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Changes in protein expression during honey bee larval development Chan, Queenie W; Foster, Leonard J Oct 29, 2008

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Open Access2008Chan and FosterVolume 9, Is ue 10, Article R156ResearchChanges in protein expression during honey bee larval developmentQueenie WT Chan and Leonard J FosterAddress: Centre for High-Throughput Biology, Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada. Correspondence: Queenie WT Chan. Email: queeniecwt@shaw.ca© 2008 Chan and Foster; 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.AbstractBackground: The honey bee (Apis mellifera), besides its role in pollination and honey production,serves as a model for studying the biochemistry of development, metabolism, and immunity in asocial organism. Here we use mass spectrometry-based quantitative proteomics to quantify nearly800 proteins during the 5- to 6-day larval developmental stage, tracking their expression profiles.Results: We report that honey bee larval growth is marked by an age-correlated increase ofprotein transporters and receptors, as well as protein nutrient stores, while opposite trends inprotein translation activity and turnover were observed. Levels of the immunity factorsprophenoloxidase and apismin are positively correlated with development, while otherssurprisingly were not significantly age-regulated, suggesting a molecular explanation for why beesare susceptible to major age-associated bee bacterial infections such as American Foulbrood orfungal diseases such as chalkbrood. Previously unreported findings include the reduction ofantioxidant and G proteins in aging larvae.Conclusion: These data have allowed us to integrate disparate findings in previous studies to builda model of metabolism and maturity of the immune system during larval development. This publiclyaccessible resource for protein expression trends will help generate new hypotheses in theincreasingly important field of honey bee research.BackgroundHoney bees (Apis mellifera) have been a subject of scientificresearch for more than 2,300 years [1], yet it is only in thepast two decades that bee research has expanded beyondbehavioral or social traits to a molecular level. With the pub-lication of the honey bee genome in 2006 [2], the basic infor-mation to enable proteome-level analyses of this organism isnow available. Since then, various groups have published pro-significance in caste determination [7] and in the pathogene-sis of certain economically significant honey bee diseases,such as American and European Foulbrood.The larval development of the honey bee, which follows a 3-day period as an egg, is 5-6 days in duration and precedes thepupal (metamorphosis) and adult stages. Apart from anastounding increase in size, larval growth is relatively unre-Published: 29 October 2008Genome Biology 2008, 9:R156 (doi:10.1186/gb-2008-9-10-r156)Received: 7 July 2008Revised: 23 September 2008Accepted: 29 October 2008The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2008/9/10/R156Genome Biology 2008, 9:R156teomic analyses of whole bees or individual organs/tissues [3-6] but these studies have focused on adult animals. Larvaldevelopment in honey bees is largely unexplored, despite itsmarkable at the macroscopic level [8]. However, female beesdifferentiate into workers or queens (caste differentiation) inresponse to diet very early in larval development and the156.2http://genomebiology.com/2008/9/10/R156 Genome Biology 2008,     Volume 9, Issue 10, Article R156       Chan and Foster Racquisition of immunity to certain diseases during this 5- to6-day period suggests complex molecular biological changesare taking place.Insect development has been studied mainly using the fruitfly as the model system. Drosophila embryogenesis has his-torically attracted far more attention than any other growthstage, due to its value for studying the mechanism of spatialregulation of transcription and translation. With the excep-tion of the economically important silkworm Bombyx mori,research on larval development has been slow. For honeybees, the lack of published works is evident: the article enti-tled 'Morphology of the Honeybee Larva' published by Nelsonin 1924 [8] still remains today as one of the most citedresources on this subject. Here we have used mass spectrom-etry-based proteomics to profile the changing abundance ofindividual proteins over the first 5 days of the worker larvalstage and used these data, with the help of sequence-basedfunction prediction, to build a framework for the develop-mental processes going on in the maturing larva.ResultsIn order to obtain suitably aged larval samples for proteomicprofiling of the first 5 days of development, for each experi-ment we isolated an open-mated, laying queen on an emptyframe of brood comb for a short period of time to allow her tolay several hundred eggs (see Materials and methods). Theframe and queen were then separated by a queen excluderand workers were allowed to tend the brood. Starting on theday the eggs hatched (day 1, roughly corresponding to firstinstar) larvae were collected every day for 5 days. Hemol-ymph was separated from the remaining tissues (termed'solid tissues' henceforth) prior to protein extraction (seeMaterials and methods) and equal amounts of protein fromeach age were resolved on a reducing SDS polyacrylamide gel(Figure 1). The protein composition of solid tissues wasgrossly consistent across all ages, but varied drastically in thehemolymph. Hemolymph from 1- to 3-day old larvae show astaining pattern distinct from that of 4- to 5-day old larvae.These differences may be partially attributed to slight varia-tions in collection methods for young and old larvae but it ismore likely that these represent real biological changes occur-ring as the late larvae prepare for pupation. Most notably, a70 kDa hexamerin band emerges from day 3 and beyond andaccounts for the majority of the protein in the hemolymph, anobservation that has been made numerous times by otherresearchers [9-11]. A second observation that argues againstthese dramatic changes around day 3 being simply an artifactof sample collection is the absence of the major protein bandsin the hemolymph gel in the solid tissue gel, and vice versa.As a means for identifying and quantifying the expressionlabeling method we used employs deuterated and hydrogen-ated forms of formaldehyde to reductively dimethylate pri-mary amines in peptides, but since there are only two labelingconditions possible in this schema, we compared the expres-sion of protein from days 1, 2, 4 and 5 larvae versus that fromday 3 in order to generate an expression profile spanning thewhole development period. Three biological replicates of eachtissue type were analyzed, which resulted in the detection of12,421 non-redundant peptides (supplementary Table 10 inAdditional data file 1). After applying the cutoff criteria (seeMaterials and methods), 1,333 proteins were identified (sup-plementary Table 1 in Additional data file 1) with an estimatedfalse discovery rate of 0.97% (see Materials and methods),thus providing experimental evidence for 12.7% of the 10,517genes in the predicted honey bee gene set. In general, the pep-tide ratios showed no labeling bias and were approximatelynormally distributed (Figure 1). Among these, 790 werequantified in 2 or more days by averaging the intensity ratiofrom at least 2 of the 3 replicates (if more than 5 peptides werequantified, the top 5 most intense peptides were selected):378 (48%) of them matched this criterion in both the tissueand hemolymph, 309 (39%) were specific to solid tissue and103 (13%) were specific to hemolymph. An example of usingpeptide ratios to derive relative protein expression profiles isshown in Table 1 for the odorant binding protein 14[GenBank:94158822].A major strength of this method is the ability to track thechanging abundances of hundreds of proteins during devel-opment. Those whose levels can be traced for at least 4 out of5 days in either the tissue or hemolymph were considered tohave an informative profile, a total of 522 proteins. Approxi-mately equal numbers of tissue proteins showed an expres-sion trend either positively or negatively correlated with age,The peptide ratios within an experiment are roughly normally distributed and show no l beling biasFigur  1The peptide ratios within an experiment are roughly normally distributed and show no labeling bias. Using replicate number 1 of day 1 versus day 3 0200400600800100012001400Less th an -3.501-3.500 to  -2.5 01-2.500 to  -1.5 01-1.500 to  -0.5 01-0.500 to  0.4990.500  to 1.4991.500  to 2.4992.500  to 3.499Grea ter t han 3.500Genome Biology 2008, 9:R156profiles of proteins in developing larvae, we used a quantita-tive proteomics approach employing stable isotope labelingand liquid chromatography-tandem mass spectrometry. Thesolid tissue quantification data as an example, the peptide ratios are displayed as a histogram, sorted into natural-log unit bins (bin size = 1).156.3http://genomebiology.com/2008/9/10/R156 Genome Biology 2008,     Volume 9, Issue 10, Article R156       Chan and Foster Rbut the latter was more common for hemolymph proteins, asmight be expected from the high dynamic range of hemol-ymph as shown in Figure 2. It is crucial to note that thedecreasing trend likely does not reflect an absolute reductionin expression levels of most proteins, but is rather a phenom-enon of analyzing equal amounts of protein between two sam-ples with a very large difference in absolute protein amountscaused primarily by drastic increases in secreted hexamerins.Consequently, lower abundance proteins become harder todetect in this background. Although the protein concentra-tion in hemolymph changes only slightly beyond 1 day afterhatching, the total volume, and thus absolute protein content,increases exponentially with age (Figure 3).There is no direct functional information available for morethan 99% of honey bee proteins, so to derive some functionalinsight from the data acquired here we used BLAST2GO [12]to systematically predict function based on sequence similar-ity (supplementary Table 2 in Additional data file 1). Aftergrouping specific molecular function ontologies into broadercategories until they converged under one term (supplemen-tary Table 3 in Additional data file 1), the third-level termsTable 1An example of using peptide ratios used to derive protein relative expression results (odorant binding protein 14 [GenBank:94158822])PeptideSample Larval ages compared Replicate (1, 2, or 3) 1 2 3 4 5 Ln (peptide average) Ln (protein average)H 1, 3 1 -1.26 -1.41 -1.66 -1.82 -2.25 -1.68 -3.01H 2 -3.29 -3.39 -3.55 -3.68 -3.74 -3.53H 3 -3.76 -3.82 -3.82 -3.82 -3.82 -3.81H 2, 3 1 -0.68 -0.93 -1.28 -1.32 -1.77 -1.20 -1.35H 2 -0.85 -0.97 -1.06 -1.13 -1.62 -1.12H 3 -1.57 -1.59 -1.69 -2.11 NA -1.74H 4, 3 1 3.91 3.91 3.91 3.91 NA 3.91 2.22H 2 -0.10 0.53 0.91 1.94 3.10 1.28H 3 0.67 1.56 1.60 1.69 1.84 1.47H 5, 3 1 3.91 3.91 3.91 3.91 3.91 3.91 3.09H 2 2.16 2.32 2.50 3.04 3.04 2.61H 3 2.05 2.55 2.57 2.78 3.79 2.75T 1, 3 1 -1.29 -1.37 -1.57 -1.80 -1.80 -1.57 -0.53T 2 -1.68 -2.16 -2.16 NA NA -2.00T 3 1.47 1.83 2.59 NA NA 1.96T 2, 3 1 -0.55 -0.58 -0.78 -0.91 -0.93 -0.75 -0.72T 2 -0.34 -0.42 -0.55 -1.04 -1.42 -0.75T 3 -0.46 -0.66 -0.86 NA NA -0.66T 4, 3 1 -0.39 -0.40 -0.69 -0.70 -0.76 -0.59 -0.37T 2 -0.06 0.24 0.38 0.70 0.81 0.41T 3 -0.65 -0.95 -1.02 -1.07 NA -0.92T 5, 3 1 -0.02 -0.05 -0.27 -0.31 -0.40 -0.21 0.76T 2 0.37 0.67 0.90 1.47 NA 0.85T 3 0.80 1.53 1.67 2.04 2.08 1.62Both the honey bee larval hemolymph (H) and tissue (T) samples were collected daily for 5 days post-hatching, and peptides from days 1, 2, 4, and 5 were isotopically labeled and mixed at 1:1 (by protein amount) with day 3 peptides, which were labeled differentially from the other days. Relative Genome Biology 2008, 9:R156peptide intensities were recorded (limited at 50-fold or 3.91 in natural log (Ln)) and proteins with a minimum of 2 quantified peptides were natural log-transformed and averaged; for proteins with greater than 5 peptides, the top 5 most intense ones were selected for averaging. In samples where there were less than 5 peptides, their absence is indicated by NA (not available).156.4http://genomebiology.com/2008/9/10/R156 Genome Biology 2008,     Volume 9, Issue 10, Article R156       Chan and Foster Rwere analyzed in detail. To find whether a given function termwas developmentally regulated, an average expression profilewas generated using data from proteins belonging under eachterm and tested for significance at the p < 0.05 level (seeMaterials and methods). The slope between day 1 and day 5was calculated to approximate the directionality and strengthof temporal correlation. In the 34 terms considered, 11 ofthem had activity profiles that satisfied the significance crite-ria in at least one of either the solid tissue or hemolymphexpression profiles (Table 2; details in supplementary Table 4in Additional data file 1). Gene Ontology (GO) terms 'sub-strate-specific transporter activity' [GO:0022892] and 'trans-membrane transporter activity' [GO:0022857], both of whichwere tissue-specific activities, were very mildly positively cor-related with larval age. The majority were negatively corre-lated with age, with the most statistically significant being'structural constituent of ribosome' [GO:0003735] and'nucleic acid binding' [GO:0003676]. Others showing a simi-lar trend include 'enzyme inhibitor activity' [GO:0004857],'helicase activity' [GO:0004386], and 'nucleotide binding'[GO:0000166]. Terms that did not show regulation in eitherthe tissue or hemolymph tended to be ones with non-specificparticipation in different pathways, such as 'transferase activ-ity' [GO:0016740], 'kinase regulator activity' [GO:0019207]and 'cofactor binding' [GO:00048037].With the current lack of a thoroughly curated protein functiondatabase for the honey bee, we manually assigned functionalcategories by employing a variety of available bioinformatictools (see Materials and methods, and supplementary Table 5in Additional data file 1). This is necessary because certainmajor classes of honey bee proteins, such as hexamerins andmultiple terms, or two very similar proteins were assigned todifferent but similar terms ('nucleic acid binding'[GO:0003676], and 'translation factor activity, nucleic acidbinding' [GO:0008135]), which greatly complicates down-stream hierarchical clustering and enrichment analysis.Groups that showed a significant temporal regulation (crite-ria nearly identical to the analysis of level 3 molecular func-tion GO terms) are shown in Table 3 (details insupplementary Table 6 in Additional data file 1). A commonprotein expression pattern within a group was frequentlyobserved. Ribosomal protein levels in the tissue were consist-ently lowest at day 2 and day 5, but overall decreased in rela-tive concentration with age (p < 1e-16). Proteasome subunitsand protein-folding chaperones exhibited the same overalltrend (p < 1e-9 and p < 0.005, respectively). Energy storageproteins, including apolipoproteins and hexamerins,increased with age throughout the body but the trend wasmore dramatic in the hemolymph (p < 0.005). There were nosigns of temporal regulation of enzymes for fatty acid synthe-sis, beta oxidation, and carbohydrate metabolism. However,several groups of energy producing proteins showed varyingdegrees of positive correlation with time: tricarboxylic acidcycle proteins (p < 0.05), ATP synthase subunits (p <0.0005), and electron transport chain enzymes (p <PAGE of honey bee larvae (a) hemolymph and (b) solid tissueFigure 2PAGE of honey bee larvae (a) hemolymph and (b) solid tissue. Age is shown in days post-hatching. Molecular weight markers are shown on the left.188624938281814631      2      3      4      5 1      2      3      4      5 (a) (b)18862493828181463Larval ageLarval ageDevelopmental changes of larval hemolymphFigure 3Developmental changes of larval hemolymph. The left axis denotes the volume of hemolymph per larva (diamonds; μl) or hemolymph protein concentration (squares; μg/μl), while the right axis describes the mass of total protein per larva (triangles; μg). Measurements were made by pooling 5-120 larva (n = 3 separate pools) depending on age (x-axis, in days) and size. (Error bars represent 2 standard deviations.)0510152025303540450 1 2 3 4 5 60100200300400500600Genome Biology 2008, 9:R156odorant binding proteins, do not have high enough homologyto proteins in other better annotated organisms and wouldthus be ignored. Furthermore, most proteins were assigned to0.00005). Surprisingly, we observed a decreased expressionof antioxidant proteins, members of the Ras GTPase super-family, and ubiquitylation enzymes in the solid tissues as156.5http://genomebiology.com/2008/9/10/R156 Genome Biology 2008,     Volume 9, Issue 10, Article R156       Chan and Foster Rdevelopment progressed (p < 0.05, p < 0.01, and p < 0.05,respectively). Many typically intracellular proteins, such asribosomal proteins and proteasome subunits, were found inhemolymph as we have described previously [3] and as othershave reported in other insects [13,14].We used hierarchical clustering to further analyze the 522proteins that were profiled in either or both the solid tissue(Figure 4a) and hemolymph (Figure 4b), followed by enrich-ment analysis according to manually assigned groupings.Clusters that satisfied the criteria (see Materials and meth-ods) for significant enrichment are shown in Table 4 (com-plete dataset shown in supplementary Tables 7 and 8 inAdditional data file 1 for tissue and hemolymph, respectively).Only a few functional classes of proteins were enriched in thesame node, since expression profiles for some proteinsexhibit biological variability that causes apparent inconsist-ency with the timecourse. Transcription and chromatin-asso-ciated proteins as well tRNA synthetases - clearly related bytheir tasks - shared node 376 (correlation 0.94) with pentosephosphate pathway and ubiquitylation enzymes. Energy stor-age and beta-oxidation proteins were both concentrated innode 434 (correlation 0.86, solid tissue). Protein turnovermachinery, including ribosomes, protein folding, and protea-some, were all enriched in node 200 (correlation 0.84, hemo-lymph). Many of these clusters are also protein familiesalready noted to show significant temporal regulation, suchtion and categorization, since their regulation patterns weregrouped using independent methods.Automated and semi-automated functional annotation andcategorization effectively highlighted expression trends inlarge classes of proteins. With this approach, however, classeswith only a few members or those where particular proteinshave highly specialized function tended to fall below the sig-nificance threshold unless they were considered individually.In solid tissues, the levels of 86 proteins changed significantly(p < 0.05) over the tested period, accounting for 13% of all thequantifiable proteins in solid tissues. For example, levels ofneuropeptide Y receptor increased 46-fold from day 3 to day5. In the hemolymph, 66 of 481 (14%) quantified proteinschanged significantly during the larval stage (p < 0.05). Mostof these are intracellular proteins, yet the regulation of trulysecreted proteins is frequently far more dramatic. An imagi-nal disc growth factor [GenBank:66514614] increased morethan 13-fold from day 1 to day 5 (Figure 5a). Odorant bindingprotein 14 [GenBank:94158822] levels changed in a similarfashion, with the former displaying a 40-fold change over 5days (Figure 5b). Antimicrobial peptide apismin [Gen-Bank:58585112] (Figure 5c) and melanization enzyme proph-enoloxidase [GenBank:58585196] expression were alsopositively correlated with age.To our knowledge this is the first proteome-level descriptionTable 2Expression trends of proteins categorized under Gene Ontology termsOrgan GO ID number GO term Proteins considered t-Test of slope between day 1 and day 5 SlopeH GO:0004857 Enzyme inhibitor activity 6 1.9E-02 -0.19T GO:0004857 Enzyme inhibitor activity 6 0.007 -0.41T GO:0004386 Helicase activity 4 0.016 -0.37T GO:0016787 Hydrolase activity 80 0.002 -0.10H GO:0003676 Nucleic acid binding 18 7.2E-05 -0.24T GO:0003676 Nucleic acid binding 38 7.8E-09 -0.33H GO:0000166 Nucleotide binding 36 4.8E-03 -0.09T GO:0000166 Nucleotide binding 72 0.022 -0.08H GO:0016491 Oxidoreductase activity 19 1.8E-02 0.18H GO:0004871 Signal transducer activity 3 4.7E-02 -0.22H GO:0003735 Structural constituent of ribosome 18 1.5E-08 -0.41T GO:0003735 Structural constituent of ribosome 35 8.4E-16 -0.34T GO:0022892 Substrate-specific transporter activity 34 0.035 0.11T GO:0008135 Translation factor activity, nucleic acid binding 10 0.037 -0.24H GO:0022857 Transmembrane transporter activity 4 3.9E-02 -0.11T GO:0022857 Transmembrane transporter activity 27 0.010 0.12Proteins were categorized under third-level molecular function terms and were evaluated as a group to assess whether their expression trends were age-regulated by performing paired t-tests comparing values from day 1 and day 5 of larval development, reporting the average slope between these two days if p < 0.05 in either the solid tissue (T) or hemolymph (H). The total number of proteins belonging under a particular GO term considered in the calculation is listed under 'Proteins considered'.Genome Biology 2008, 9:R156as energy storage proteins, ATP synthases, antioxidant pro-teins, and ubiquitylation enzymes. This indirectly suggeststhat suitable assignments were made during manual annota-of honey bee larval development, so to gain additional insight,we compared our data with a previously reported develop-mental study of the fruit fly. While Drosophila and Apis are156.6http://genomebiology.com/2008/9/10/R156 Genome Biology 2008,     Volume 9, Issue 10, Article R156       Chan and Foster Rseparated by 300 million years of evolution [2], Drosophila isnonetheless the closest highly studied phylogenetic neighborof the bee. A whole body transcriptome study of the Dro-sophila melanogaster life cycle was published in 2002 [15],which included a list of genes that were significantly regulatedduring the larval period. After finding the protein homologscommon to our study and the fruit fly larval dataset (34 intotal), we calculated the slope of linear regression of expres-sion trends for both organisms (the slope of the honey bee tis-sue and hemolymph profiles were averaged when needed; seeMaterials and methods). Slope values that have oppositesigns or an absolute difference in slope of greater than 0.75were considered dissimilar, amounting to 38% (13 of 34) ofthe proteins considered (Table 5; complete dataset withBLAST homolog search results in supplementary Table 9 inAdditional data file 1). The most extreme slope reported forboth organisms is for the hexamerin 70b protein (1.5 for beesand 1.6 for flies).honey bee larvae. The most striking, by far, is the 1,500-foldincrease in weight over just 6 days [16]. In our proteomicanalysis of the solid tissue, the most abundant organs are bestrepresented, namely the fat body (accounts for 65% of themass [17]), followed by the midgut and larval tubules. Thehemolymph fraction reflects the secretory activities of allthese tissues and also the molecules associated with intercel-lular communication and regulation. The presence of intrac-ellular proteins suggest that hemolymph plays a major role inclearing apoptotic cells, in line with observations of the equiv-alent connective tissue in mammals (that is, blood) [18]. Nodissection of specific larval organs was performed becausemany do not develop until the late stages, making direct com-parisons of organ development by quantitative proteomicsimpossible.We have found both automated (BLAST2GO) and semi-auto-mated annotation (manual selection of descriptions providedby automated tools and manual categorization) to be very val-uable for maximizing available information on an organismTable 3Expression trends of manually annotated and categorized proteinsOrgan Class Proteins considered t-Test of slope between day 1 and day 5 SlopeT Adaptor 2 0.002 -0.43T Aldo-keto reductase superfamily 3 0.041 -0.22T Antioxidant 15 0.017 -0.20T ATP synthase 10 1.4E-04 0.21H Carbohydrate metabolism 15 0.003 0.20T Cuticle 7 0.036 0.19T Electron transport chain 14 1.1E-05 0.22H Energy storage 4 0.004 1.20T Energy storage 5 0.028 0.56T Kinases or phosphatases 2 0.044 0.24H Pentose phosphate pathway 4 0.001 0.06H Peptidase 15 0.045 0.14H Proteasome 9 1.4E-04 -0.23T Proteasome 18 8.6E-10 -0.32T Protein folding 34 0.001 -0.17T Ras superfamily 10 0.009 -0.27T Ribonucleoprotein 4 0.024 -0.43H Ribosome 20 2.1E-09 -0.40T Ribosome 38 4.7E-17 -0.34T Tricarboxylic acid cycle 21 0.033 0.10T Translation 14 0.015 -0.25T Ubiquitination 3 0.021 -0.35H Uncategorized 21 0.029 0.18Proteins were categorized manually by function and evaluated as a group to assess whether their expression trends were age-regulated by performing paired t-tests to compare values from day 1 and day 5. Significant (p < 0.05) groups in either the solid tissue (T) or hemolymph (H) are shown.Genome Biology 2008, 9:R156DiscussionThe data presented here, at the level of the whole proteome,documents the dramatic changes occurring in developingwith otherwise very little functional annotation. While auto-mated ontological methods were reliable and bias-free, out-puts might be too generic (for example, 'ion binding'156.7http://genomebiology.com/2008/9/10/R156 Genome Biology 2008,     Volume 9, Issue 10, Article R156       Chan and Foster R[GO:0043167]), or failing to accurately represent several veryimportant protein families of the honey bee (for example,hexamerins and odorant binding proteins), highlighting theneed for manual intervention. Cluster analysis is an indispen-sable tool for spotting expression trends, but given that thesoftware for rigorous statistical enrichment analysis isdesigned specifically for popular model organisms such asmouse, worm, and yeast, the descriptive statistical approachused here was nevertheless able to provide credible insightsabout larval developmental biology or led to conclusions con-The major behaviour during the larval stage is feeding as itprepares itself for the subsequent pupal stage when no feed-ing occurs. Based on various data acquired over the past cen-tury, it has been proposed that the larval fat body undergoestwo phases, beginning with a high rate of protein synthesisand poor uptake of hemolymph substances, followed by aphase of low cellular synthesis and improved uptake and stor-age of hemolymph proteins [19]. Our data now allows us toclarify this model and provide molecular-level detail of thesechanges. One of the most remarkable events in a growingTable 4Enrichment analysis of protein classes following hierarchical clusteringNode number Correlation Proteins in this node Protein class Class total Percent class enrichmentSolid tissue265 0.98 5 Helicase 3 67277 0.98 8 Hormone synthesis 4 50376 0.94 79 Transcription 3 100376 0.94 79 Chromatin-associated protein 3 67376 0.94 79 tRNA synthetase 3 67376 0.94 79 Pentose phosphate pathway 4 50376 0.94 79 Ubiquitylation 4 50377 0.94 5 Food 6 50411 0.90 108 Aldo-keto reductase superfamily 3 67419 0.89 137 Proteasome 24 67419 0.89 137 Antioxidant 16 50419 0.89 137 Protein receptor 4 50421 0.89 29 ATP synthase 10 60427 0.88 34 Small molecule receptor 4 50434 0.86 51 Energy storage 5 80434 0.86 51 Beta-oxidation 8 50438 0.83 7 Cuticle 7 57439 0.83 146 Ras superfamily 10 50Hemolymph128 0.97 35 Translation 7 57150 0.96 6 Short-chain dehydrogenase family 4 50180 0.93 21 Small molecule receptor 4 50183 0.92 23 Food 8 63183 0.92 23 Glycolipid metabolism 3 67185 0.92 9 Ubiquitylation 4 50190 0.89 63 Amino acid metabolism 8 50197 0.86 29 Energy storage 4 100200 0.84 81 Proteasome 10 90200 0.84 81 Protein folding 20 60200 0.84 81 Ribosome 31 81203 0.82 21 Tricarboxylic acid cycle 4 75Proteins that were manually categorized by function were subjected to average-linkage clustering (Figure 3). Separate analyses were done for solid tissue and hemolymph. Only proteins that were quantified for at least 4 out of 5 days and protein classes that had at least 5 members were considered for enrichment analysis. Classes with at least 50% enrichment in nodes with a correlation of >0.8 are considered significant and shown.Genome Biology 2008, 9:R156firmed by other information. larva is the substantial synthesis of hexamerins and lipopro-teins in the fat body, followed by their appearance in the156.8http://genomebiology.com/2008/9/10/R156 Genome Biology 2008,     Volume 9, Issue 10, Article R156       Chan and Foster RAverage-linkage clustering of proteins quantified in the honey bee larvaeFigure 4 Average-linkage clustering of proteins quantified in the honey bee larvae. Proteins that were quantified in either or both the (a) tissue or (b) hemolymph for at least four out of five tested days were arranged by hierarchical clustering using software described in [46]. All expression values, shown relative to Correlation0 0.8 1.0-1.0Correlation0 0.8 1.0-1.012345Days-4 0 4(a) (b)12345DaysNo dataGenome Biology 2008, 9:R156day 3 (= 0, black), have been natural log-transformed (>0, red; <0, green; no data, grey). These proteins, which have been manually annotated with a function and category, are calculated for enrichment within a node (results in Table 3) if the node correlation value is >0.8 (see thick bar on scale).156.9http://genomebiology.com/2008/9/10/R156 Genome Biology 2008,     Volume 9, Issue 10, Article R156       Chan and Foster Rhemolymph near the end of this developmental stage(reviewed extensively in [20]). While the age-dependent pro-duction of these abundant storage proteins is well known,here we provide evidence of a concomitant up-regulation oflow copy transmembrane transporters [GO:0022857] thatmay facilitate the export, including a porin [Gen-Bank:66521459]. Paradoxically, this astounding rate of pro-tein production and export is paired with an opposite trend inprotein synthesis machinery and accessories, which had beensuspected in two reports in the 1960s [21,22]. Now we haveevidence for these previous suggestions, including the clearage-associated decrease of more than 50 detected ribosomalsubunits, coupled with an increase of two transcriptionrepressors (although at p < 0.1 these did not satisfy signifi-cance criteria) to support this former notion.Fat accumulation is an important purpose of the rapid larvalgrowth, clearly indicated by the size of the fat body tissue rel-ative to the whole organism, as well as the buildup of lipo-phorins. Lipids in larval food is only 4% by weight [23],meaning that de novo synthesis must account for the bulk ofstored fat. Fatty acid synthase [GenBank:66515350] was oneof the most abundant proteins throughout the entire testedperiod based on absolute protein expression estimates [24],yet to our surprise we did not observe significant temporalregulation in the expression of this enzyme with age. It isworth noting that 'fat body' is somewhat of a misnomer, giventhat it is involved in protein and glycogen storage, as well asfat [19,25]. To drive these endergonic biosynthetic processes,the demands for ATP must therefore be great. Not only do weobserve significant age-associated increases in ATP synthasesubunits, but also enzymes in energy-producing pathwayssuch as the tricarboxylic acid cycle and the electron transportchain components. This may be attributed to an increase inmitochondria size or numbers; however, there are at least tworeports that claim the number of mitochondria decreases asthe larva approaches pupation in other insects [26,27].Proteins with high copy number, including the many dis-cussed above, are always the first to be investigated in anyorganism. The difficulties in studying proteins in honey beelarva have multiple sources: the abundant storage proteinsbroaden the dynamic concentration range, obscuring the rareproteins; the clean dissection of larval organs presents a tech-nical challenge since the fat body is large and is difficult toremove; and finally, the lack of available antibodies againsteven the most common proteins makes many conventionalbiochemistry experiments, such as immunoprecipitation andwestern blotting, impossible. These reasons have especiallyhindered the study of fine larval organs such as the nervoussystem and low abundance proteins related to immunity orpathway regulation.cation has been observed in old larvae [28,29]. Odorant bind-Expression profiles of four selected proteins during larval developmentFigure 5Expression profiles of four selected proteins during larval development. Expression levels (y-axis, expressed in natural log scale) over 5 days of larval growth (x-axis) are shown for 3 proteins discussed in the text: (a) imaginal disc growth factor [GenBank:66514614], (b) odorant binding protein 14 [GenBank:94158822], (c) apismin [GenBank:58585112]. Error bars represent one standard deviation.-4-20241 2 3 4 5-4-20241 2 3 4 5-4-20241 2 3 4 5(a)(b)(c)Genome Biology 2008, 9:R156The ability of larvae to respond to external stimuli and inter-nal regulatory cues increases with time, a trend that is clearlyreflected in our data. For example, odorant-based communi-ing protein 14 [GenBank:94158822] was detected even on thefirst day after hatching, showing upregulation with age (Fig-ure 5b). This suggests that younger larvae may have the capa-156.10http://genomebiology.com/2008/9/10/R156 Genome Biology 2008,     Volume 9, Issue 10, Article R156       Chan and Foster RTable 5Comparing expression levels in honey bee versus fruit fly larvaeHoney bee accession numberProtein description Slope difference (honey bee minus fruit fly)Expression trend: different or same?GenBank:48126476 Translation: initiation factor 3f 0.01 SameGenBank:110749015 Short-chain dehydrogenase family: oxidoreductase0.22 SameGenBank:110756656 Protein methylation: arginine methyltransferase0.10 SameGenBank:110759433 Ribonucleoprotein: ribonucleoprotein 0.10 SameGenBank:110761364 Cytoskeleton: alpha-actinin 0.19 SameGenBank:66547531 Pentose phosphate pathway: 6-phosphogluconate dehydrogenase0.24 SameGenBank:66509442 Peptidase: dipeptidyl aminopeptidase 0.02 SameGenBank:110764347 Amino acid metabolism: enolase-phosphatase E1 (methionine salvage pathway)0.50 SameGenBank:110762382 Transcription: spermidine synthase 0.51 SameGenBank:110763730 Antioxidant: glutathione S transferase 0.32 SameGenBank:66504249 Uncategorized: protein kinase c substrate 0.33 SameGenBank:110755309 Protein receptor: high density lipoprotein binding protein0.33 SameGenBank:110750855 Unknown function: unknown function 0.36 SameGenBank:94158626 Electron transport chain: cytochrome p4500.51 SameGenBank:58585148 Energy storage: hexamerin 70b 0.15 SameGenBank:48095159 Peptidase: serine protease 0.34 SameGenBank:66524124 Peptidase: carboxypeptidase B 0.07 SameGenBank:66509812 Peptidase: angiotensin converting enzyme 0.28 SameGenBank:110762229 Peptidase: chymotrypsin 0.47 SameGenBank:66510448 Glycolipid metabolism: beta-glucosidase (glucocerebrosidase)0.10 SameGenBank:110766932 Uncategorized: mannosidase, lysosomal 0.50 SameGenBank:66513481 Ubiquitination: ubiquitin-activating enzyme E10.48 DifferentGenBank:66522467 Uncategorized: juvenile hormone inducible protein0.95 DifferentGenBank:48104663 Protein receptor: protein kinase C receptor0.60 DifferentGenBank:110758189 Uncategorized: carboxylesterase 0.94 DifferentGenBank:110756254 Ribonucleoprotein: ribonucleoprotein 0.67 DifferentGenBank:66522232 Uncategorized: isochorismatase 0.43 DifferentGenBank:66535270 Uncategorized: oxoacidtransferase 0.42 DifferentGenBank:66521459 Membrane transporter: porin 0.59 DifferentGenBank:110764660 Helicase: RNA helicase 0.56 DifferentGenBank:110762902 ATP synthase: ATP synthase component 0.77 DifferentGenBank:66525867 Small molecule carrier: solute carrier 0.82 DifferentGenBank:58531215 Membrane transporter: translocase, ATP 0.87 DifferentGenBank:110759569 Apoptosis: beta-hexosaminidase 1.14 DifferentGenes in a life-cycle transcriptomic analysis of D. melanogaster [15] were compared to honey bee larval proteomics data in this report by finding homologs common to these studies. Significant matches (see Materials and methods for criteria) were assessed by comparing the slope values calculated between days 1 and 4: a protein is marked 'same' if the sign of the slope was the same and had a difference no greater than 0.75; Genome Biology 2008, 9:R156otherwise, they are marked as 'different'.156.11http://genomebiology.com/2008/9/10/R156 Genome Biology 2008,     Volume 9, Issue 10, Article R156       Chan and Foster Rbility to bind certain odorant molecules, but whether thattranslates into pheromonal communication is entirely specu-lative. The positive temporal regulation of antimicrobial pep-tide apismin [GenBank:58585112] (Figure 5c) and themelanization enzyme prophenoloxidase [Gen-Bank:58585196] in the hemolymph, which have clear roles indefense [30-33], matches the observed susceptibility to dis-eases such as foulbroods of the young larvae, suggesting thatone or both of these may be the factor responsible for success-ful defense against foulbroods in older larvae. However, a C-lectin [GenBank:110750008] and a complement factor [Gen-Bank:66508940] actually have no observable expressiontrends, indicating that they may have alternative roles differ-ent from homology-based function predictions. The 46-foldincrease of a neuropeptide Y receptor [GenBank:110764421],which controls appetite and fat storage, is reasonable giventhe feeding activity of the larvae. An imaginal disc growth fac-tor [GenBank:66514614] increased by 40-fold over the courseof the experiment (Figure 5a) presumably gears the larva forpupal development where specific limbs and organs will growfrom imaginal discs containing highly differentiated cells.Proteomics is generally a discovery method and is thus anexcellent mechanism for hypothesis generation. We were ableto find several peculiar proteins supported by a number ofhigh quality mass spectra but no plausible explanation for itspresence or degree of age-dependent regulation. A proteinannotated as 'PREDICTED: similar to CG15040-PA' [Gen-Bank:110749732] was consistently found only in the hemol-ymph of older larvae (up to 24-fold higher in 5-day oldcompared to 3-day old larvae), yet it has no likely homologsor discernable functional domains, bearing only a vagueresemblance to a protein [GenBank:124512744] from Plas-modium falciparum 3D7, found by PSI-BLAST [34,35].ConclusionTo study honey bees, individual, environmental, and socialfactors must be considered. The larval developmental stagehas been shown to be a highly complex period of biochemicalregulation. The proteomics data presented here are able tosupport a model for energy metabolism and storage, as well asreveal unexpected expression trends for proteins thatrespond to external and internal stimulus, such as pherom-ones, pathogens, and oxidants.Materials and methodsReagentsAll salts and chemicals were of analytical grade or better andwere obtained from Sigma-Aldrich (St. Louis, MO, USA)unless otherwise indicated. All solvents were of high perform-ance liquid chromatography grade and were obtained fromtrypsin, Promega (Nepean, Ontario, Canada); loose ReproSil-Pur 120 C18-AQ 3 μm, Dr Maisch (Ammerbuch-Entringen,Germany); 96-well full skirt PCR plates, Axygen (Union City,CA, USA); fused silica capillary tubing, Polymicro (Phoenix,AZ, USA); 5 μl Microcap pipettes for hemolymph collection,Drummond (Broomall, PA, USA); soft forceps for holdingbees, BioQuip (Rancho Dominguez, CA, USA); proteaseinhibitor mixture, Roche Applied Science (Basel, Switzer-land); precast 4-12%, 1 mm thick NuPAGE Novex BisTris2Gels, Invitrogen (Carlsbad, CA, USA).Obtaining larvae of known agesHoney bee (A. mellifera ligustica) larvae were obtained fromcolonies kept at the University of British Columbia, Vancou-ver, BC, Canada. Samples were collected in the summer andearly autumn. To acquire larvae of known ages, a queen wasisolated on an empty frame of dark comb bracketed by twoframes approximately 50% filled with honey and pollen for 16h inside a nucleus colony with several hundred worker bees.The brood frame with newly laid eggs was then replaced intothe original hive, along with the queen, workers and two sup-porting frames. The queen was separated from the newly laideggs using a queen excluder to prevent additional eggs frombeing deposited. Three days after reintroducing the eggs intothe colonies, larvae were collected for five consecutive days.In this system the maximum error in larval age would be 16 h.Empirical testing with shorter times did not yield enougheggs/larvae to sample the same population over all five daysof development. Before proceeding with protein collection, alllarvae were washed three times in phosphate buffered salineto reduce royal jelly contamination.Protein collectionFor 1- to 3-day old larvae, hemolymph was collected by pierc-ing the larval skin, taking care not to cause organ damage byavoiding deep cuts. For 4- and 5-day old larvae, hemolymphwas collected by inserting a disposable 5 μl glass Microcappipette two-thirds of the way down one side of the larva,drawing liquid by capillary action. All hemolymph sampleswere centrifuged for 10 minutes at 16,100 relative centrifugalforce (r.c.f.) at 4°C to pellet cells and debris, which wereadded to the tissue samples. The tissues were homogenized bya bead mill using a tungsten bead in each 2 ml self-lockingtube (Eppendorf, Hamburg, Germany) at 30 Hz for 5 minutesin 50 μl of phosphate buffered saline containing a proteaseinhibitor cocktail tablet solution (Roche) at 8 times the sug-gested concentration. Lysis buffer (100 μl of NP-40, and soon) was added before the sample was homogenized by 10strokes through a syringe tipped with a 25 G needle. The sam-ple was clarified for 10 minutes at 16,100 r.c.f. at 4°C and thepelleted debris was discarded. The Coomassie Plus ProteinAssay (Pierce, Rockford, IL, USA) was used to determine pro-tein concentrations of the tissue lysates and the clarifiedGenome Biology 2008, 9:R156ThermoFisher Scientific (Waltham, MA, USA). The followingmaterials were obtained as indicated: endopeptidase Lys-Cfrom, Wako Chemicals (Osaka, Japan); porcine modifiedhemolymph according to the manufacturer's instructionsbefore they were stored at -20°C until used.156.12http://genomebiology.com/2008/9/10/R156 Genome Biology 2008,     Volume 9, Issue 10, Article R156       Chan and Foster RDenaturing protein gel electrophoresisTissue and hemolymph proteins were resolved on precast(Invitrogen) 4-12% NuPAGE gels (30 μg/lane) in reducingconditions with MES buffer according to the manufacturer'sinstructions. Blue-silver stain [36] was used to visualize pro-tein bands.Sample preparation for mass spectrometry analysisLarval tissue or hemolymph protein were aliquoted to pro-vide 20 μg per sample before they were precipitated using theethanol/acetate method as described [37]. The insoluble pro-teins were pelleted and temporarily stored at 4°C after a 10-minute centrifugation at 16,100 r.c.f. The ethanol superna-tant was vacuum-dried, solubilized in sample buffer (3% ace-tonitrile, 1% trifluoroacetic acid, 0.5% acetic acid), andpurified using the C8 flavor of STop And Go Extraction(STAGE) tips [38] to remove contaminants such as lipids,nucleic acids, and protease inhibitors. Bound proteins wereeluted using 100% acetonitrile and vacuum-dried before add-ing 0.5 μl of 1.5 M Tris-HCl, pH 8.8. The bulk protein pelletand C8 purified proteins were digested using LysC and trypsinas described [37]. Peptides were desalted using C18 STAGEtips and the eluted solution was dried by vacuum centrifuga-tion. For proteome profiling by relative quantification, binaryanalysis between timepoints was performed by chemicaldimethylation of peptides from different timepoints usingeither light (CH2O) or heavy (CD2O) isotopologues of formal-dehyde. For both the hemolymph and tissue samples, 3-dayold larvae were used as a reference for all other timepoints,such that their peptides were always labeled with the oppos-ing form of formaldehyde from days 1, 2, 4, and 5 before mix-ing the differentially labeled samples. Samples werefractionated on C18-SCX-C18 STAGE tips using a 10-stepammonium acetate elution gradient [39] and dried peptidesamples were resuspended in 1% trifluoroacetic acid, 3% ace-tonitrile, 0.5% acetic acid prior to analysis on a linear trap-ping quadrupole-Orbitrap hybrid mass spectrometry(ThermoFisher Scientific, Waltham, MA, USA) as describedin [3].Raw data processingFollowing liquid chromatography-mass spectrometry analy-sis, peak lists were extracted from the raw data usingExtract_MSN.exe (ThermoFisher Scientific) and DTA Super-charge [40] as described [41]. Results were searched usingMascot (v2.2) against a database containing the proteinsequences of: Honey Bee Official Gene Set 1 [42], commonexogenous contaminants (human and sheep keratins) andadditives (porcine trypsin, lysyl endopeptidase C), the poly-protein of the deformed wing virus (common and oftenasymptomatic [43]), and the reversed sequences of all of theabove as a decoy for reporting false discovery rates. The fol-lowing Mascot parameters were used: trypsin (allowing up tothe peptides' amino termini and lysine ε-amino groups, 10ppm peptide tolerance; 0.8 Da tandem mass spectrometrytolerance, and electrospray ionization-Trap fragmentationcharacteristics. Results were saved in Peptide Summary for-mat with the 'Require Bold Red' option checked, applying ascore cutoff corresponding to p < 0.05, which is 27 whereresults were limited to tryptic peptides, and 47 where noenzymes were specified. Since each sample was fractioned,generated files were combined using the in-house script Pick-letrimmer.pl. MSQuant [40] was used to semi-automaticallyextract chromatographic peak volumes in both the light andheavy isotopologues of each detected peptide. Only peptideswith an absolute calibrated mass error of <5 ppm were con-sidered further. For protein quantification, the file was parsed(in-house script: QC_msqfa.pl) to obtain natural logarithm(Ln)-transformed heavy/light peptide volume ratios, whichwere median-normalized before they were averaged to calcu-late a relative protein ratio of day 3 larvae/day × larvae(where x = 1, 2, 4, or 5). From the three biological replicatesof each binary comparison, proteins quantified with at leasttwo quantified peptides from two or more replicates wereaveraged. Proteins whose relative quantities could be trackedfor at least 4 of 5 days in either the tissue or hemolymph wereconsidered to be profiled. For protein identification, theabove peptides and unquantified sequences were extractedfrom MSQuant outputs. After removing redundant entries(in-house script: QC_remduplicate.pl), each was matched totheir respective protein (in-house script: finalist.pl),excluding hits that were verified by equal to or less than twopeptides of at least six or more residues. The false discoveryrate was estimated by dividing the sequence-reversed pro-teins that failed to be eliminated after applying the abovecriteria.Automated protein annotation to Gene Ontology termsAll identified proteins were matched to GO [44] terms usingBLAST2GO [12], following their standard procedure of per-forming BLAST searches for each protein (BLASTp, nr data-base, high scoring segment pair (HSP) cutoff length 33, report20 hits, maximum e-value 1e-10), followed by mapping andannotation (e-value hit filter 1e-10, annotation cutoff 55, GOweight 5, HSP-hit coverage cutoff 20). After generating adirected acyclic graph (sequence filter 2, score alpha 0.6,node score filter 0) of molecular function terms (not shown),which groups specific terms into broader categories, ontolo-gies on the third level of this graph were further analyzed bystatistical testing (see below). The term 'protein binding'[GO:0005515] was omitted because this included the mostnumber of proteins, most of which belonged under anothermore informative term.Semi-automated protein annotation and manual Genome Biology 2008, 9:R156one missed cleavage) or no enzyme specificity (in separatesearches); carbamidomethyl as a fixed modification, variablemodifications of dimethylation by both hydrogen isotopes atcategorizationProtein descriptions were taken from several sources or tools,all of which are sequence homology-based derivations. Offi-156.13http://genomebiology.com/2008/9/10/R156 Genome Biology 2008,     Volume 9, Issue 10, Article R156       Chan and Foster Rcial protein names given in the Official Gene Set 1 [42] wereused if the name was informative. BLAST2GO-deriveddescriptions were used where protein function was not clearfrom the official name (for example 'hypothetical protein'). Ifan appropriate name was still not derived, searches againstthe Conserved Domain Database (NCBI) were performed andconsidered matched for e-values <1e-10. As a final measurefor matching a protein with a functional name, proteinsdescriptions were copied from Blast2seq [45] results(accessed via BLink in NCBI) if matches had >25% sequenceidentity and an e-value of <1e-10 over the aligned region. Ifnone of these steps provided useful information, the proteinwas labeled and categorized with 'unknown function.' Pro-teins with descriptions but that did not fit under a specific cat-egory were classified as 'uncategorized' (supplementary Table5 in Additional data file 1). Proteins that were not manuallyannotated are marked with 'NA' in the 'Description' column ofsupplementary Table 1 in Additional data file 1.Statistical analysisTo each class of manually assigned proteins, a pairwise, two-tailed t-test was performed using each protein in that class bytaking the relative ratio in day 1 and comparing to day 5.Groups with p < 0.05 were considered to be temporally regu-lated, and their directionality of regulation was calculated byaveraging the slopes of individual proteins within a groupusing day 1 and day 5 timepoints. Third-level GO molecularfunction terms were analyzed in the same manner, except allthe proteins considered were quantified over all five daystested in at least one of the tissue or hemolymph datasets. Toindividual proteins, the same criteria for significance wasused, taking values from each biological replicate as a datapoint in a pairwise comparison between the earliest and latestday the protein was quantified. We also performed averagelinkage clustering of the protein expression levels for proteinsthat were quantified over at least 4 days in either the tissue orhemolymph using Cluster and visualized by Treeview [46].The grouping sizes ranged from 2 to 55 proteins. To normal-ize this variation, the number of proteins in a given class isreported as a percentage of the total class size (percentenrichment, using in-house script QC_nodeenrichment.pl).Only nodes that included at least 50% of all the proteins inthat class and had a Pearson's correlation coefficient ofgreater than 0.8 were considered to be within the same clus-ter. Protein families with three or fewer members wereincluded as part of the tree diagram, but were not consideredfor whether they formed a significant cluster.Comparison to DrosophilaProteomic profiles resulting from this work were compared tothe transcriptomic profiles of previously published Dro-sophila homologs [15] for the timepoints matching mostclosely to days 1 to 4 of the honey bee larval stage (h = 24, 49,Bee Official Gene Set 1, which were defined as matches havinge-values <1e-10 with at least 25% identity within the alignedregion. Timepoints of the Drosophila dataset were normal-ized to the h = 72 timepoint and Ln transformed. To comparethe expression trend between the two organisms, the slope ofthe line-of-best-fit for proteins (bees) or genes (flies) was cal-culated: expression trends with slopes that differed in signageor had an absolute difference of greater than 0.75 were con-sidered to be dissimilar. Slopes whose absolute Pearson's cor-relation coefficient value was <0.5 were consideredinsignificant and, therefore, not considered. In instanceswhere a significant slope could be calculated for a protein inboth the tissue and hemolymph samples, the slopes wereaveraged.AbbreviationsGO: Gene Ontology; Ln: natural logarithm; r.c.f.: relative cen-trifugal force; STAGE: STop And Go Extraction.Authors' contributionsQWCT and LJF jointly conceived of the study, authored thescripts used in the data analysis and wrote the manuscript.QWCT conducted all the experimental work, mass spectro-metric analysis and bioinformatics. LJF supervised the workand helped to troubleshoot throughout.Additional data filesThe following additional data are available with the onlineversion of this paper. Additional data file 1 includes supple-mentary Tables 1-10.Additional data file 1Supplement ry Tables 1-10. 1: relative quantification of bee larval pro-teome. Supplementary Table 2: Gene Ontology terms assigned to honey be  larv l proteins. Suppleme tary Table 3: Gene O tol gy categoriz tion o  r tei s y molecular function using direc ed acyclic graphs. Suppl mentary Tabl  4: Gene Ontology 'mol cularfun t ' vocabul ies as igned to pro e ns n l ve  3 of a dir cted. S l mentary Table 5: ma ual y a si d pr tein i   functi nal class. Suppl m tary Tabl  6: averagesl  val es of pro eins w hin ma u lly ssi ned function l ass s. Su plementary Table 7: nrich e t ly s h ra hi-l ust ring of prot in  profil d fro  th  h y s lid issue. Sup le entary Tab e 8: enrich e t a alysi  of h era ch cal t ri g f prot ns profil d fro  t e o e  b e larv l h m -ymph 910  ptid  s que c  dat .Click her f ilAcknowledgementsThe authors wish to thank Nikolay Stoynov for technical assistance. Oper-ating funds for this work came from the Natural Sciences and EngineeringResearch Council (NSERC) of Canada. The mass spectrometry hardwareand software were funded by the Canadian Foundation for Innovation andthe Michael Smith Foundation for Health Research through the BritishColumbia Proteomics Network. QWTC is supported by an NSERC PGS-D award and LJF is the Canada Research Chair in Organelle Proteomics anda Michael Smith Foundation Scholar.References1. Haldane JBS: Aristotle's account of bees' 'dances'.  J HellenicStudies 1955, 75:24-25.2. Consortium THGS: Insights into social insects from thegenome of the honeybee Apis mellifera.  Nature 2006,443:931-949.3. Chan QW, Howes CG, Foster LJ: Quantitative comparison ofcaste differences in honeybee hemolymph.  Mol Cell Proteomics2006, 5:2252-2262.4. Scharlaken B, de Graaf DC, Goossens K, Peelman LJ, Jacobs FJ: Dif-ferential gene expression in the honeybee head after a bac-terial challenge.  Dev Comp Immunol 2008, 32:883-889.5. 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