
Hairless H 1638568_s_at 1.12 D - EPN specific
relative of woc row 1638839_at 1.12 D - EPN specific
ubiquitin-like protein-specific protease 1 Ulp1 1639435_at 1.12 D - EPN specific
vestigial vg 1641470_s_at 1.12 D - EPN specific
Enhancer of decapping 3 Edc3 1641685_at 1.12 D - EPN specific
shifted shf 1635403_at 1.11 2 C – shared EPN+wasps
Argonaute-1 AGO1 1632602_s_at 1.11 0.828827339 C – shared EPN+wasps
Transposon.32 SD01615P 1624543_s_at 1.11 D - EPN specific
nucampholin ncm 1628866_at 1.11 D - EPN specific
combgap cg 1630717_s_at 1.11 D - EPN specific
quaking related 58E-1 qkr58E-1 1631768_at 1.11 D - EPN specific
Activated Cdc42 kinase-like Ack-like 1637111_a_at 1.11 D - EPN specific
unc-104 ortholog unc-104 1637684_at 1.11 D - EPN specific
prominin-like CG7740 1641333_s_at 1.11 D - EPN specific
Dmel_CG17836 Xrp1 1632712_s_at 1.1 2.17 G – shared EPN+pathogenic G- bacteria
withered whd 1626147_s_at 1.1 D - EPN specific
ariadne ari-1 1628016_s_at 1.1 D - EPN specific
skywalker sky 1631094_s_at 1.1 D - EPN specific
Verprolin 1 Vrp1 1634562_s_at 1.1 D - EPN specific
polychaetoid pyd 1637428_a_at 1.1 D - EPN specific
capsuleen csul 1640862_a_at 1.1 D - EPN specific
semaphorin 2a Sema-2a 1629819_s_at 1.09 2.452724324 E - shared EPN+bac. injection
Enhancer of bithorax E(bx) 1625243_a_at 1.09 D - EPN specific
sloppy paired 2 slp2 1627053_at 1.09 D - EPN specific
egghead egh 1631621_s_at 1.09 D - EPN specific
Laminin B2 LanB2 1632666_at 1.09 D - EPN specific
sec24 sec24 1640083_at 1.09 D - EPN specific
Protein kinase related to protein kinase N Pkn 1640417_a_at 1.09 D - EPN specific
Posterior sex combs Psc 1631095_at 1.08 0.927774215 C – shared EPN+wasps
lethal (3) persistent salivary gland 2 l(3)psg2 1622931_at 1.08 D - EPN specific
Additional sex combs Asx 1628901_at 1.08 D - EPN specific
Transport and Golgi organization 6 Tango6 1636939_at 1.08 D - EPN specific
Bric-a-brac interacting protein 2 bip2 1639551_at 1.08 D - EPN specific
antiapoptosis clone 11 Aac11 1623100_at 1.07 D - EPN specific
Dorsal switch protein 1 Dsp1 1624304_s_at 1.07 D - EPN specific
polymerase eta DNApol-η 1626494_at 1.07 D - EPN specific
Meltrin Meltrin 1627649_at 1.07 D - EPN specific
crumbs crb 1628146_at 1.07 D - EPN specific
jim jim 1636911_at 1.07 D - EPN specific
Myb-interacting protein 130 mip130 1639185_at 1.07 D - EPN specific
fat facets faf 1640541_at 1.07 D - EPN specific
crooked legs crol 1626018_a_at 1.06 D - EPN specific
tonalli tna 1626730_s_at 1.06 D - EPN specific
Negative elongation factor A Nelf-A 1631550_at 1.06 D - EPN specific
Reduction in Cnn dots 5 Rcd5 1632420_at 1.06 D - EPN specific
Son of sevenless Sos 1633335_at 1.06 D - EPN specific
capicua cic 1635909_at 1.06 D - EPN specific
Hexokinase A Hex-A 1625638_a_at 1.05 7.469895514 1.735 F - missing in wasp infection
Furin 1 Fur1 1628952_s_at 1.05 D - EPN specific
Distal-less Dll 1630237_a_at 1.05 D - EPN specific
Spinophilin Spn 1633021_s_at 1.05 D - EPN specific
lola like lolal 1640311_s_at 1.05 D - EPN specific
drongo CG3365 1623693_a_at 1.04 1.93 G – shared EPN+pathogenic G- bacteria
leonardo 14-3-3ζ 1625148_s_at 1.04 D - EPN specific
Cyclin B3 CycB3 1626454_at 1.04 D - EPN specific
RhoGEF3 RhoGEF3 1626756_a_at 1.04 D - EPN specific
Trithorax-like Trl 1628275_at 1.04 D - EPN specific
Prosap Prosap 1629160_s_at 1.04 D - EPN specific
bves bves 1633387_at 1.04 D - EPN specific
wallenda wnd 1633727_s_at 1.04 D - EPN specific
hephaestus heph 1637478_s_at 1.03 0.98616031 3.42258756 B - missing in pathogenic bacteria
worniu wor 1631502_at 1.03 0.990381437 C – shared EPN+wasps
zipper zip 1627840_a_at 1.03 0.938871223 C – shared EPN+wasps
Suppressor of Cytokine Signaling at 16D Socs16D 1636122_at 1.03 0.857330289 C – shared EPN+wasps
embargoed emb 1622939_at 1.03 0.797073236 C – shared EPN+wasps
scalloped sd 1625515_a_at 1.03 0.755818539 C – shared EPN+wasps
echinoid ed 1627506_at 1.03 0.318416425 E - shared EPN+bac. injection
HERC2 HERC2 1629145_at 1.03 D - EPN specific
Kruppel homolog 1 Kr-h1 1631481_a_at 1.03 D - EPN specific
Nijmegen breakage syndrome nbs 1632161_at 1.03 D - EPN specific
mastermind mam 1632457_s_at 1.03 D - EPN specific
Bre1 Bre1 1624301_at 1.02 D - EPN specific
RNA-binding protein 1 Rbp1 1638486_at 1.02 D - EPN specific
Serpin 88Eb Spn88Eb 1636946_at 1.01 12 3.468032833 B - missing in pathogenic bacteria
mitochondrial RNA polymerase mtRNApol 1626738_at 1.01 D - EPN specific
domeless dome 1632381_at 1 1.558303521 C – shared EPN+wasps
pebbled peb 1622949_at 1 0.839048644 C – shared EPN+wasps
coronin pod1 1641620_s_at 1 2.009326839 E - shared EPN+bac. injection
Dmel_CG1233 CG1233 1625945_a_at 1 D - EPN specific
scaf6 scaf6 1627184_at 1 D - EPN specific
ballchen ball 1628342_s_at 1 D - EPN specific
piopio pio 1638681_at 1 D - EPN specific
Zinc finger AT-hook protein D19A 1640054_at 1 D - EPN specific
escargot esg 1641639_at 1 D - EPN specific
Spermidine Synthase SpdS 1630830_a_at -1 2.10757595 E - shared EPN+bac. injection
Dmel_CG13018 CG13018 1629541_at -1 D - EPN specific
Dmel_CG33169 CG33169 1632606_a_at -1 D - EPN specific
Glutathione S transferase E3 GstE3 1637129_at -1.01 6.271921203 E - shared EPN+bac. injection
Dmel_CG7607 CG7607 1626684_at -1.01 D - EPN specific
Dmel_CG7231 CG7231 1633333_a_at -1.01 D - EPN specific
Bekka Bka 1627349_at -1.03 D - EPN specific
mitochondrial ribosomal protein L10 mRpL10 1624012_at -1.04 D - EPN specific
Jonah 25Bii Jon25Bii 1639196_at -1.07 1.19702279 C – shared EPN+wasps
cornichon related cnir 1638047_at -1.07 D - EPN specific
Dmel_CG6041 CG6041 1627432_at -1.11 1.282119657 C – shared EPN+wasps
mitochondrial ribosomal protein L36 mRpL36 1624765_at -1.12 D - EPN specific
garnysstan gny 1636630_s_at -1.13 1.093143152 C – shared EPN+wasps
technical knockout tko 1626534_at -1.13 D - EPN specific
mitochondrial ribosomal protein S18C mRpS18C 1628621_at -1.23 D - EPN specific
ZnT35C CG3994 1639619_a_at -1.24 D - EPN specific
Allatostatin C Ast-C 1634436_at -1.26 D - EPN specific
fuseless fusl 1640365_s_at -1.29 D - EPN specific
Niemann-Pick type C-2f Npc2f 1634158_at -1.31 D - EPN specific
Lcp65APsi CR18775 1631002_at -1.32 1.338640275 C – shared EPN+wasps
Dmel_CG34172 CG34172 1625901_s_at -1.33 D - EPN specific
Cyp9f3Psi CG17875 1640584_at -1.35 D - EPN specific
selenocysteine methyltransferase CG10621 1626086_at -1.36 3.322991509 0.77 B - missing in pathogenic bacteria
Tetraspanin 42Er Tsp42Er 1634977_at -1.37 1.68 G – shared EPN+pathogenic G- bacteria
Tak1-like 2 Takl2 1632359_at -1.45 D - EPN specific
Glutathione S transferase E7 GstE7 1640065_at -1.46 6.081492362 E - shared EPN+bac. injection
Dmel_CG14610 CG14610 1636683_at -1.48 11 C – shared EPN+wasps
Juvenile hormone esterase Jhe 1637833_at -1.49 D - EPN specific
Ugt86Dj Ugt86Dj 1634029_at -1.51 D - EPN specific
Dmel_CG11825 CG11825 1635263_at -1.56 1.36 G – shared EPN+pathogenic G- bacteria
Dmel_CG17571 CG17571 1635878_s_at -1.59 D - EPN specific
Insulin-like peptide 3 Ilp3 1627943_at -1.61 D - EPN specific
Cuticular protein 67Fb Cpr67Fb 1638742_at -1.7 1.19722855 C – shared EPN+wasps
Jonah 65Ai Jon65Ai 1636460_at -1.75 0.48 G – shared EPN+pathogenic G- bacteria
Ecdysone-induced protein 63F 2 Eip63F-2 1639350_at -1.85 D - EPN specific
Dmel_CG8560 CG8560 1639641_at -1.87 1.215745914 0.73 H - missing in bac. Injection
Adipokinetic hormone Akh 1631816_at -2.26 D - EPN specific
Dmel_CG3344 CG3344 1632584_at -2.31 1.177934444 0.865 H - missing in bac. Injection
Glutathione Synthetase GS 1627321_x_at -2.35 D - EPN specific
Gene identified by more transcripts in italics.
KEGG ID KEGG path FDR P-value
dme04914 Progesterone-mediated oocyte maturation 2.42e-05 3.84e-07
dme04310 Wnt signaling pathway 0.00467 0.000148
dme04120 Ubiquitin mediated proteolysis 0.00988 0.000471
dme04630 Jak-STAT signaling pathway 0.0197 0.00156
dme04340 Hedgehog signaling pathway 0.0197 0.00153
dme04320 Dorso-ventral axis formation 0.0209 0.00199
dme04512 ECM-receptor interaction 0.0276 0.00306
dme03450 Non-homologous end-joining 0.0582 0.00739
dme03018 RNA degradation 0.0718 0.0103
dme00310 Lysine degradation 0.073 0.0116
dme03040 Spliceosome 0.0852 0.0149
dme03420 Nucleotide excision repair 0.104 0.0198
dme03440 Homologous recombination 0.112 0.0231
dme04013 MAPK signaling pathway - fly 0.161 0.0357
dme03013 RNA transport 0.179 0.0425
Arefin, Supplementary table 4
Arefin, Table S5

Name Fold (compared to Function Product

naive larval
expression)
CG4250 no naive larval circadian cycling LPS-induced tumor
expression in heads necrosis factor alpha
type
Lectin24Db no naive larval galactose binding C-type lectin-like
expression lectin
Lectin28C 59.62 galactose binding C-type lectin-like

lectin
Eater 43.67 cell adhesion, bacterial cell surface

recognition, binding protein
phagocytosis,
Scavenger 25.44 scavenger Complement control

receptor class C, receptor module
type 1 (SR-C1)
Glutactin 19.67 extracellular Carboxylesterase type

matrix B
glycoprotein
SPARC 13.75 ligand binding or SPARC/ostenectin-

carrier like

Arefin Table S6
MicroArrays
EPN Assay
EPN assay
(log2 FC)
(log2 FC)
qRT-PCR
mutant
RNAi
Gene Name Abbreviation Detected in larval immune response
Immune induced molecule 3 IM3 8.99 9.59 ns A - all infections
Diptericin B DptB 7.29 5.33 ns A - all infections
Candidates from our Microarray study
GNBP-like CG13422 7.1 3.73 FB B - missing in pathogenic bacteria

Immune induced molecule 2 IM2 6.84 ns B - missing in pathogenic bacteria
Drosomycin Drs 6.02 7.27 ns A - all infections
Metchnikowin Mtk 5.5 ns A - all infections
Attacin-A AttA 5.44 3.55 FB A - all infections
Peptidoglycan recognition protein SB1 PGRP-SB1 5.27 3.77 ns A - all infections
Thioester-containing protein 1 Tep1 2.66 2.82 C – shared EPN+wasps
Dmel_CG6639 SPH93 2.47 ns A - all infections
starry night stan 2.32 ns D - EPN specific
Immune induced molecule 18 CG10332/IM18 1.83 FB D - EPN specific
A kinase anchor protein 200 Akap200 1.2 ns F - missing in wasp infection
Dmel_CG7607 CG7607 1.01 3.78 D - EPN specific
Argonaute 2 AGO2 0,79* ns E - shared EPN+bac. injection
Spermidine Synthase SpdS -1 -1.23 E - shared EPN+bac. injection
Peptidoglycan recognition protein LF PGRP-LF 1.8 0,44* sensitive D - EPN specific
Thioester-containing protein 2 Tep2 1.99 2.47 ns B - missing in pathogenic bacteria
Recognition
Thioester-containing protein 4 Tep4 0,73* ns B - missing in pathogenic bacteria

molecules
Thioester-containing protein 3 Tep3 sensitive

Gram-negative bacteria binding protein 3 GNBP3 ns bac. injection
Peptidoglycan recognition protein SA PGRP-SA ns shared wasp+bac. injection
Peptidoglycan recognition protein LC PGRP-LC ns
Peptidoglycan recognition protein LE PGRP-LE ns
Glutactin Glt 1.29 1.96 Hem sensitive G – shared EPN+pathogenic G- bacteria
enriched genes
dSR-Cl CG4099 ns wasp specific

Hemocytes
Eater CG6124 ns
Lectin28C CG7106 ns
SPARC CG6378 ns
Lectin24DB CG2958 ns
Dmel_CG4250 CG4250 ns wasp specific
larval translucida ltl ns
Wrinkled W ns
* - statistically significant but |log2FC|<1

FB - significantly sensitive to EPN infection after knockdown in fat body (driver PPL-Gal4)
Hem - significantly sensitive to EPN infection after knockdown in hemocytes (driver He-Gal4)
ns - non-significant response to EPN
Table S1. Supplementary Material and Methods: A: Gene-specific primers used
for confirmation of the array data by qPCR: see Figure 3 for results. B:
Drosophila stocks used for nematode infections (see Fig. 1, 4-6 for results)
Table S2. The 100 most strongly regulated genes are enriched in immune genes.
Gene set enrichment analysis for the 100 most highly regulated genes in Fig 2 A was
performed using Amigo (http://amigo.geneontology.org/cgi-
bin/amigo/term_enrichment?session_id=).
Table S3. Complete set of transcripts that are regulated in different infection
models. The differentially expressed transcripts in Drosophila larvae after infection
with common G- and G+ bacteria [3,4], pathogenic G- bacteria [4], wasps [5-7] and
nematodes (this work) are shown.
Table S4. Significantly enriched KEGG pathways after gene set enrichment
analysis (GSEA). Enriched pathways after Fisher test for upregulated subset of
significantly changed transcripts (logFC cutoff > 0.4 (TRUE ), Q-VALUE cutoff
0.05:left column; and P-value at cutoff 0.05: right column) are shown.
Table S5: Hemocytes-enriched genes and their function. Genes were analyzed for
enriched expression using previously published data [3].
Table S6: Comparison of functional analysis results and qPCR results with
microarray data. Complete set of genes used in our functional analysis and qPCR
compared to microarray results.

Supplementary References
1 Mace KA, Pearson JC, McGinnis W: An epidermal barrier wound repair
pathway in drosophila is mediated by grainy head. Science (New York, NY
2005;308:381-385.
2 Quinones-Coello AT, Petrella LN, Ayers K, Melillo A, Mazzalupo S, Hudson
AM, Wang S, Castiblanco C, Buszczak M, Hoskins RA, Cooley L: Exploring
strategies for protein trapping in drosophila. Genetics 2007;175:1089-1104.
3 Irving P, Ubeda JM, Doucet D, Troxler L, Lagueux M, Zachary D, Hoffmann
JA, Hetru C, Meister M: New insights into drosophila larval haemocyte
functions through genome-wide analysis. Cell Microbiol 2005;7:335-350.
4 Vodovar N, Vinals M, Liehl P, Basset A, Degrouard J, Spellman P, Boccard F,
Lemaitre B: Drosophila host defense after oral infection by an
entomopathogenic pseudomonas species. Proc Natl Acad Sci U S A
2005;102:11414-11419.
5 Wertheim B, Kraaijeveld AR, Schuster E, Blanc E, Hopkins M, Pletcher SD,
Strand MR, Partridge L, Godfray HC: Genome-wide gene expression in
response to parasitoid attack in drosophila. Genome Biol 2005;6:R94.
6 Schlenke TA, Morales J, Govind S, Clark AG: Contrasting infection strategies
in generalist and specialist wasp parasitoids of drosophila melanogaster. PLoS
pathogens 2007;3:1486-1501.
7 Lee MJ, Mondal A, Small C, Paddibhatla I, Kawaguchi A, Govind S: A
database for the analysis of immunity genes in drosophila: Padma database.
Fly (Austin) 2011;5:155-161.

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Research Article

Genome-Wide Transcriptional Analysis of

Key Words ute to controlling nematobacterial infections encode: a

© 2013 S. Karger AG, Basel Dr. Ulrich Theopold

2 J Innate Immun Arefin /Kucerova /Dobes /Markus /Strnad /

DOI: 10.1159/000353734 Wang /Hyrsl /Zurovec /Theopold

Nematode Infection of Drosophila J Innate Immun 3

4 J Innate Immun Arefin /Kucerova /Dobes /Markus /Strnad /

DOI: 10.1159/000353734 Wang /Hyrsl /Zurovec /Theopold

Nematode Infection of Drosophila J Innate Immun 5

6 J Innate Immun Arefin /Kucerova /Dobes /Markus /Strnad /

DOI: 10.1159/000353734 Wang /Hyrsl /Zurovec /Theopold

Table 1. Gene ontology analysis of genes regulated by nematobacterial infection

Region Gene Category Biological process

A 17 all GO:0019731 antibacterial humoral response

Nematode Infection of Drosophila J Innate Immun 7

Fold change (log2)

Knockdown in fat body Knockdown in hemocytes

Normalized mortality (fold)

starry night 51382 GD

starry night 1665 GD

Attacin-A 50320 GD Non-inf.

starry night 107993 KK

a Immune molecules Development Neg. contro l b NIG RNAi

8 J Innate Immun Arefin /Kucerova /Dobes /Markus /Strnad /

DOI: 10.1159/000353734 Wang /Hyrsl /Zurovec /Theopold

Normalized mortality (fold)

Fig. 5. Candidate screen for Drosophila 2

was scored as in figure 4. * p < 0.05; ** p <

Nematode Infection of Drosophila J Innate Immun 9

Discussion maturation pathway, Wnt signaling and ubiquitin-me-

10 J Innate Immun Arefin /Kucerova /Dobes /Markus /Strnad /

DOI: 10.1159/000353734 Wang /Hyrsl /Zurovec /Theopold

Nematode Infection of Drosophila J Innate Immun 11

12 J Innate Immun Arefin /Kucerova /Dobes /Markus /Strnad /

DOI: 10.1159/000353734 Wang /Hyrsl /Zurovec /Theopold

130.237.94.172 - 11/23/2013 12:01:37 PM

Nematode Infection of Drosophila J Innate Immun 13

Inf. PC Inf. Vkg‐GFP Inf. Merge

artificial wound made by a needle (125 micrometer in diameter) is shown as control

Fig. S3. Damage of the basement membrane after nematode infection of

boundary in green channel (Vkg-GFP), PC: phase contrast, ).

Fig. S4. No significant changes in mortality are observed among Drosophila

infection with Heterorhabditis bacteriophora at a dose 25 IJs per larva at 25° C.

for further details).

Gene Primer name Sequence Annealing

1 Attacin A CG10146 9287 GD 1 viable 3 yes Strongly induced

Hemocytes enriched genes (fig. 6)

Mutant lines (Fig.5)

From Bruno Lemaitre lab (Fig.5)

From Dominique Ferrandom lab (Fig.5)

Injection of G+ and G- bacteria (FC)

Pathogenic G- Bacteria (FC)

Name Fold (compared to Function Product

Lectin28C 59.62 galactose binding C-type lectin-like

Eater 43.67 cell adhesion, bacterial cell surface

Scavenger 25.44 scavenger Complement control

Glutactin 19.67 extracellular Carboxylesterase type

SPARC 13.75 ligand binding or SPARC/ostenectin-

GNBP-like CG13422 7.1 3.73 FB B - missing in pathogenic bacteria

Thioester-containing protein 4 Tep4 0,73* ns B - missing in pathogenic bacteria

Thioester-containing protein 3 Tep3 sensitive