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The innate immune and systemic response in honey bees to a bacterial pathogen, Paenibacillus larvae Chan, Queenie W; Melathopoulos, Andony P; Pernal, Stephen F; Foster, Leonard J Aug 21, 2009

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ralssBioMed CentOpen AcceBMC GenomicsResearch articleThe innate immune and systemic response in honey bees to a bacterial pathogen, Paenibacillus larvaeQueenie WT Chan1, Andony P Melathopoulos2, Stephen F Pernal2 and Leonard J Foster*1Address: 1Centre for High-Throughput Biology and Department of Biochemistry & Molecular Biology, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada and 2Agriculture and Agri-Food Canada, Beaverlodge Research Farm, PO Box 29, Beaverlodge, AB, T0H 0C0, CanadaEmail: Queenie WT Chan - queeniecwt@shaw.ca; Andony P Melathopoulos - melathopoulosa@agr.gc.ca; Stephen F Pernal - PernalS@AGR.GC.CA; Leonard J Foster* - ljfoster@interchange.ubc.ca* Corresponding author    AbstractBackground: There is a major paradox in our understanding of honey bee immunity: the highpopulation density in a bee colony implies a high rate of disease transmission among individuals, yetbees are predicted to express only two-thirds as many immunity genes as solitary insects, e.g.,mosquito or fruit fly. This suggests that the immune response in bees is subdued in favor of socialimmunity, yet some specific immune factors are up-regulated in response to infection. To explorethe response to infection more broadly, we employ mass spectrometry-based proteomics in aquantitative analysis of honey bee larvae infected with the bacterium Paenibacillus larvae. Newly-eclosed bee larvae, in the second stage of their life cycle, are susceptible to this infection, butbecome progressively more resistant with age. We used this host-pathogen system to probe notonly the role of the immune system in responding to a highly evolved infection, but also what othermechanisms might be employed in response to infection.Results: Using quantitative proteomics, we compared the hemolymph (insect blood) of five-dayold healthy and infected honey bee larvae and found a strong up-regulation of some metabolicenzymes and chaperones, while royal jelly (food) and energy storage proteins were down-regulated. We also observed increased levels of the immune factors prophenoloxidase (proPO),lysozyme and the antimicrobial peptide hymenoptaecin. Furthermore, mass spectrometry evidencesuggests that healthy larvae have significant levels of catalytically inactive proPO in the hemolymphthat is proteolytically activated upon infection. Phenoloxidase (PO) enzyme activity wasundetectable in one or two-day-old larvae and increased dramatically thereafter, paralleling veryclosely the age-related ability of larvae to resist infection.Conclusion: We propose a model for the host response to infection where energy stores andmetabolic enzymes are regulated in concert with direct defensive measures, such as the massiveenhancement of PO activity.Published: 21 August 2009BMC Genomics 2009, 10:387 doi:10.1186/1471-2164-10-387Received: 15 April 2009Accepted: 21 August 2009This article is available from: http://www.biomedcentral.com/1471-2164/10/387© 2009 Chan et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 9(page number not for citation purposes)BMC Genomics 2009, 10:387 http://www.biomedcentral.com/1471-2164/10/387BackgroundHoney bees, Apis mellifera, face a number of niche-specificpathogens such as the endospore-forming bacteriumPaenibacillus larvae, the causative agent of American Foul-brood (AFB) [1]. Bees are only susceptible to P. larvae dur-ing the first 48 h following eclosion (egg hatching), intheir first and second instar developmental stages. Itremains unclear why larvae acquire immunity against P.larvae after the third instar, whereas the ingestion ofmerely 10 spores can cause systemic infection and deathin the previous instars [2]. It was thought that P. larvaespores germinate in the larval midgut and enter the epi-thelium by phagocytosis [3,4] but recent data suggest thatthe bacteria follow a paracellular route to breach the epi-thelial wall [5]. The effectiveness of the antimicrobial pep-tide (AMP) defensin against P. larvae was documented ingrowth inhibition assays [6,7] using fractionated royaljelly (honey bee food). However, Evans et al. found nochanges in defensin gene expression in larvae fed P. larvaespores and, paradoxically, that abaecin (another AMP)gene expression was greatest in newly eclosed larvae [8,9],the most susceptible stage. More recently, the same groupshowed that infection caused elevated expression of Toll-like receptor, MyD88 and IκB [10]. Thus, even thoughthey do not always respond as expected, honey bees haveall the components of an innate immune system. Here weexplore the response of this system to a physiologicallyrelevant infection in a natural setting.To this end, hemolymph (arthropod blood) is well-suitedfor studying insect immunity; it is especially relevant inthe case of P. larvae as the bacterium contacts hemolymphas soon as it breaches the gut epithelium. This fluid con-tains antimicrobial factors produced largely by the fatbody and, to a lesser extent, hemocytes. These cells canalso respond to infectious particles by phagocytosingthem or by autolysis, which is part of an encapsulationpathway used to inhibit growth of microorganisms. Like-wise, as the connective tissue responsible for transportingvarious molecules throughout the body, it is also opti-mally suited for monitoring systemic changes in otherpathways. Previously, we have examined how hemol-ymph changes during normal larval development [11]. Inthat study we observed that most immune factors werenot significantly altered during development, with onlythe AMP apismin and the monooxygenase prophenoloxi-dase showing any age-related changes in expression. Thus,based on our earlier work and that of others [8-12], weexpected that a P. larvae infection should induce, in hemo-lymph, elevated levels of at least some AMPs, as well asother antibacterial enzymes such as lysozyme and proph-enoloxidase. To address these predictions, we use massspectrometry (MS)-based proteomics to measure changesto convey immunity to older larvae and adults must beexpressed at extremely low levels in the susceptible earlylarval instars. Using a functional assay, we demonstratehow one potentially critical player in host defense, phe-noloxidase (PO), correlates with larval resistance to infec-tion.ResultsDifferent strains of P. larvae produce equivalent outcomesIn order to test the response of worker larvae to P. larvaeinfection, we spray-inoculated a small section of combcontaining one-day-old worker larvae with either (A) ahomogenate of scale from natural infections of P. larvae(PL-Scale) or (B) a laboratory-cultured strain of P. larvae(NRRL B-3650, PL-Lab). We used two sources of P. larvaeas we had no a priori knowledge regarding their relativepathogenicity. Four days post-infection we harvestedhemolymph from 5-day larvae and compared the proteinexpression in the two infected conditions versus an unin-fected control using a quantitative proteomics approach[11]. Using an ultra-high accuracy/resolution LTQ-Orbit-rapXL, we identified a combined total 331 proteins (listedin Additional Files 1 and 2) with an estimated false dis-covery rate of 0.30%. Protein regulation differed betweenbees infected with the PL-Lab strain compared to the PL-Scale strain (Fig 1a, p < 0.0001) but among the 25 pro-teins quantified in both infections (Fig 1b) that were sig-nificantly different from control (p < 0.05), 40% (10)were higher in PL-Scale and 60% (15) were higher in PL-Lab. Thus, while there were differences between the inoc-ulums, they were not consistently in one direction and sothere was not enough evidence to reject the null hypothe-sis that regulation among these shared proteins differedbetween the two strains (Wilcoxon matched-pairs signed-ranked test, p = 0.87).Diseased honey bee larvae express higher levels of mitochondrial metabolic enzymesA total of 33 proteins, out of 179 quantified, were regu-lated by a magnitude of at least 2-fold for either one orboth inoculums (p < 0.05 or p < 0.01, two-tailed, non-par-ametric Wilcoxon matched-pairs signed-ranked statisticaltest, full results listed in Additional File 3). Among themost up-regulated of all quantified proteins were severalmitochondrial metabolic enzymes (Fig 2a). One malatedehydrogenase (MDH) [GI:66513092], for example,showed about a 14-fold increase by both infection meth-ods (p < 0.01). This bee MDH is 67% identical to humanmitochondrial MDH2 [GI:12804929], implying its directparticipation in the mitochondrial matrix and the tricar-boxylic acid (TCA) cycle, instead of the malate-aspartateshuttle that is carried out by human MDH1[GI:66506786]. Further to this point, the levels of aspar-Page 2 of 9(page number not for citation purposes)in hemolymph protein levels in larvae challenged with P.larvae. Furthermore, we predict that a protein that is abletate aminotransferase, another major enzyme of this shut-tle, showed no change. An aldehyde dehydrogenaseBMC Genomics 2009, 10:387 http://www.biomedcentral.com/1471-2164/10/387(ALDH) [GI:66530423], a homolog of the human mito-chondrial isoform [GI:118504] was up-regulated 25-foldacyltransferase [GI:48097100], which participates in beta-oxidation and the mevalonate pathway, was significantly(p < 0.01) up-regulated at 14- and 9-fold in PL-Scale andPL-Lab, respectively.Infected larvae deplete their energy stores during infectionClearly the metabolic capacity of larvae is undergoing amassive change in response to infection (Fig 2a), suggest-ing that concerted changes may also be occurring in theirenergy stores. Food proteins, which comprise a familycalled the major royal jelly proteins (MRJPs), are consist-ently depleted (Fig 2b), except in one case that was not sig-nificant at the p < 0.05 level. At the same time, 5 d larvaeshould be accumulating enormous levels of hexamerin(HEX) proteins in the hemolymph [11] as an amino acidsource for later growth in the pupal stage. However,HEX110 [GI:110761029], HEX70b [GI:58585148] andHEX70c [GI:66549815] have a modest but significant (p< 0.01) 2- to 3-fold decrease under PL-Lab infection con-ditions (Fig 2c). Similar reductions were seen for two lipidcarriers, retinoid- and fatty-acid binding protein[GI:110758758] and apolipophorin III [GI:66557660],while a putative neuropeptide Y (NPY) receptor[GI:110764421] that may regulate food intake wasstrongly up-regulated by the PL-Lab infection (p < 0.05)The protein-folding/quality control machinery is over-expressed in response to infectionProtein-folding chaperones and heat-shock proteins(HSPs) have been implicated in disease responses due tostress associated with tissue damage [13], with evidencethat they also have roles in signal transduction in immunepathways [14]. Twenty-six molecular chaperones weredetected in larval hemolymph, with many being up-regu-lated 3- to 20-fold in diseased larvae (Fig 2d). Amongthem are three proteins with multiple domains homolo-gous to disulfide isomerases ([GI:110768510],[GI:66546657], [GI:66531851]), a 90 kDa heat shockprotein HSP90 [GI:110758921], a 60 kDa heat shock pro-tein HSP60 [GI:66547450] and a heat shock cognate 5homolog [GI:66501507]. In human studies, heat shockproteins such as HSP60 have been repeatedly linked tomacrophage activation [15,16]. Hemocytes, being some-what similar to macrophages in their phagocytic capacity,have been noted to undergo morphological changes dur-ing AFB infection, while at the same time populations ofother hemolymphic cells increase [17] so these effectsmay be linked with the HSP up-regulation observed here.Lysozyme and hymenoptaecin levels increase with bacterial challengeWe were able to identify four low molecular weightdefense proteins: lysozyme [GI:66565246], hymenoptae-Peptide ratios from two different infection methodsFigur 1Peptide ratios from two different infection methods. Hemolymph was collected from infected and healthy 5-day old larvae. In (a), ratios of the 1207 peptides concomitantly quantified two different infection methods is shown. In (b), twenty-five proteins quantified with a minimum of 95% confi-dence are shown. All values are shown in natural log scale, relative to the control hemolymph (PL-Scale, x-axis, PL-Lab, y-axis). Linear regression is represented by the diagonal lines: (a) slope = 0.69, y-intercept = 0.28, R2 = 0.29; (b) slope = 0.85, y-intercept = 0.041, R2 = 0.76.-100PL-ScalePL-ScalePL-LabPL-LabABPage 3 of 9(page number not for citation purposes)(p < 0.01) in PL-Lab-infected samples, and the same trendwas observed in infection with PL-Scale, although it didnot reach statistical significance (p < 0.1). Acetyl-CoAcin [GI:58585174], apidaecin 22 [GI:58585226], anddefensin [GI:58585176]. We observed a 13-fold increaseof lysozyme in PL-Lab infections (p < 0.01) and a 16-foldBMC Genomics 2009, 10:387 http://www.biomedcentral.com/1471-2164/10/387Page 4 of 9(page number not for citation purposes)American Foulbrood-induced changes in protein expression regulation in selected functional familiesFigu e 2American Foulbrood-induced changes in protein expression regulation in selected functional families. Pro-teomes of honey bee hemolymph from larvae (5 days after hatching) infected using methods PL-Scale (Sc) or PL-Lab (Lb) were compared with healthy controls. Relative levels are expressed in the natural log scale (y-axis), with the level from uninfected bees defined at 0. Data points are relative peptide ratios pooled from 3 biological replicates, with the horizontal bar represent-ing the median level of protein regulation. Those with the median beyond 2-fold (outside of the shaded box) and meets statis-tical significance, as calculated by the two-tailed Wilcoxon matched-pairs signed-ranked test are marked by a single (*, p < 0.05) or double (**, p < 0.01) asterisk. Selected proteins and functional families are shown: (a) highly regulated metabolic proteins, (b) major royal jelly proteins, (c) energy storage proteins, (d) protein folding chaperones, and (e) immunity-related proteins. NA = unquantifiable proteins. Protein abbreviations in alphabetical order (name, accession number): ACAT (acetyl-CoA acyl-transferase, [GI:48097100]); ALDH (aldehyde dehydrogenase, [GI:66530423]); apoLIII (apolipophorin III, was "hypothetical protein", [GI:66557660]); Eater (a homolog identified by [10], [GI:110763407]); ECHD (enoyl-Coa hydratase, [GI:110773271]); ERp60 (a homlog of protein disulfide isomerase, [GI:66546657]); FABP (retinoid- and fatty acid binding protein, [GI:110758758]); Gly93 (glycoprotein 93, a homolog of HSP90, [GI:110758921]); GNBP1 (Gram-negative binding protein 1, [GI:110755978]); H70/90 (HSP70/90 organizing protein, [GI:110756123]); HEX110 (hexamerin 110, was "larval serum protein 2", [GI:110761029]); HEX70b (hexamerin 70b, [GI:58585148]); HEX70c (hexamerin 70c, was "hexamerin 2 beta", [GI:66549815]); HSC5 (heat shock protein cognate 5, [GI:66501507]); HSC70Cb (heat shock cognate 70Cb, [GI:66505007]); HSP1 (heat shock protein 1, [GI:110749824]); HSP60 (heat shock protein 60, [GI:66547450]); HSP8 (heat shock protein 8, [GI:66537940]); HSP90 (heat shock protein 90, [GI:66512625]); Hympt (hymenoptaecin, [GI:58585174]); Lys (lysozyme, [GI:66565246]); MDH (malate dehydrogenase, 66513092); MRJP1 (major royal jelly protein 1, [GI:58585098]); MRJP2 (major royal jelly protein 2, [GI:58585108]); MRJP3 (major royal jelly protein 3, [GI:58585142]); PDI (disulfide isomerase, [GI:110768510]); PDI (disulfide isomerase, [GI:66531851]); pPO (prophenoloxidase, [GI:58585196]); pPO-a (prophenoloxi-dase-activating factor, [GI:110758534]); Serpin (serine protease inhibitor 5, [GI:66566441]); TCP1 (a homlog of chaperonin, [GI:66560172]).MDH ALDH ACAT ECHD MRJP1 MRJP2 MRJP3 FABP HEX110 HEX70b HEX70c apoLIIIpPOpPO-a Serpin GNBP1 Hympt Lys EaterHSP1 H70/90 Gly93 PDI HSC5 HSC70 HSP90 PDI HSP8 ERp60 HSP60 TCP1Sc Lb Sc Lb Sc Lb Sc Lb Sc Lb Sc Lb Sc Lb Sc Lb Sc Lb Sc Lb Sc Lb Sc LbSc Lb Sc Lb Sc Lb Sc Lb Sc LbSc Lb Sc LbSc Lb Sc Lb ScLb Lb Sc LbSc Lb Sc LbSc Lb Sc Lb Sc ScSc Lb LbSc Lb-4-3-2-101234-4-3-2-101234-4-3-2-101234-4-3-2-101234-4-3-2-101234NANA NA NA NA NA NA**** ** ************ ** **** ** ******************** ** ****** **A B CD EBMC Genomics 2009, 10:387 http://www.biomedcentral.com/1471-2164/10/387increase of hymenoptaecin (Fig 2e) but there were too fewpeptides detected for apidaecin 22 and defensin to meetour criteria for quantitation (see Materials and Methods).Other immune factors that were identified but did notappear to be regulated by infection include a Gram-nega-tive bacteria binding protein [GI:110755978], peptidogly-can recognition protein (PGRP) SA [GI:110765019] andPGRP SC2 [GI:66522804], suggesting that the responseseen for lysozyme and hymenoptaecin is a specificresponse to P. larvae infection.ProPO expression and proteolytic activation are enhanced during infectionThe melanization cascade, which leads to the encapsula-tion of infectious agents, is one of the most importantdefensive mechanisms of insect innate immunity. One ofthe central steps in this mechanism is the cleavage ofproPO to PO, the active form of the monooxygenase. ThePO enzyme, which is activated by proteolytic action, catal-yses a key step in the synthesis of melanin and plays a cru-cial role in melanotic encapsulation of invaders [18]. Weobserved a 4-fold increase (p < 0.01) in the expression ofproPO in PL-Scale-infected larvae and a 5-fold increase (p< 0.01) in PL-Lab-infected larvae (Fig 2e). The tryptic pep-tide SVATQVFNR, whose C-terminus is the predictedpropeptide cleavage site [19], was elevated by about 10-fold compared to tryptic peptides found in the remainderof the protein (Fig 3). The higher ratios for this propeptideversus the other peptides of the protein suggest that theincreased PO response during infection is largely due tothe proteolytic activation of an existing pool of PO in thehemolymph and only partially attributable to up-regu-lated expression.Phenoloxidase activity is not found in the first two days of larval development, but increases sharply afterwardsAlthough PO is well-known for its activity against patho-gens, there is little indication so far that its expressionlevel affects the outcome of infection by P. larvae. Recentdata from our group suggests that proPO levels correlatepositively with age [11], but we had been unable to estab-lish a full profile of proPO levels during the entire courseof larval development due to the low absolute levels ofexpression. A PO activity assay [20], used to test for theoxygenation of monophenols to diphenols and diphenolsto quinones [21], should be more sensitive than massspectrometry and so was employed here to detect POactivity in developing, healthy larvae. PO activity was eas-ily detected in crude hemolymph from fourth- to fifth-instar honey bee larvae but there are at least two geneproducts in the honey bee genome that could function inthis assay based on domain comparisons [22]. To deter-mine which of the two possible proteins is responsible foranion exchange chromatography and the PO activity ineach fraction was correlated (Fig 4a) with the abundanceof each protein [23]. The measurement of relative proPOlevels was accomplished using differential isotopic labe-ling of peptides in each fraction, selecting one fraction asthe reference to compare against the others (Fig 4b). ThePO activity profile matched very closely to the levels of thegene product annotated as 'prophenoloxidase'[GI:58585196] across the chromatographic fractions andmatched very poorly to HEX70b, which also has a putativephenoloxidase catalytic domain.Conceivably, older larvae can boost PO activity inresponse to infection, but could a lack of PO activity in theearly larval stages explain the susceptibility of young lar-vae to P. larvae infection? Cell-free hemolymph wasextracted from healthy larvae one to five days aftereclosion and tested for PO activity and, indeed, there wasno detectable activity in the first two days of development,with some activity detected in day three and substantialactivity thereafter (Fig 5).DiscussionHere we have used a quantitative proteomics approach tocompare the proteomes of healthy and P. larvae-infectedA. mellifera larvae, leading to the discovery that theinfected state is associated with an elevated expression ofimmunity proteins, chaperones, certain metabolic pro-teins with an accelerated consumption of energy stores.One particular immune factor, proPO, was particularlyup-regulated in response to infection. Intriguingly, theactivity level of this enzyme during development of larvaeMass spectrometry-based peptide analysis for prophenoloxi-dase (proPO)Figure 3Mass spectrometry-based peptide analysis for proph-enoloxidase (proPO). Protein domains [22] of proPO are shown in row I. Notable regions [19] and protein length are shown in row II. Row III describes the average quantity of three peptides in infected larval hemolymph, represented by a color scale to depict fold-differences relative to healthy controls (black). Peptides used in averages and their statisti-cal significances using the two-tailed, paired t-teset: n = 3 for SVATQVFNR (p < 0.05), n = 3 for GLDFTPR (p < 0.1), n = 5 for SSVTIPFER (p < 0.05). Averages were generated by con-sidering values from both infection methods (PL-Scale and .....................................................................................................I II III IVprohemocyanin_Mhemocyanin_N hemocyanin_C6931IIIIII0(control)10SVATQVFNR GLDFTPR SSVTIPFERPage 5 of 9(page number not for citation purposes)the PO activity in hemolymph, hemolymph from healthy,fourth and fifth instar larvae was fractionated by strongPL-Lab) together.BMC Genomics 2009, 10:387 http://www.biomedcentral.com/1471-2164/10/387appears to correlate very tightly with susceptibility of thelarvae to infection. Our data support a model where thehost larva responds to infection not only by producingproteins that can fight the infection directly, but also byengaging its metabolic pathways and energy resourcesrequired to support the effort.The observed depletion of energy stores in the form ofMRJPs, hexamerins and lipid transporters suggest that theobserved up-regulation of metabolic enzymes is, at leastin part, tied to energy production. In larvae infected withE. coli, which is not a natural pathogen of bees, MRJP-1,MRJP-7/MRJP-2 were mildly lowered in expression com-pared to the mock-infected control [12]. The negative cor-relation between energy availability and infection survivalhas also been observed in other insects such as the butter-fly Pieris rapa [24] and the bumblebee Bombus terrestris[25]. In further support of the increased energy demandsof the infected state, the putative bee neuropeptide Yreceptor was also up-regulated in infected larvae. Whilethe ligand for this receptor in bees remains unknown, thereceptor and its cognate ligand in mammals control feed-ing and appetite in mammals [26].The most obvious class of proteins expected to increase inresponse to infection are those involved in the innateimmune response. Lysozyme's primary known function isto degrade the peptidoglycan shell of Gram-positive bac-teria [27] and is therefore expected to have a significantrole in inhibiting P. larvae. Interestingly, the C-type lys-ozyme [GI:110762174] that has been previously shownto be up-regulated upon infection [10] is not the lysozymewe have identified here, which is also known as the desta-bilase-lysozyme [GI: 6656246]. Because these two formsare drastically different (e.g., the best-matched region isonly 50 amino acids long and shares only 20% sequenceidentity), it is clearly not a case of the peptides identifiedby MS/MS being shared by both enzymes. The 13- to 16-fold up-regulation of destabilase-lysozyme suggests that itcan be important in host defense, which is also supportedby the observation that its homolog has antimicrobialactivity in the medicinal leech Hirudo medicinalis [28]. TheAMPs comprise another humoral-based defense mecha-nism, killing Gram-negative and positive bacteria alike[12,29]; many of them work by forming pores in the bac-terial cell wall. Among those known in bees, we were onlyable to quantify hymenoptaecin in the hemolymph, andits dramatic up-regulation suggests that it plays a crucialrole in defending against P. larvae, a conclusion that issupported by other reports [10,12]. Conspicuously absentin our data, however, are defensin and abaecin, whichhave both been implicated in the larval response to P. lar-vae [6-10]. Although we detected peptides from defensin,Hemolymph fractionationFigure 4Hemolymph fractionation. (a) Hemolymph from fourth- and fifth-instar larvae was fractionated by strong anion exchange using a step gradient of increasing salt. Each frac-tion (A-F from 0.04 M to 0.24 M salt in 0.04 M increments, plus DS = desalted hemolymph, and FT = flowthrough) was normalized by protein concentration and was subjected to a PO assay (see Methods). Activity is represented by relative reaction rates to DS (left axis, bars). Using mass spectrome-try, proPO levels were measured relative to the fraction containing the highest activity (Fraction D), shown on a natu-ral log scale (N = 3). This was accomplished by averaging the ratio of at least two peptides for proPO in a differentially label mixture of peptides from Fraction F versus the remain-ing fractions. Error bars = 2 standard deviations. As an exam-ple in (b), two spectra of the differentially labeled (+28Da and +32Da) peptide FSDTIVPR is shown at a 1:1 mixture of peptides from (I) Fraction D and sample DS and (II) Fraction D and E.AB0136Relative PO activity (bars)Elution (increasing [NaCl])-2.0-1.0-3.0DS FT BA C D E F-4.00Relative proPO level (line)DDSED481 483 485m/z481 483 485m/z100%100%IIIPage 6 of 9(page number not for citation purposes)BMC Genomics 2009, 10:387 http://www.biomedcentral.com/1471-2164/10/387the signal:noise ratios in the MS1 spectra were not highenough to allow accurate quantitation; no abaecin pep-tides were detected. Our inability to detect these twoAMPs with sufficient signal suggests that their concentra-tion is likely much lower than hymenoptaecin, which isconfirmed by a recent one-dimensional (1D) gel electro-phoresis study of larval hemolymph [12].The consistent up-regulation of proPO in both infectionmethods is in agreement with the well-characterized anti-microbial activity of this enzyme. The ability of larvae toemploy melanization as a defense mechanism has beenquestioned because the proPO levels are low compared toadults, to the point of being undetectable on a stained 1Dgel [12]. In our own experiments, even with ion-exchangefractionation prior to LC-MS/MS analysis on an LTQ-OrbitrapXL, one of the most sensitive systems available, itwas difficult to detect throughout most of larval develop-ment except for the oldest samples (5 d post-hatching)[11]. However, we are clearly able to detect PO activity asearly as three days after hatching, where the absence ofactivity in the earlier timepoints (days 1 and 2) preciselymatch the period of maximum susceptibility of the larvaeto AFB [1,30]. Thus, our data argue that older larvae havesignificant levels of PO and that they are indeed capableof utilizing the PO pathway to fight infection.Conclusiontions. Unlike most other systems, proteins in larvae notonly play major roles in immune defense but also consti-tute one of their primary stores of energy. Studying such aresponse in most other systems with more conventionalenergy reserves (e.g., lipids) would necessitate a wide vari-ety of tools in order to monitor energy usage, immune fac-tor production and metabolic flux all at the same time. Bymonitoring all these aspects simultaneously, our dataclearly demonstrate that host defense against bacterialchallenge is a concerted response involving proteins thatkill the microbes directly, as well as metabolic and cell/protein repair enzymes that indirectly support this defen-sive effort. By using proteomics techniques on this uniquemodel organism where immunity and protein energy fluxare tightly coupled, we have been able to build a morecomprehensive picture of the insect innate immuneresponse.MethodsHoney bees and infection experimentsAll infection experiments were conducted at Beaverlodge,AB, Canada during July and August of 2005. Three five-frame nucleus colonies ('nucs') were prepared with threeframes of bees and open brood and with newly-mated sis-ter queens. In each nuc, 100 by 100 mm patches of firstinstar larvae were selected and sprayed with 20 mL of oneof the following: 1) a 6.0E+06 spores/mL suspension ofspores isolated from naturally occurring AFB 'scale' col-lected in 2004 (PL-Scale), 2) a 4.4E+06 spores/mL suspen-sion of spores from NRRL B-3650 (PL-Lab), a virulentlaboratory strain of P. larvae (courtesy of Jay Evans), and3) phosphate-buffered saline (PBS).Sample collection and processing for MSFour days after infection larvae (estimated to be in latefourth or fifth instar) within each marked square (PL-Scale, PL-Lab and control) were extracted using soft for-ceps (Bioquip, Rancho Dominguez, CA) and bled asdescribed [11]. Hemolymph was processed as describedfor larvae in [31]. In each case, we compared 20 μg of pro-tein from infected hemolymph with the control by differ-ential labeling of tryptic peptides using light (C1H2O) andheavy (C2H2O) isotopologs of formaldehyde prior toanalysis by liquid chromatography-tandem mass spec-trometry (LC-MS/MS) using a linear trapping quadrupole-OrbitrapXL exactly as described [11].MS data analysisRaw data processing to arrive at peptide ion volume ratioswas performed exactly as described [11]. Data for eachinfection method were pooled, and proteins with five ormore quantified peptides from any one or all of the repli-cates were considered quantified. This approach operatesPhenoloxidase activity assaysFigure 5Phenoloxidase activity assays. Protein concentration-normalized hemolymph was collected over the first five days of larval development. The PO activity assay (see Methods) was conducted on the samples, where activity is represented here by the maximum A520 attained by the samples. All PO assay measurements were performed in triplicate. Error bars = 2 standard deviations.Day post-hatching1 2 3 4 500.51.0Maximum A520Page 7 of 9(page number not for citation purposes)The larval stage of a honey bee represents a unique systemfor applying proteomics to probe host-pathogen interac-on the underlying assumption that each peptide ratio is atechnical replicate, which is different from most publica-BMC Genomics 2009, 10:387 http://www.biomedcentral.com/1471-2164/10/387tions where averages are made at the protein, not the pep-tide level. The conventional method implies that a proteinquantified from averaging over a large number of quanti-fied peptides has the same statistical power than onequantified with the bare minimum. To circumvent thisdisadvantage, we took single peptides from three biologi-cal replicates as individual data points, which reasonablyaccounts for the greater statistical power afforded by well-detected proteins with many quantified peptides. Theaverage level of protein regulation is represented by themedian peptide ratio. We employed the two-tailed Wil-coxon matched-pairs signed-ranked test on these proteinsusing Analyse-It (v2.12, http://www.analyse-it.com/),with peptides as data points to assess whether the expres-sion level of each protein was significantly changed byinfection at 95% and 99% confidence [32]. For proteinswith more than 100 peptides, the 100 most intense[M+nH]n+ ions (heavy and light combined, n ≥ 2) wereselected for analysis. The same test was used on peptidesquantified in both infection methods to assess whetherthe two methods yielded the same effects on proteinexpression. Experimentally or bioinformatically-inferredevidence of protein functions and names discussedthroughout this report is provided in Additional File 4.Hemolymph collection for PO activity assayWe collected honey bee larvae and estimated their age indays by size. Animals at each age were pooled to collect atleast 8 μL of hemolymph per replicate for three replicates– the number of larvae required varied from approxi-mately 150 for the very young larvae, and 2–5 for the old-est larvae tested (five days old); 18 fourth to fifth instarlarvae were pooled and used for the fractionation experi-ment and processed as described for larvae in [31]. Pro-tein concentrations were assayed by Coomassie Plus(except in the protein fractionation experiment, seebelow) and were normalized across all samples usingAssay Buffer (20 mM Tris-HCl, pH 8).Hemolymph fractionationHemolymph was desalted using a mini Zeba column(Pierce) according to the manufacturer's instructions. Ofthe 75 μL total volume, 25 μL was reserved for MS analy-sis. The remainder was applied to a mini strong anionexchange column (Pierce), and washed with 100 μL ofAssay Buffer between step-elutions of a 10-step sodiumchloride gradient prepared in Assay Buffer: 0.04, 0.08,0.12, 0.16, 0.20, 0.24, 0.28, 0.32, 0.36, 0.40, 0.50, 1.0, 2.0M. Protein concentrations of all the fractions, includingthe flow-through and desalted hemolymph, were esti-mated by absorbance at 280 nm. Fractions eluted from0.28 M or higher salt had negligible amounts of proteinand were not further analyzed. The protein concentrationFor each fraction we then measured the PO activity usingan enzyme assay (see below) and levels of each proteinrelative to the 0.16 M NaCl fraction. At least 2 peptides ofPO were used for calculating the average PO level in eachfraction. In cases where peptide ratios were above 50-foldand likely beyond the linear dynamic range of the ratiomeasurements, the high ratios were arbitrarily given thesame value as the next highest ratio value below 50-fold.In solution phenoloxidase assayConducted as described [20], substrate (8 μL of 5 mM 4-methylcatechol (Sigma) and 8 μL of 40 mM 4-hydroxy-proline ethyl ester) was added to 8 μL of hemolymph tostart the reaction, except for the experiment with larvae ofdifferent ages where the hemolymph:subtrate ratio was4:1. Absorbance readings at 520 nm were taken immedi-ately using a Nanodrop spectrophotometer (ND-1000,ThermoFisher Scientific) to calculate the initial reactionrate (ΔA520/ΔT). For larval aging experiments, instead ofthe rate, the maximum A520 value was recorded after thehighest level was reached in approximately 40 min.Authors' contributionsAPM and SFP designed and performed the colony infec-tion experiments. APM and LJF collected the hemolymphsamples in Beaverlodge. QWTC and LJF designed the pro-teomic analyses and wrote all the scripts used in the dataanalysis. QWTC performed all the proteomic, biochemi-cal and bioinformatic analyses. QWTC and LJF wrote theinitial version of the manuscript.Additional materialAdditional file 1This file contains the list of proteins considered identified by mass spectrometry-based sequencing and the peptide sequences of each pro-tein. Protein accession numbers preceded by "999" are proteins that have been falsely discovered by matches reversed peptide sequences.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-10-387-S1.xls]Additional file 2This file contains the list of quantified peptides and their relative expression values.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-10-387-S2.xls]Additional file 3This file contains the list of proteins, their median averaged values based on peptide relative expression.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-10-387-S3.xls]Page 8 of 9(page number not for citation purposes)of the other fractions was equalized using Assay Buffer.Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central BMC Genomics 2009, 10:387 http://www.biomedcentral.com/1471-2164/10/387AcknowledgementsThe authors thank the other members of the Cell Biology Proteomics group for fruitful discussions and advice. In particular, we thank Nikolay Stoynov for technical assistance. Operating funds for the work described here came from a Discovery Grant from the Natural Sciences and Engi-neering Research Council (NSERC) to LJF and from the Alberta Crop Industry Development Fund to SFP. The apiary at UBC is supported in part by the Boone-Hodgson-Wilkinson Fund. Infrastructure used in this project was supported by the Canada Foundation for Innovation, the British Columbia (BC) Knowledge Development Fund and the Michael Smith Foundation through the BC Proteomics Network (BCPN). LJF is the Can-ada Research Chair in Organelle Proteomics and a Michael Smith Founda-tion Scholar. QWTC is supported by an NSERC post-graduate scholarship doctoral (PGS-D) award.References1. Shimanuki H: Bacteria.  In Honey bee pests, predators, and diseasesEdited by: Morse RA, Flottum K. Medina: A.I. Root Co; 1997:35-54. 2. Brødsgaard CJ, Ritter W, Hansen H: Response of in vitro rearedhoney bee larvae to various doses of Paenibacillus larvae lar-vae spores.  Apidologie 1998, 29:569-578.3. Gregorc A, Bowen ID: Histopathological and histochemicalchanges in honeybee larvae (Apis mellifera L.) after infectionwith Bacillus larvae, the causative agent of American foul-brood disease.  Cell Biol Int 1998, 22:137-144.4. Davidson EW: Ultrastructure of American foulbrood diseasepathogenesis in larvae of the worker honey bee, Apis mellif-era.  J Invertebr Pathol 1973, 21:53-61.5. Yue D, Nordhoff M, Wieler LH, Genersch E: Fluorescence in situhybridization (FISH) analysis of the interactions betweenhoneybee larvae and Paenibacillus larvae, the causativeagent of American foulbrood of honeybees (Apis mellifera).Environ Microbiol 2008, 10:1612-1620.6. Bilikova K, Wu G, Simuth J: Isolation of a peptide fraction fromhoneybee royal jelly as a potential antifoulbrood factor.  Api-dologie 2001, 32:275-283.7. Bachanova K, Klaudiny J, Kopernicky J, Simuth J: Identification ofhoneybee peptide active against Paenibacillus larvae larvaethrough bacterial growth-inhibition assay on polyacrylamidegel.  Apidologie 2002, 33:259-269.8. Evans JD: Transcriptional immune responses by honey beelarvae during invasion by the bacterial pathogen, Paenibacil-lus larvae.  J Invertebr Pathol 2004, 85:105-111.9. Evans JD, Lopez DL: Bacterial probiotics induce an immuneresponse in the honey bee (Hymenoptera: Apidae).  J EconEntomol 2004, 97:752-756.10. Evans JD, Aronstein K, Chen YP, Hetru C, Imler JL, Jiang H, KanostM, Thompson GJ, Zou Z, Hultmark D: Immune pathways anddefence mechanisms in honey bees Apis mellifera.  Insect MolBiol 2006, 15:645-656.11. Chan QW, Foster LJ: Changes in protein expression duringhoney bee larval development.  Genome Biol 2008, 9:R156.12. Randolt K, Gimple O, Geissendorfer J, Reinders J, Prusko C, MuellerMJ, Albert S, Tautz J, Beier H: Immune-related proteins inducedin the hemolymph after aseptic and septic injury differ inhoney bee worker larvae and adults.  Arch Insect Biochem Physiol14. Asea A: Chaperokine-induced signal transduction pathways.Exerc Immunol Rev 2003, 9:25-33.15. Henderson B, Allan E, Coates AR: Stress wars: the direct role ofhost and bacterial molecular chaperones in bacterial infec-tion.  Infect Immun 2006, 74:3693-3706.16. Coggins CC, Gumbardo DJ: The nursing process applied to staffdevelopment.  J Nurs Staff Dev 1991, 7:196-200.17. Zakaria ME: The cellular responses in teh haemolymph ofhoney bee workers infected by american foulbrood disease(AFB).  Journal of Applied Science Research 2007, 3:56-63.18. Cerenius L, Soderhall K: The prophenoloxidase-activating sys-tem in invertebrates.  Immunol Rev 2004, 198:116-126.19. Lourenco AP, Zufelato MS, Bitondi MM, Simoes ZL: Molecularcharacterization of a cDNA encoding prophenoloxidase andits expression in Apis mellifera.  Insect Biochem Mol Biol 2005,35:541-552.20. Pye AE: Microbial activation of prophenoloxidase fromimmune insect larvae.  Nature 1974, 251:610-613.21. Mason HS: Oxidases.  Annu Rev Biochem 1965, 34:595-634.22. Marchler-Bauer A, Anderson JB, Cherukuri PF, DeWeese-Scott C,Geer LY, Gwadz M, He S, Hurwitz DI, Jackson JD, Ke Z, et al.: CDD:a Conserved Domain Database for protein classification.Nucleic Acids Res 2005, 33:D192-196.23. Foster LJ, de Hoog CL, Zhang Y, Zhang Y, Xie X, Mootha VK, MannM: A Mammalian Organelle Map by Protein Correlation Pro-filing.  Cell 2006, 125:187-199.24. Asgari S, Schmidt O: Isolation of an imaginal disc growth factorhomologue from Pieris rapae and its expression followingparasitization by Cotesia rubecula.  J Insect Physiol 2004,50:687-694.25. Moret Y, Schmid-Hempel P: Survival for immunity: the price ofimmune system activation for bumblebee workers.  Science2000, 290:1166-1168.26. Naslund E, Hellstrom PM: Appetite signaling: from gut peptidesand enteric nerves to brain.  Physiol Behav 2007, 92:256-262.27. Masschalck B, Michiels CW: Antimicrobial properties of lys-ozyme in relation to foodborne vegetative bacteria.  Crit RevMicrobiol 2003, 29:191-214.28. Zavalova LL, Yudina TG, Artamonova II, Baskova IP: Antibacterialnon-glycosidase activity of invertebrate destabilase-lys-ozyme and of its helical amphipathic peptides.  Chemotherapy2006, 52:158-160.29. Casteels P, Ampe C, Jacobs F, Tempst P: Functional and chemicalcharacterization of Hymenoptaecin, an antibacterialpolypeptide that is infection-inducible in the honeybee (Apismellifera).  J Biol Chem 1993, 268:7044-7054.30. Woodrow AW, Holst EC: The mechanism of colony resistanceto American foubrood.  Journal of Economic Entomology 1942,35:892-895.31. Chan QW, Howes CG, Foster LJ: Quantitative comparison ofcaste differences in honeybee hemolymph.  Mol Cell Proteomics2006, 5:2252-2262.32. McCornack R: Extended Tables of the Wilcoxon Matched PairSigned Rank Statistic.  Journal of the American Statistical Associatioin1965, 60:864-871.Additional file 4This file contains the list of proteins discussed in the paper with direct mention of their known or putative function, and the evidence or resource for this information.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-10-387-S4.xls]yours — you keep the copyrightSubmit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.aspBioMedcentralPage 9 of 9(page number not for citation purposes)2008, 69:155-167.13. Ranford JC, Henderson B: Chaperonins in disease: mechanisms,models, and treatments.  Mol Pathol 2002, 55:209-213.

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