� larvae collected 88 hours after egg-laying� infection with H. bacteriophora harbouring GFP

expressing P. luminescens in microtiter plates(100 IJs/larva)

� after 2 hours larvae were rinsed in water andtransferred to vials with fly food

� after additional 6 hours larvae were scored for aGFP signal

� infected larvae used for RNA isolation� Affymetrix GeneChip® Drosophila Genome 2.0

Array System was used for the microarrayanalysis

1 Department of Animal Physiology and Immunology, Institute of Experimental Biology, Masaryk University, Kotlářská 2, 61137 Brno, Czech RepublicContact e-mail: hyrsl@sci.muni.cz

2 Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 10691 Stockholm, Sweden3 Biology Centre of the AS CR, Institute of Entomology, Branišovská 1160/31, 37005 České Budějovice, Czech Republic

Pavel Hyršl1, Pavel Dobeš1, Badrul Arefin2, Lucie Kučerová3, Robert Markus2, Zhi Wang2, Michal Žurovec3, Ulrich Theopold2

New insights to insect response to the infection by nematobacterial complex

Top 100 upregulated genes sorted according to their functions

We compared the transcripts that weresignificantly regulated uponnematobacterial infection to previouslypublished microarrays of the fly larvaeinfected with common Gram negative (G-)and Gram positive (G+) bacteria (Irvinget al. 2005, Vodovar et al. 2005),pathogenic G- bacteria (Vodovar et al.2005) and wasps (Wertheim et al. 2005,Schlenke et al. 2007, Lee et al. 2011).A core compartment of 17 genes wasinduced under all conditions (e.g. genescoding IMs or attacin-C).

One of the first steps of the infection by the nematobacterial complex is an invasionof nematode that is used by EPB as a vector to get past the mechanical barriers of the hostbody. Using DDC-GFP-expressing (activation of DOPA decarboxylase coupled with productionof GFP) D. melanogaster larvae we observed activation of the DDC reporter mainly around thewounds located in the cuticle and gut of larvae (Fig. 6, 8) infected by complex H. bacteriophoraand P. luminescens. This suggests that the nematode invades its insect host mainly throughthe anus and mouth by penetrating the gut wall or by penetrating the cuticle. Wounds weremostly located between body segments and melanized (Fig. 5, 7). Arrows indicate woundsites.

Once inside the host, EPN release their symbiotic bacteria and they both are recognizedby the insect immune system. We looked at the response of hemocytes that are normallylocalized in sessile compartments of D. melanogaster larvae as visible in hml-GAL4 x UAS-RFP larvae (Fig. 9). After the immune challenge – infection by the nematobacterial complex –hemocytes are released to circulation (Fig. 11) simultaneosly with the expansion of EPBin hemocoel (Fig. 12). Arrows indicate wound sites and arrowheads P. luminescens TT01in infected larva enlarged at the inset.

We performed a genome-wide analysis of the D. melanogaster transcriptome responseto nematobacterial infection at the time point at which the nematodes reached the hemolymph.

Genome-wide analysis of host response to nematobacte rial infection

First steps of nematobacterial infection under the microscope

We detected 642 geneswhose expression wassignificantly influenced bynematobacterial infection;most of them (518)upregulated upon infectionincluding highly inducedgenes involved inantimicrobial response anddevelopment.Based on Gene Ontologyannotation we identifiedseveral factors and pathwayssuch as Wnt, Jak-STAT orHedgehog which could beinvolved in sealing andrepairing of wounds causedby invading nematodes aswell as a number of immunemolecules that were enrichedafter nematode infection.

We use data from microarrays as a starting point for functional tests. D. melanogaster mutants and RNAi lines for differentialy regulated genes combined with nematode infection assay help us toidentify factors that are critical during the infection. We have already identified several key players in the immune response and further investigation is likely to characterize the infection by nematodesand their symbiotic bacteria in more detail improving our understanding of the insect immune response.Our research is supported by research grants from the Swedish Research Council (VR-NT 2010-5118), the Swedish Foundation for International Cooperation in Research and Higher Education(STINT), by grant from Ministry of Agriculture of Czech Republic (NAZV-KUS QJ1210047) and by the Program of „Employment of Newly Graduated Doctors of Science for Scientific Excellence“(CZ.1.07/2.3.00/30.009) co-financed from European Social Fund and the state budget of the Czech Republic.

What about the other pathogens?

P-value Sample frequency Background frequency Genes

6.56e-05 9/76 (11.8%) 136/13716 (1.0%) AttC IM4 Mtk IM3 edin DptB lola Rel Tep2

3.53e-04 6/76 (7.9%) 50/13716 (0.4%) AttC Mtk IM3 DptB Rel Tep2

6.99e-04 10/76 (13.2%) 234/13716 (1.7%) AttC IM4 Mtk IM3 edin DptB lola Rel Tep2 dome

1.10e-03 30/76 (39.5%) 2225/13716 (16.2%) ato Cpsf160 Trn Sin3A IM4 CG7394 Taf2 CG31122 Pdcd4 CG3690 Ssrp CG6686 sa Ptp4E klu rst SA

Vm26Aa sqz lea lola Rel Tie CG1532 pav CG7376 CG14762 Taf4 dome trn

1.74e-03 29/76 (38.2%) 2144/13716 (15.6%) ato Cpsf160 Trn Sin3A CG7394 Taf2 CG31122 Pdcd4 CG3690 Ssrp CG6686 sa Ptp4E klu rst SA Vm26Aa

sqz lea lola Rel Tie CG1532 pav CG7376 CG14762 Taf4 dome trn

2.02e-03 22/76 (28.9%) 1328/13716 (9.7%) ato Cpsf160 Trn Sin3A CG7394 Taf2 Ssrp CG6686 Ptp4E klu rst SA sqz lea lola Rel CG1532 pav CG7376 CG14762 Taf4 trn

2.56e-03 10/76 (13.2%) 270/13716 (2.0%) AttC IM4 Mtk Tsf1 IM3 edin DptB Rel IM1 Tep2

2.62e-03 11/76 (14.5%) 337/13716 (2.5%) AttC Sin3A IM4 Mtk IM3 edin DptB lola Rel Tep2 dome

2.95e-03 7/76 (9.2%) 111/13716 (0.8%) AttC Mtk IM3 DptB lola Rel Tep2

5.42e-03 50/76 (65.8%) 5489/13716 (40.0%) pkaap ato Cpsf160 Trn Sin3A IM4 CG7394 Taf2 CG31122 Rcd5 Pdcd4 CG3690 Ssrp CG6686 Tsf1 sa

Ptp4E CG31229 IM3 klu mre11 rst Nup358 SA Vm26Aa ALiX Acn CG15744 sqz lea vfl Akh BRWD3 ana3 glu lola Rel CG8108 Tie dalao CG1532 pav

Fancd2 CG7376 CG14762 Taf4 Tep2 dome trn dgt5 6.50e-03 17/76 (22.4%) 892/13716 (6.5%) AttC IM4 Rcd5 CG14650 Mtk Tsf1 IM3 mre11 ALiX

edin BRWD3 DptB glu Rel Fancd2 IM1 Tep2

6.99e-03 15/76 (19.7%) 706/13716 (5.1%) ato Taf2 Pdcd4 klu ALiX lea vfl BRWD3 lola Rel CG8108 dalao pav Taf4 dome

7.24e-03 7/76 (9.2%) 127/13716 (0.9%) AttC Mtk IM3 edin DptB Rel Tep2

7.40e-03 5/76 (6.6%) 48/13716 (0.3%) ato Taf2 ALiX BRWD3 CG8108

7.85e-03 23/76 (30.3%) 1558/13716 (11.4%) ato Cpsf160 Trn Sin3A CG7394 Taf2 Ssrp CG6686 Ptp4E klu rst SA sqz lea glu lola Rel CG1532 pav

CG7376 CG14762 Taf4 trn

Biological Process

GO Term

GO:0006959

humoral immune response GO:0019731

antibacterial humoral response GO:0006955

immune response GO:0048869 cellular

developmental process GO:0030154 cell differentiation

GO:0022008 neurogenesis

GO:0006952

defense response GO:0002376 immune system

process GO:0019730 antimicrobial

humoral response GO:0044763 single-organism cellular

process

GO:0006950 response to stress

GO:0048518 positive regulation of biological process GO:0042742

defense response to bacterium GO:0010740

positive regulation of intracellular protein kinase

cascade GO:0007399 nervous system

development

W−

2N

W−

1

W−

3

WI−

1

WI−

2N

WI−

3

1622961_atCG33013

CG141411634680_at

1633228_at

AkhCG15155

CG33054

CG15706CG14270

1641547_atCG13516

CG33170

CG7394Vm26Aa

1640584_at

CG13038CG31229

CG36901623489_at

CG10470

1638826_atCG5174

CG14463

CG1532CG42335

CG14695Ssrp

CG18596

Nf −YCPtp4E

Nup358

CG10990CG8108

Cpsf160CG6686

Fancd2

Sox21bMagi

AcnCG4951

CG9915

mre11lola

CG3605

CG9839Tie

vf lSin3A

fs (1)h

PGRP−SB1CG30026

IM1

Att CDptB

pavdome

MTA1−lik e

edinTsf1

CG15065

IM4IM3

slp2klu

Nelf−A

trnCG7376

ana3

CG16711HE RC2

falCG14762

CG42232

CG14650CG31122

CG8366

lola1622892_s _at

Reldgt5

rst

TepIIALiX

Mtk

Taf2SA

Taf4Hmt 4−20

dalao

CG8160pkaap

BRW D3CG15744

lea

Rc d51636492_at

glu

sqz1626147_s _at

HM: contrast E3_WI_W , 100 / 642 probes (RMB), to p100

−1 0 1Row Z−Score

00.

20.

40.

6

Color Keyand Density Plot

Den

sity

WWI

C1 C2 C3 I1 I2 I3

Fig. 2

Fig. 3

Fig. 5

Fig. 4

Fig. 6

Fig. 8 – gutFig. 7 – gut

DAPIGFP

GFP

GFPnon-infected non-infected

16 after infection16 after infection

16 after infection16 after infection

Fig. 9

Fig. 12

Fig. 10

Fig. 11

GFP

GFPRFP

RFP

16 after infection 16 after infection

non-infectednon-infected

� Irving P, Ubeda JM, Doucet D, Troxler L, Lagueux M, Zachary D, Hoffmann JA, Hetru C, Meister M. Cell Microbiol 2005;7:335-350. Lee MJ, Mondal A, Small C, Paddibhatla I, Kawaguchi A, Govind S: Fly (Austin) 2011;5:155-161. Schlenke TA, Morales J, Govind S, Clark AG: PLoS pathogens 2007;3:1486-1501. Vodovar N, Vinals M,Liehl P, Basset A, Degrouard J, Spellman P, Boccard F, Lemaitre B: Proc Natl Acad Sci USA 2005;102:11414-11419. Wertheim B, Kraaijeveld AR, Schuster E, Blanc E, Hopkins M, Pletcher SD, Strand MR, Partridge L, Godfray HC. Genome Biol 2005;6:R94.

Entomopathogenic nematodes (EPN) Heterorhabditis bacteriophora are obligate and lethalinsect parasites. These EPN are symbiotically associated with entomopathogenic bacteria(EPB) Photorhabdus luminescens creating the highly pathogenic nematobacterial complexthat is able to kill the host within 24 to 48 hours. H. bacteriophora with its bacterial symbiontsare able to infect a broad spectrum of insect species including e.g. larvae of flies or bees(Fig. 1). Symbiotic bacteria help to digest host tissues and provide nutrients for themselvesand developing nematodes.

For successful development within the host, EPN and their symbiotic EPB must overcomeinsect defences including cellular and humoral immune responses (Fig. 2). We used the well-established tripartite model (Drosophila melanogaster, nematodes and their symbioticbacteria), DNA chips and bioinformatic tools to compare gene expression in non-infected andinfected fly larvae. We focused on the early time point of nematode infection when EPBestablish themselves in the hemolymph after release from their nematode vector.

Fig. 1 – Bee larvae infected by nematobacterialcomplex Heterorhabditis – Photorhabdus.Cadavers have the typical coloration caused bypigments produced by EPB (A) and melanizedwounds caused by nematode invasion are visible inthe cuticle (B).

A B