An Integrated Proteomics Reveals Pathological Mechanism ofHoneybee (Apis cerena) Sacbrood DiseaseBin Han,§ Lan Zhang,§ Mao Feng, Yu Fang, and Jianke Li*

Institute of Apicultural Research/Key Laboratory of Pollinating Insect Biology, Ministry of Agriculture, Chinese Academy ofAgricultural Science, Beijing, China

*S Supporting Information

ABSTRACT: Viral diseases of honeybees are a major challenge for the global beekeepingindustry. Chinese indigenous honeybee (Apis cerana cerana, Acc) is one of the major Asianhoneybee species and has a dominant population with more than 3 million colonies. However, Accis frequently threatened by a viral disease caused by Chinese sacbrood virus (CSBV), which leadsto fatal infections and eventually loss of the entire colony. Nevertheless, knowledge on thepathological mechanism of this deadly disease is still unknown. Here, an integrated gel-based andlabel-free liquid chromatography−mass spectrometry (LC−MS) based proteomic strategy wasemployed to unravel the molecular event that triggers this disease, by analysis of proteomics andphosphoproteomics alterations between healthy and CSBV infected worker larvae. There were 180proteins and 19 phosphoproteins which altered their expressions after the viral infection, of which142 proteins and 12 phosphoproteins were down-regulated in the sick larvae, while only 38proteins and 7 phosphoproteins were up-regulated. The infected worker larvae were significantlyaffected by the pathways of carbohydrate and energy metabolism, development, proteinmetabolism, cytoskeleton, and protein folding, which were important for supporting organ generation and tissue development.Because of abnormal metabolism of these pathways, the sick larvae fail to pupate and eventually death occurs. Our data, for thefirst time, comprehensively decipher the molecular underpinnings of the viral infection of the Acc and are potentially helpful forsacbrood disease diagnosis and medicinal development for the prevention of this deadly viral disease.

KEYWORDS: Chinese sacbrood virus, Chinese honeybee, 2-DE, label-free LC−MS, proteome, phosphoproteome

1. INTRODUCTION

The honeybee is a most important pollinator in the ecosystem.Of the main crops for human consumption, 70% rely uponhoneybee pollination services.1 The pollinator decline will havesevere consequences for food security.2 China has over 8.7million honeybee colonies, of which the Chinese indigenoushoneybee (Apis cerana cerana, Acc) represents over 3 million.3

The Acc has a long apicultural history and has strongerbiological characteristics in resisting Varroa destructor, wasps,extreme climates (cold/hot weather) and adverse conditions.4,5

Therefore, it plays significant roles both for the pollination ofplants and crops, as well as the maintenance of the vegetationcover and biodiversity.6,7 In addition, the Acc is a major honeyproducer and its honey is a popular bee-product in the Asianmarket. Unfortunately, Acc has been stricken by a fatal viraldisease caused by Chinese sacbrood virus (CSBV), whichresults in severe and deadly infections of the colony andeventually losses of the entire colony.8 This has become a hugechallenge for the Chinese and even for the Southeast Asianbeekeeping industry.The CSBV is a small RNA virus (picorna-like virus) that has

an icosahedral virion with a diameter of 26−30 nm, and itsgenome consists of a single positive-strand RNA molecule with8.8 kb.9 Although the CSBV shows a close genetic relationshipto its western counterpart, sacbrood virus (SBV), no crossinfection has been reported yet.10 The CSBV primarily affects

honey bee worker larvae aged 1−3 days, but the apparentsymptoms are exhibited at their prepupal stage. Once thecolonies get infection, the nurse bees can distinguish and uncapthe sealed sick larvae in the comb cells.11 The most obvioussymptom of the CSBV infection is that diseased larvae fail topupate and further take a sac-like appearance because theecdysial fluid is accumulated between the prepupal and pupalskins.12 In addition, the color of the infected larvae changesfrom pearly white to pale yellow, to light brown and finally,dark brown.9,13 Shortly after death, the larvae become wrinkled,forming a gondola-shaped scale. In addition, the CSBV can alsoreduce the lifespan and foraging activities of adult bees.14,15

Since this viral disease broke out in 1972 in southern China,some efforts have been made to develop diagnostic methodssuch as electron microscopy,16 enzyme-linked immunosorbentassay (ELISA) and reverse transcription-polymerase chainreaction (RT-PCR).17 Recently, attempts have been made touse RNA interference to treat this disease.18 However, there isstill a scarcity of information regarding the pathological changesof this fatal honeybee disease at the molecular level. Similar toall other insects, the honeybees lack a classically adaptiveimmune system as in the case of mammals. To survive, theyhave evolved cellular and humoral immune responses

Received: December 31, 2012Published: February 19, 2013

Article

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© 2013 American Chemical Society 1881 dx.doi.org/10.1021/pr301226d | J. Proteome Res. 2013, 12, 1881−1897

constituted of the defense mechanisms to cope with microbialinfections.19 Therefore, the in vitro reared honeybee larvaecould respond with a prominent humoral reaction to asepticinjury, and Gram-negative bacteria challenged bee larvae canincrease synthesis of antimicrobial peptides.20

Two-dimensional gel electrophoresis (2-DE) based proteo-mics technology permits quantitative and multicolor fluores-cence detection of phosphoproteins and total proteins within asingle gel electrophoresis experiment. This has been widelyused in unraveling the pathological mechanism in humandiseases.21 Meanwhile, the label-free LC−MS based proteomicsstrategy is able to precisely and reproducibly quantify proteinexpression directly without using any labeling, which has beenbroadly applied to the proteomic profiling, biomarker discoveryand disease diagnostics.22−24 However, despite rapid develop-ment in proteomics technologies and their application inhoneybee developmental biology,25−27 no such study has beenconducted on protein expression and phosphorylation changesassociated with the pathological mechanism in the honeybeedisease. Therefore, the current study employed gel-based (2-DE) and shotgun proteomic (label-free LC−MS based)strategies, which have complementary natures, to gain an in-depth understanding of the pathological mechanism of the fatalviral disease of the Acc by comparison of the proteome-widechange of the healthy and sick worker larvae. This may bepotentially helpful for early diagnosis and development of amore specific target medicine to treat this disease and facilitatethe investigation of sacbrood disease at cellular level with theavailability of immortalize honey bee cell lines.28

2. MATERIALS AND METHODS

2.1. Chemical Reagents

All the chemicals used for 2-DE were purchased from Sigma(St. Louis, MO) except for Biolyte and immobilized pHgradient (IPG) strips that were from Bio-Rad (Hercules, CA).Modified sequencing grade trypsin was from Roche (Mis-sissauga, ON, Canada). Chemicals used but not specified hereare noted with their sources in the text. All reagents used wereanalytical grade or better.2.2. Biological Samples

The honeybee larvae (Acc) infected by Chinese sacbrood viruswith typical symptom (A shot brood pattern in the comb, thesick larvae found in the uncapped cells with raised mouth parts.When taking the infected larva out of cell it presents a sac-likeappearance) (Supporting Information Figure 1) were collectedfrom five bee colonies maintained in the apiary of the Instituteof Apicultural Research, Chinese Academy of AgriculturalSciences in Beijing. The infection of the CSBV virus wasconfirmed by morphological observation under electronicmicroscopy and RT-PCR test according to Chen et al.29 andYan et al.30

For purification of the virus, CSBV-infected larvae werehomogenized in 5 mL of NT buffer (100 mM NaCl, 10 mMTris, pH 7.4) and the mixture was centrifuged at 1000g for 10min. The supernatant was extracted with an equal volume of1,1,2-trichlorotrifluoroethane before the aqueous phase waslayered over a discontinuous CsCl gradient (1.5 and 1.2 g/cm3)and centrifuged at 270 000g for 1 h in an SW50 rotor(Beckman Coulter, Los Angeles, CA). The material at the CsClinterface was harvested. The purified virus was added into sugarsyrup, which was made of 1 part sugar to 1 part water byvolume, and then the sugar syrup was fed to five honeybee

colonies, which was the infected group. Five other control(healthy) colonies were feed only with sugar syrup.The healthy and infected larvae samples were collected at the

prepupal stage (nine days after egg hatching) from fivehoneybee colonies. Briefly, to ensure the exactly the same ageof the larvae to be sampled, the egg laying queen bee wasconfined to a single wax comb frame containing worker cells for5 h with a cage made of a queen excluder, through whichworkers but not the queen could pass. Subsequently, the queenwas removed, and the fertilized eggs contained in the framewere maintained in the honeybee colony for further develop-ment. At the experimental time point, each 100 larvae of theCSBV infected and healthy larvae were sampled and frozen at−80 °C until use. Each 100 larvae were pooled as one biologicalreplicate, and three independent biological replicates wereproduced.

2.3. Protein Extraction and 2-DE

Larval protein extractions were carried out according to ourpreviously described method with some modifications.31

Briefly, the larvae were homogenized with lysis buffer (LB, 8M urea, 2 M thiourea, 4% CHAPS, 20 mM Tris-base, 30 mMdithiothreitol (DTT), and 2% Biolyte pH 3−10). The mixturewas homogenized for 30 min on ice and sonicated 20 s per 5min during this time, then centrifuged at 12 00g and 4 °C for 10min, and further centrifuged at 15 000g and 4 °C for 10 min.Three volumes of ice-cold acetone was added to the collectedsupernatants, and then the mixture was kept on ice for 30 minfor protein precipitation and desalting. Subsequently, themixture was centrifuged twice at 15 000g and 4 °C for 10min. The supernatant was discarded and the pellets wereresolved in LB; then, the mixture was homogenized for 5 minon ice and sonicated for 2 min. Protein concentration wasdetermined according to the Bradford method using BSA as thestandard and the absorption was measured at 595 nm(spectrophotometer DU800, Beckman Coulter, Los Angeles,CA).A volume of 450 μg of each sample was resuspended in LB

and then mixed with rehydration buffer [8 M urea, 2% CHAPS,0.001% bromophenol blue, 45 mM DTT, 0.2% Biolyte pH 3−10]. The mixture was loaded onto a 17 cm IPG strip(immobilized pH gradient, pH 3−10, linear, Bio-Rad).Isoelectric focusing (IEF) was performed at 18 °C accordingto manufacturer’s instructions (Protean IEF Cell, Bio-Rad).The equilibration of IPG strips and the second-dimensionelectrophoresis were carried out as previously described.28

2.4. Image Acquisition and Statistical Analysis

After 2-DE, Pro-Q Diamond (Invitrogen, Eugene, OR) wasused for phosphoprotein stain and Comassiee Blue Brilliant(CBB, G-250) was applied to detect total proteins. In brief, the2-DE gels were fixed overnight in 40% (v/v) ethanol and 10%(v/v) acetic acid and washed three times with ultra pure water(15 min per wash). To stain the phosphoproteins, the 2-DEgels were incubated in Pro-Q Diamond solution in the dark for3 h followed by destaining with three successive washes ofdestaining solution (20% acetonitrile (ACN) in 50 mM ofsodium acetate, pH 4.0, (30 min per wash). After destaining,the gels were again washed two times in deionized water for 5min per wash in order to reduce the possible corrosion onimages due to destaining solution. Three independent 2-DE gelimages from triplicate samples of the CSBV infected andhealthy larvae were scanned for phosphoprotein spots using aPharos FX plus system (Bio-Rad, Hercules, CA) at an

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excitation of 532 nm with a 610 BP 30 band-pass emissionfilter. Subsequently, the total proteins were stained with CBBG-250, and the gels were digitized using an ImageScanner (GEHealthcare, Waukesha, WI) at 16 bit and 300 dpi resolution.Gel images were imported into Progenesis SameSpots (v 4.1,

Nonlinear Dynamic, U.K.) for analysis. All gels were matchedwith one of the selected reference gel. The match analysis wasperformed in an automatic mode, and further manual editingwas performed to correct the mismatched and unmatchedspots. The expression level of a given protein spot wasexpressed in terms of the volume of the spot. The software wasused to perform gel alignment, spot averaging and normal-ization. The differences of protein spots were considered to bestatistically significant with p < 0.05 and at least 2-fold changes.The q-value which determines adjusted p-values for each testwas calculated by the Samespot software to estimate falsepositive results.

2.5. Trypsin Digestion and Protein Identification by MassSpectrometry (MS)

The differentially expressed proteins spots were manuallyexcised from the CBB-stained gels of healthy and CSBVinfected larval samples and distained for 30 min using 100 mLof ACN (50%) and 25 mM NH4HCO3 (pH 8, 50%) until thegels were transparent. The gels were dehydrated for 10 min anddried for 30 min using a Speed-Vac system (RVC 2-18, MarinChrist, Germany). Then, 10 μL of trypsin solution (finalconcentration 10 ng/μL) was pipetted on each dried proteinspot. Protein digestion and peptide extraction were doneaccording to our previously established protocol.32

The digested protein spot was analyzed by LC−MS systemequipped with a 1200 Series nanoflow HPLC (high perform-ance liquid chromatography) system (Agilent Technologies,Santa Clara, CA) interfaced with a Chip-cube (G4240A, AgilentTechnologies) to a 6520 Q-TOF (quadruple time-of-flighttime-of-flight, Agilent Technologies). Peptides were separatedby reversed phase chromatography using a microfluidic Chipcomprised of an analytical column (75 μm i.d., 150 mm lengthwith a 300 Å C18 stationary phase) and a 160 nL trap column(5 mm). All data were acquired in the positive ionization modewithin mass to charge ratio (m/z) range of 300−2000. Themass spectrometry were operated in auto MS/MS acquisitionmode and the top three most intense precursor ions wereselected for MS/MS. The peptides were loaded in 0.1% formicacid at 4 μL/min and then resolved at 500 nL/min for 15 min.Elution from the analytical column was performed by a binarysolvent mixture composed of water with 0.1% formic acid(solvent A) and ACN with 0.1% formic acid (solvent B). Thefollowing gradient program was used: from 3 to 8% B in 1 min,from 8 to 40% B in 5 min, from 40 to 85% B in 1 min and 85%B for 1 min.MS/MS Peaks were retrieved using in-house Mascot Distiller

(v. 2.3, Matrix Science, U.K.) and searched (in-house Mascot, v.2.3, Matrix Science, U.K.) against a sequence databasegenerated from protein sequences of Apis (downloaded May2011) augmented with sequences from Drosophila melanogaster(downloaded May 2011), Sacharomyces cerevisiae (downloadedMay 2011) and common repository of adventitious proteins(cRAP, from The Global Proteome Machine Organization,downloaded May 2011), totaling 72 672 entries. Searchparameters: Carbamidomethyl (C) was selected fixed mod-ification and Oxidation (M) was selected as variablemodifications. The other parameters used were the following:

Taxonomy, all entries; Enzyme, trypsin; Missed cleavages, 1;Precursor ion mass tolerance, ±50 ppm; Fragment ion masstolerance, ±0.05 Da. When the identified peptides matchedmultiple members of a protein family or a protein appearsunder the same name and accession number, the match wasconsidered in terms of a higher Mascot score, the putativefunction, and differential patterns of protein spots on 2-DE gels.Protein identification was accepted if they contained at leasttwo identified peptides having both minimal cutoff Mascotscore of 24 and probability of 95% correct match.

2.6. Label-Free LC−MS Based Proteome Quantification

Label-free LC−MS proteome profiling and quantification wasperformed with three replicate injections of each sample on theQ Exactive mass spectrometer (Thermo Fisher Scientific,Germany). A 50 μL sample of protein was subjected to 200 μLof ice-cold acetone, and then the mixture was kept on ice for 30min for protein precipitation and desalting. Subsequently, themixture was centrifuged at 15 000g and 4 °C for 5 min and theresulting supernatants were discarded, and the pellets weredried. The dried pellets were dissolved in 100 mM NH4HCO3,and the proteins were reduced with 10 mM DTT and alkylatedwith 50 mM iodoacetamide. Proteins were digested usingtrypsin at a 1:50 enzyme/protein concentration at 37 °C for 14h. After digestion, 1 μL of formic acid was added into thesolution to stop the reaction, and then dried using a Speed-Vacsystem.Reverse phase chromatography was performed using the

Thermo EASY-nLC 1000 with a binary buffer system consistingof 0.5% acetic acid (buffer A) and 80% ACN in 0.5% acetic acid(buffer B). The peptides were separated by a linear gradient ofbuffer B up to 40% in 120 min with a flow rate of 250 nL/minin the EASY-nLC 1000 system. The following gradient programwas used: from 3 to 8% B in 8 min, from 8 to 20% B in 78 min,from 20 to 30% B in 16 min, from 30 to 70% B in 5 min, from70 to 90% B in 3 min and 90% B for 10 min. The LC wascoupled to a Q Exactive mass spectrometer via the nano-electrospray source (Proxeon Biosystems, now Thermo FisherScientific). The Q Exactive was operated in the data dependentmode with survey scans acquired at a resolution of 70 000 at m/z 300. Up to the top 10 most abundant isotope patterns withcharge ≥2 from the survey scan were selected with an isolationwindow of 1.6 Th and fragmented by HCD with normalizedcollision energies of 25. The maximum ion injection times forthe survey scan and the MS/MS scans were 20 and 60 ms,respectively, and the ion target value for both scan modes wasset to 1 × 106. Repeat sequencing of peptides was kept to aminimum by dynamic exclusion of the sequenced peptides for30 s.The acquired MS data (profile mode) was processed with

Progenesis LC−MS (v.2.6 Nonlinear Dynamics, U.K.) programaccording to developer’s guidelines. The quantify-then-identifyapproach taken by Progenesis LC−MS quantified all detectedpeaks and identifications retrieved later once they exhibitedexpression alteration (>2-fold change and p < 0.02) using one-way ANOVA that the q-value was used to estimate false positiveresults in the multiple test (Progenesis LC−MS). Proteins withdifferential expression were identified on the basis of tandemMS data using in-house Mascot search engine (v. 2.3 MatrixesScience, U.K.). Searching parameters were same as the 2-DEprotein spot identification, expect a precursor ion masstolerance was ±15 ppm and fragment ion mass tolerance was±20 mmu. The decoy search was performed to estimate the

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false discovery rate (FDR). For label-free LC−MS basedprotein quantitation, Mascot results were imported intoProgenesis LC−MS. Similar proteins were grouped and onlynonconflicting features were used for quantitation.

2.7. Bioinformatics Analysis

The identified proteins were annotated by searching against theUniprot database (http://www.uniprot.org/) and Flybase(http://flybase.org/) and grouped on the basis of theirbiological process of GO terms.To enrich the identified proteins to specific GO functional

terms, the protein list was analyzed by CluoGo (v. 1.4, aCytoscape plugin) software applying to the Drosophila databasedownloaded from the GO database (release date, February 28,2012). Ontology was selected as a biological process.Enrichment analysis was done by right-side hypergeometricstatistical testing and the probability value was corrected byBonferroni method. The results are represented visually ingraphical form.For the protein−protein interaction network analysis, the

protein list of the differential expression of the healthy and

CSBV infected larvae from both 2-DE and label-free LC−MSanalysis were further analyzed by the Interologous InteractionDatabase (I2D) v1.9I2D (http://ophid.utoronto.ca/i2d),33,34

which integrated known and predicted mammalian andeukaryotic PPI data sets from D. melonogaster sources andmapped them to fly protein orthologs. PPI networks wereannotated, visualized and analyzed using NAViGaTOR v2.2.1(http://ophid.utoronto.ca/navigator/). Only protein nodeswith more than three interaction degrees were considered.

2.8. Quantitative Real-Time PCR

Total RNA was extracted from the five respective healthy andCSBV infected larvae using TRIzol regent (Takara Bio, Kyoto,Japan). Each sample was analyzed individually and processed intriplicate. Nineteen differentially expressed proteins wereexamined to detect the corresponding mRNA levels byquantitative real-time PCR, based on the sequences inhoneybee cDNA library. Gene names, accession number,forward and reverse primer sequence were listed andglyceraldehyde 3-phosphate dehydrogenase (GAPDH) wasused as the reference gene (Supporting Information Table 1).

Figure 1. 2-DE images of healthy larvae and Chinese sacbrood virus infected worker larvae of ACC. (A) The 2-DE gels stained with the ComassieeBlue Brilliant (CBB, G-250). (B) The 2-DE gels stained with the phosphoprotein-specific fluorescent dye (Pro-Q Diamond). Proteins are separatedon 17 cm IPG gel strips (pI 3−10 linear) with 450 μg of sample loading, followed by 12.5% SDS-PAGE on a vertical slab gel. Differentiallyexpression protein spots of known identity are labeled with color codes, where red indicates up-regulation and blue indicates down-regulation at eachdevelopmental stage.

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Reverse transcription was performed using a RNA PCR Kit(Takara Bio, Kyoto, Japan), according to the manufacturer’sinstructions. Real-time PCR amplification was conducted oniQ5Multicolor Real-Time PCR Detection System (Bio-Rad,Hercules, CA) as previously described.35 Gene expression datawas normalized by GAPDH. After verifying amplificationefficiency of the selected genes and GAPDH in approximatelyequal levels, the differences in gene expression were calculatedusing 2−ΔΔCt method.36 The statistical analysis of geneexpression was performed by one-way ANOVA (SPSS version16.0, SPSS, Inc.) using Duncan’s multiple-range test. An errorprobability p < 0.05 was considered statistically significant.

2.9. Western Blot

To further verify the variation tendency of differentiallyexpressed proteins identified by the proteomic approaches,heat shock protein (Hsp)60, Hsp90, actin, and tubulin wereselected for Western blot analysis by the method we describedpreviously with some modifications.35 Briefly, equal amount ofprotein sample (12 μg/lane) were separated by stacking (4%)and separating (12%) SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis) gels and then transferred toa nitrocellulose transfer membrane (0.2 μm pore size)(Invitrogen, Eugene, OR) using the iBlot apparatus (Invitrogen,Eugene, OR). After blocking, the membranes were incubatedfor 1.5 h at room temperature with primary rabbit polyclonalantibodies of anti-Hsp60, Hsp90, actin and tubulin antibodies(Abcam, Cambridge, MA) at a dilution of 1:1000. Followingthree washes, the membranes were further incubated withhorseradish peroxidase-conjugated rabbit anti-goat secondaryantibody at a dilution of 1:8000 for 1.5 h. Immunoreactiveprotein bands were detected using the ECL Western BlottingSubstrate (Pierce, Rockford, IL) and quantified by densitom-etry using Quantity-one image analysis system (Bio-Rad,Hercules, CA). GAPDH was detected simultaneously asloading control of the analysis.

3. RESULTS

3.1. 2-DE Analysis of Differential Proteome andPhosphoproteome

To investigate proteomics and phosphoproteomics alterationsbetween healthy and CSBV infected Acc worker larvae undernatural conditions, first a multifluorescent stain approach based2-DE was used to detect proteins and phosphoproteinssimultaneously. Figure 1A is a representative 2-DE gel imageof total proteins stained with CBB. Overall, 421 and 409protein spots were reproducibly detected in the healthy andCSBV infected larvae. By contrast, 137 and 129 phosphopro-tein spots were visualized by Pro-Q Diamond dye in the healthyand CSBV infected larvae, respectively (Figure 1B). In general,approximately 32% of the total proteins were modified byphosphorylation, which generally agrees with the notion thatabout 1/3 of proteins are phosphorylated in a Eukaryoticorganism.37

Quantitatively, 92 total protein spots and 32 phosphoproteinspots showed significant changes of expression (>2-fold and p <0.05). Of these, 77 total protein spots and 20 phosphoproteinspots were successfully identified (Supporting InformationTables 2 and 3). Of these, 51 and 26 total protein spots, 13 and7 phosphoprotein spots were up-regulated in the healthy andsick larvae, respectively.The remaining proteins and phosphoproteins were not

identified either because of their abundance was too low toproduce enough spectra or because the database search scorescan not yield unambiguous results (>95%). Moreover, some ofthe highly sensitive fluorescent stained proteins were present inlow abundance and under the detection limit of CBB stainduring spot excision.3.2. Label-Free LC−MS Analysis of Differential Proteome

For the purpose of a more comprehensive evaluation of larvalproteome changes by CSBV infection, a label-free LC−MSbased approach was accomplished as a verification andcomplement of the 2-DE results. The analysis of healthy andinfected larvae was achieved by three replicate injections of

Figure 2. Comparison of the nonredundant protein numbers identified by both 2-DE and label-free LC−MS analysis. A Venn diagram shows thetaxonomical distribution of 180 nonredundant proteins. The pie chart on the right represents proteins identified by label-free LC−MS basedtechnique and the pie chart on the left represents proteins identified by 2-DE technique. Bold numbers indicate the protein numbers in eachpartition.

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both trypsin digested samples on the Q Exactive LC−MS/MSsystem followed by data processing using Progenesis LC−MSsoftware. In the two-dimensional feature maps created by thesoftware, a total of 120 480 features corresponding to morethan 500 proteins (FDR < 1%, at least two unique peptides)were aligned within the retention time. Then, 28 058 features

(with p < 0.05) were exported for database searching. Thesearch result was reimported into the software, and 1518peptides (Mascot score >28 and p < 0.05) were used forprotein identification and quantification. Eventually, 152proteins were identified as being differentially expressed (foldchange >2 and p < 0.05) (Supporting Information Table 4).

Figure 3. Qualitative comparison of the up-regulated protein numbers in healthy and infected worker larvae of ACC. A total of 180 nonredundantproteins are grouped into eight categories according to their biological functions. Color codes are protein numbers identified by different analysis.

Figure 4. Categorization of the identified phosphoproteins from the healthy and infected worker larvae of Acc. Color codes represent differentprotein functional groups. The percentage of each functional group is obtained based on the number of proteins under each of the functional groupof the total number of identified phosphoproteins in Supporting Information Table 3.

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Among these, 123 and 29 proteins were found up-regulated inthe healthy and sick larvae, respectively.

3.3. Qualitative and Quantitative Comparisons ofDifferentially Expressed Proteins and Phosphoproteins

In total, of the 180 nonredundant proteins which weredifferentially expressed between two samples by both 2-DEand label-free LC−MS based techniques, 28 and 119 proteinswere exclusively identified and 33 proteins were overlapped(Figure 2). Notably, proteins up-regulated in the healthy larvaerepresented 79% (142 proteins), meaning the diseased larvaedown-regulated this number, and only 21% (38 proteins) wereup-regulated in the sick larvae. They were mainly related tocarbohydrate and energy metabolism (22), development (44),protein metabolism (43), cytoskeleton (20), protein folding

(20), lipid metabolism (21), antioxidant activities (6) and silkprotein (4) (Figure 3). In particular, the healthy larvaeoverexpressed more proteins in almost all the functionalcategories expected for lipid metabolism. More interestingly,the proteins involved in cytoskeleton, antioxidation, and silkproteins were up-regulated only in the healthy larvae. Of the 20differentially regulated phosphoproteins, those involved incytoskeleton were the most represented (9, or 45%), followedby metabolism (3, or 15%) and protein folding (3, or 15%),development (2, or 10%) and antioxidant activates (2, or 10%).Only one protein (5%) was involved in carbohydrate andenergy metabolism (Figure 4).For a better understanding of the biological significance of

protein expression levels in physiological changes after a viral

Figure 5. Quantitative comparisons of differentially expressed proteins and phosphoproteins. The ratio of the protein abundance is healthy larvae toinfected larvae. The positive value indicates higher expression in healthy larvae and negative values denote higher expression proteins in infectedlarvae. The ratio is limited to 10, and error bar is standard deviation. (A) Quantitative comparison of differentially expressed proteins identified by 2-DE. (B) Differentially expressed phosphoproteins identified by 2-DE. (C) Quantitative comparison of differentially expressed proteins identified bylabel-free LC−MS analysis.

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challenge, the expression level of each protein between the twosamples was compared. In 2-DE analyses, 13 proteins showed>10-fold higher expression of the 51 total protein spots up-regulated in the healthy larvae (Figure 5A). On the other hand,of the 26 protein spots up-regulated by infected larvae, fourproteins showed >10-fold changes (Figure 5A). As for thephosphoproteins, four of them showed stronger expression by>10-fold in healthy larvae (Figure 5B). For the label-free LC−MS proteome analyses, 37 proteins showed >10 fold up-regulation in the healthy larvae, while only five proteins werehighly expressed (>10-fold) in the infected larvae (Figure 5C).

3.4. GO (Gene Ontology) Functional Term Enrichment

To enrich the identified proteins to specific functional GOterms and elucidate the possible different biological eventsbehind the proteomic data, the two protein lists, proteins up-regulated in the healthy and CSBV infected larvae, wereseparately analyzed by ClueGo software. Accordingly, theproteins up-regulated in healthy larvae were significantlyenriched into five major functional groups, i.e., carbohydrateand energy metabolism, development, protein metabolism,cytoskeleton and protein folding (Figure 6A). The functionalleading term (with lowest statistical p value) in carbohydrateand energy metabolism was “generation of precursormetabolites and energy”. And the leading term in developmentwas “cell redox homeostasis”. In the third functional group,cytoskeleton, “cytoskeletal organization” was the leading term.The leading term in protein metabolism was “proteinpolymerization”. Finally, “’de novo’ protein folding” was theleading term in protein folding. By comparison, the proteinsup-regulated in the CSBV infected larvae were significantlyenriched in three major functional groups, i.e., small molecule

catabolic process, cellular chemical homeostasis and proteinfolding (Figure 6B). And the leading terms of these threefunctional groups were “small molecule catabolic process”,“cellular cation homeostasis”, and “response to heat”,respectively. The proteins significantly enriched in the leadingterm of each functional group were listed in SupportingInformation Table 5.

3.5. Protein−Protein Interaction (PPI) Analysis

In a living cell, proteins perform functions cooperativelythrough interactions by forming PPI networks, and the study ofthese synergies is fundamental to the understanding ofbiological events in a systemic way. Therefore, of all theidentified proteins, 89 proteins were linked in the PPI networkwith variation of interaction degree measured by the number ofedges (interaction between the corresponding proteins)adjacent to each protein node (Figure 7). Therefore, proteinmetabolism was the most abundant in the network (28, or31.5%). Of these, 18 proteins were up-regulated in the healthylarvae, and 10 proteins were up-regulated in the infected larvae.Developmental proteins were the second most represented (18,or 20.2%) in the PPI network, in which 15 proteins were up-regulated in the healthy larvae and three were up-regulated inthe sick larvae. On the other hand, of the 15 proteins (16.9%)networked in protein folding, eight and seven proteins were up-regulated in the healthy and infected larvae, respectively.Likewise, from 13 (14.6%) proteins associated with carbohy-drate and energy metabolism, 11 were up-regulated in thehealthy larvae, whereas two were up-regulated in the CSBVinfected larvae. Four (4.5%) proteins implicated with lipidmetabolism were associated with equal numbers of proteins up-regulated in the healthy and sick larvae. Particularly, all eight

Figure 6. Functional enrichment analysis of the differentially expressed proteins. Single (*) or double (**) asterisk indicate significant enriched GOterms at the p < 0.05 and p < 0.01 statistical levels, respectively. (A) Functional enrichment result of proteins up-regulated in healthy larvae; (B)functional enrichment result of proteins up-regulated in CSBV infected larvae.

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(8.9%) cytoskeletal proteins and three (3.4%) antioxidantproteins in the network were up-regulated in the healthy larvae.On the basis of the interaction degree, 89 proteins linked in thePPI network with 19 proteins had more than 100 degrees ofinteraction (Supporting Information Table 6), i.e., RPLP2, eIF-5A, Rpn11, EF-2, EF-1-alpha, PSMD4 and RPS6 in proteinmetabolism; CCT-zeta, HSC70-5, HSC70-4, HSP90 andHSP60 in protein folding; TPS in carbohydrate and energymetabolism; SUMO-3 and 14-3-3zeta in development; tubulin,ADF and actin in cytoskeleton group; SOD involved inantioxidant group.

3.6. Test of Differentially Expressed Proteins by qRT-PCR

To test the tendency of protein expression between itsencoding gene at the transcript level, 20 key node proteinswith high degrees in the PPI network from six major functionalgroups (carbohydrate and energy metabolism, development,protein metabolism, protein folding, cytoskeleton and anti-oxidation) were selected for qRT-PCR analysis. The trend ofmRNA expression showed that 17 genes, rplp2, eif-5A, rpn11,ef-2, ef-1-alpha, cct-zeta, pdi, hsc70-5, hsp90, hsp60, tps,enolase, 14-3-3zeta, tctp, adf, tubulin, and sod, were consistentwith the protein expression. However, three genes, atpb, sumo-

3, and ddx24, showed mRNA-protein expression inconsistency,which may be due to the lack of a direct relationship, orunsynchronized gene transcription and translation (Figure 8).3.7. Western Blot Analysis

Since both proteomic results and bioinformatic analysistargeted some important key node proteins which are closelyrelated to CSBV infection, i.e., up-regulation of HSPs anddown-regulation of cytoskeletal proteins in the infected larvae,Western blot analysis was conducted to verify the expression ofHsp60, Hsp90, actin and tubulin. The results revealed that theexpression of Hsp60 and Hsp90 were significantly increased inthe sick larvae. On the other hand, actin and tubulin weresignificantly down-regulated after infection (Figure 9). Theseare consistent well with the proteomic data.

4. DISCUSSIONThe viral diseases of honeybees have a detrimental influence onthe development of the beekeeping industry, and cause seriouseconomic losses worldwide both of bee-products andpollination services. So far, over 18 viruses have been reportedto trouble the honeybee.38 As one of the major honeybeespecies in Asia and China, the Acc are the most susceptible to

Figure 7. Protein−protein interaction (PPI) network. PPI was predicted using I2D and Navigator software. PPI was predicted using I2D andNavigator software.33,34 Triangles represent differentially expressed proteins connected in the network with more than three interaction degrees(Supporting Information Table 6), the regular triangles stand for proteins up-regulated in healthy larvae and the inverted triangles stand for proteinsup-regulated in infected larvae. Blue lines indicate interactions between proteins. The intensity of the interaction degree is indicated by color gradientas noted on the key bar on the down right side of the figure.

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CSBV infections that have caused a deadly loss of honeybeecolonies recently. Usually, the viral infection causes cellapoptosis, tissue damage and even functional disorder of thewhole organism and all these changes can be further reflected inproteome alteration.39 To understand the underlying molecularpathological mechanism of the CSBV infection, gel-based andlabel-free LC−MS based proteomic strategies were conductedto analyzes the proteome and phosphoproteome alterationsbetween the healthy and the CSBV infected Acc larvae in vivo.Accordingly, 180 nonredundant proteins were identified asbeing differentially expressed between the healthy and diseasedlarvae. Of these, 142 were up-regulated in the healthy larvaeand only 38 proteins were up-regulated in the sick larvae(Figure 2). Proteins up-regulated in the healthy larvae weremainly enriched in carbohydrate and energy metabolism,development, protein metabolism, cytoskeleton and foldingfunctions (Figure 6A). This is believed to coincide with the

large repertoire of protein demands of the healthy larvae tosatisfy their normal development and metabolism.40 Whereasthe normal metabolism of the honeybee larvae is terriblydisturbed by the down-regulation of the above-mentionedpathways under infectious conditions, at same time, proteinsrelated to small molecule catabolic process, cellular chemicalhomeostasis and folding activities have a stronger response tofight against the viral challengers (Figure 6B).As one of the most common post-translational modifications

(PTMs), protein phosphorylation participates in almost allaspects of cell life.41 In this study, the identified differentiallyexpressed phosphoproteins are mainly associated withcytoskeleton, protein metabolism and protein folding. Bychanging the phosphorylation status, they either play essentialroles in metabolism, differentiation, cytoskeleton arrangementand cell cycle for the normal larva growth,42,43 or modulation of

Figure 8. Test of the 20 differentially expressed proteins at mRNA level by quantitative real time PCR analysis. The black and the gray bars representfold changes (healthy to infected honeybee worker larvae) of protein and mRNA, respectively. The positive values indicate higher expression in thehealthy larvae and negative values denote higher expression proteins in the infected larvae. Error bar is standard deviation.

Figure 9. Western blot analysis of Hsp60, Hsp90, actin, tubulin. Proteins samples of the healthy and Chinese sacbrood virus infected larvae ofhoneybee worker were subjected to SDS-PAGE followed by Western blot analysis. HSP60, HSP90, actin, and tubulin were detected usingcorresponding antibodies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as reference control. (A) The Western blot images ofHsp60, Hsp90, actin, tubulin and GAPDH. (B) The relative fold change of Hsp60, Hsp90, actin, tubulin (normalized by GAPDH). The gray barsrepresent relative fold change of protein expression, positive values indicates higher expression in the healthy larvae, and negative values denotehigher expression in the infected larvae. Error bar is standard deviation.

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both viral replication21,22 and interactions between viral andhost proteins during infection.44

Our data indicates that the viral infection has triggered anessential proteome and phosphoproteome change of the larvae,which derail the larvae into a deadly developmental trajectoryby collapse of several pathways such as energy supply, proteinbiosynthesis, hormonal regulation and antioxidant defenses.This gains a new molecular-level insight into the pathologicalmechanism of the CSBV diseases.

4.1. Viral Infection Disrupting Carbohydrate Metabolismand Energy Production

As a necessary nutrient substance, carbohydrates are paramountimportant energy fuels for the fast growth of honeybee larvae.Young worker larvae less than three days old are fed solely withroyal jelly, which is a protein-rich glandular secretion with thesugar content (fructose and sucrose) of only 18%. Duringdevelopment, the old larvae are fed with a blend of honey androyal jelly based diet in which the sugar content increases up to45% in the last two days before pupation.45 This programmedfood change is the biological demand of the fast growing olderlarvae to satisfy their astounding energy requirement from thecarbohydrates. However, the target of CSBV is the goblet cellsof the midgut epithelium, and the virus multiplies in thecytoplasm of goblet cells as a prelude to systemic infection.46

Hence, the viral infection may affect nutritional assimilation inthe midgut and influence the physiological metabolism oflarvae. In this study, 18 proteins (8 with >10-fold decrease)related to carbohydrate and energy metabolism were down-regulated in the CSBV infected larvae. For instance, 6-phosphofructokinase is involved in glycolytic pathways; ATP-citrate synthase and NADP-dependent malic enzyme (NADP-ME) are important components in the TCA cycle, and NADP-ME also implicated in the glyoxylate cycle;47 ATP synthasecatalyzes the synthesis of ATP that provides energy for thecell.48 All these enzymes coordinate in the network ofcatalyzing energy generation to gratify the demand for thelarval development under normal physiological conditions. Onthe contrary, the viral infection probably severely hinderedcarbohydrate uptake in the sick larvae, and the carbohydratemetabolism enzymes mentioned above were down-regulated asa consequence. In addition, some other carbohydratemetabolism enzymes were still down-regulated in the infectedlarvae. For example, glucosamine-6-phosphate deaminase,catalyzing the formation of glucosamine-6-phosphate depend-ent upon fructose-6-phosphate,49 is an active precursor of chitinin arthropods. It plays a crucial role in construction of insectcuticle and peritrophic matrix that serves as a physical barrier topathogens in the midgut.50 Trehalose-phosphate synthase isresponsible for trehalose synthesis, and trehalose has beenshown to enhance anoxia tolerance of fruit fly larvae.51 Argininekinase, a crucial enzyme in promoting the efficient synthesis ofATP,52 could fulfill the long periods of energy demand in themidgut.53 Together, the down-regulation of these enzymes isthought to weaken the larvae’s defense capability to the viralchallenge and facilitate further infection or cut down energysupply of the larvae, thus further damaging the regulardevelopment of the larvae.However, some enzymes related to metabolism of carbohy-

drates such as enolase and aldolase were up-regulated in theinfected larvae. Enolase is a crucial enzyme in the glycolyticpathway catalyzing the dehydration of 2-phosphoglycerate tophosphoenolpyruvate.54,55 Eukaryotic enolases, however, are

involved in a variety of cell functions besides their glycolyticactivities, and play significant roles in disease processes andmany others,56 such as growth control,57 cytoskeletal binding,58

temperature and salt stress tolerance.59 Aldolase, a glycolysisenzyme, has an abundant expression in the hemolymph ofDrosophila larvae following both fungal exposure and bacterialinfection60 as well as white spot syndrome virus (WSSV)invasion.61 Therefore, the escalated expression of aldolase andenolase might suggest their roles in modulation of the stressresponse by virus infection as heat shock proteins do.62

Meanwhile, since an insect virus could jeopardize oxygendelivery, the up-regulation of these enzymes is possiblyresponsible for enhancing anaerobic metabolism, and providinga special pathway for the larvae to get energy underpathological conditions.63

4.2. Viral Infection Hampering Larval Development

During the larval−pupal metamorphosis of holometabolic(completely metamorphosing) insects, the larvae go throughremarkable physiological changes to prepare themselves forpupation and metamorphosis, and most of the organs andtissues are remolded.64 Accordingly, the proteins related todevelopment play vital roles in this moulting process. At theprepupal stage, although the larvae are still in their old form,radical changes are already in progress. For example, the cuticleof the young pupa has already formed, which is enveloped inthe larval cuticle.64 However, the virus infection could inhibitpupation to facilitate its replication and transmission.65 To thisend, the down-regulation of seven cuticle proteins in the sicklarvae with four decreased >10-fold is believed to be the majorreason that the CSBV infected larvae failed to pupate. Alongwith the formation of pupate cuticle, a part of the larval musclesshould be disintegrated and then remodeled with the imaginalmyoblasts,64 and this process is also disturbed by infection. Inline with this, some proteins implicated in regulating musclecell proliferation and muscle contraction had decreasedexpression, including myophilin, muscle-specific protein 20,calreticulin and calumenin.66−68 Larval ontogenesis and cellproliferation heavily rely on the mitosis; lamin Dm0, nuclearmigration protein NudC and small ubiquitin-related modifierhave been documented to have direct mitogenic activity.69−71

The decreased expression of these proteins is supposed tohamper the larval development in this regard. Also, IDGF4 is agrowth factor that is produced by the fat body and transportedthrough the insects’ hemolymph. Its down-regulation in theinfected larvae suggests the weakened roles in stimulatingproliferation and polarization of imaginal disc cells.72 The 14-3-3 protein family contributes to a wide variety of importantsignal transduction pathways that control cell cycle, apoptosis,and programmed gene expression.73 Its down-regulation in thesick larvae suggests the modulation roles in larval to pupalmetamorphosis are suppressed. Notably, the phosphorylationlevel of 14-3-3 zeta was higher in the sick larvae. Thephosphorylation in specific residues of 14-3-3 proteins hasimportant regulatory roles,74 and cellular stress induced 14-3-3zeta phosphorylation makes cells more susceptible to apoptoticsignals.75 Hence, increased expression of this phosphorylatedprotein may directly catalyze the viral infection.On the other hand, three development related proteins were

up-regulated in the CSBV infected larvae, i.e., transitionalendoplasmic reticulum ATPase (TER94), translationallycontrolled tumor protein (TCTP) and cyclin-dependent kinase6 (CDK6). TER94 (p97 or VCP in mammals), an ATPase

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associated with various cellular activities, is required forexporting misfolded proteins from the endoplasmic reticulum(ER) into the cytosolic and the prteasome for degradation.76,77

It is proposed to be highly modulated by phosphorylation thatfacilitates its function of transporting and releasing ubiquiti-nated proteins in ubiquitin/proteasome-dependent proteolyticpathways78 and determining its role in the repairing of DNAdamage.79 TCTP, an evolutionarily conserved multifunctionalprotein, is implicated in the protection of cells against variousstress conditions and apoptosis.80 In addition, it could protectthe cell from death during the viral infection.81 CDK6 is animportant cell-cycle regulator in the progression of a cellthrough G1 phase and the G1/S transition. And many virusesarrest the host cell division cycle to favor their own growth.82,83

Consequently, the stronger expressions of these proteinscollectively suggest the virus infected larvae recruit them as aremedy for crisis.

4.3. Viral Infection Leading to Protein Degradation

The development of a multicellular organism has a stake in theprotein anabolism and catabolism.84 The developing larvaerequire continuous protein synthesis, especially duringmetamorphosis; the viral invasion, however, interrupts regularprotein metabolism.85 Ribosome is the organelle in charge ofprotein biosynthesis and many other translational factorsfunction in this process.86 Consequently, the down-regulationof nine ribosomal proteins and six eukaryotic translationinitiation factors in the sick larvae is likely projecting that thedestroyed protein biosynthesis system cannot guarantee thenormal growth of larvae. Specifically, as a source of amino acidsfor tissue reconstruction during pupal development, hexamerinsare first synthesized by a larval fat body and released into thehemolymph where they accumulate to extraordinarily highconcentrations. Then, before the initiation of metamorphosis,hexamerins are selectively sequestered by fat body cells viareceptor-mediated endocytosis, and used to build adultstructures.87 Nevertheless, the high level of hexamerins in theCSBV infected larvae until the prepupal stage suggests that thevirus infection blocked the utilization of hexamerins. This isconsistent with a higher stock of hexamerin 110 presented atstarved larvae than nonstarved larvae, and the starved larvaecannot reach the cocoon-spinning stage.88

On the other hand, following the infection, the expression ofsome proteins involved in protein degradation was increased.As we know, the proteasome is a multisubunit enzyme complexthat plays a central role in eliminating of ubiquitinated proteinsin the cell.89 The ubiquitin−proteasome system mediated viralprotein degradation constitutes a host defense process againstsome RNA viral infections.90 Moreover, proteasome activityand assembly are regulated by cAMP-dependent protein kinaseA induced phosphorylation.91,92 Thereby, the up-regulation oftwo proteasome subunits (β2 and β7) and two phosphorylatedsubunits (α1 and α3) suggests the participation of proteasomesin the protein degradation that occurs during the viral infection.

4.4. Viral Infection Collapsing Cytoskeletal Structure

The cytoskeleton generates cell mitosis, powers the movementsof motile cells and provides mechanical support for the cell.93

The actin assembles into long, fiber-like filaments, which play adominant role in many cellular functions including cytokinesis,phagocytosis and muscle contraction.94 In response tointracellular and extracellular signals that stimulate cell divisionand differentiation, the actin protein is dynamically remodeled,and this reorganization is regulated by actin-binding proteins.95

The cofilin/actin-depolymerizing factor and myosin aremembers of the actin-binding protein family. Moreover,tubulin, a basic subunit of the microtubule, can providestructural support of cells and functions as a motional path oforganelles.96 However, the functions of the cytoskeleton areseriously disturbed once the virus invades such that severedisruption of microtubule organization and centrosomefunction occurs.97 The destruction of cytoskeletal filaments ormicrotubules could further facilitate both virus replication98 andrelease of virus particles.99 Furthermore, the CSBV is thoughtto likely cause cell death by induction of apoptosis like otherpicornaviruses family members such as rhinovirus, poliovirusand foot-and-mouth disease virus,100,101 and apoptosis is alwaysaccompanied by a reduction in actin content and thedissolution of the cellular cytoskeleton.102,103 In addition, thefunction of tropomyosin in nonmuscle cells is to modulate theinteraction between actin and actin-binding proteins to stabilizethe stress fibers and protect them from disassembly.104

Therefore, reduction of tropomyosin may also lead to thedisruption of both cytoskeletal and extracellular architecture.105

This is likely the reason that the sick larvae eventually take asack like shape.Certainly, the up-regulation of these cytoskeletal proteins in

the healthy larvae is believed to be important to accomplishtheir roles in supporting dramatic cell and tissue rebuilding atthe period before pupation. In addition, the larger number andstronger expression of phosphorylated cytoskeletal proteinsseems to be enhancing the metamorphosis processes. Forinstance, phosphoryaltion of myosin regulatory light chain(MRLC) controls its accumulation during cytokinesis.106 Also,phosphorylation regulates the function of cofilin/actin-depolymerizing factor (ADF) that influences actin cytoskeletaldynamics.107 So, the increased phosphorylation level of theseproteins indicates the significance of phosphorylation in theregulation of larval development in cellular processes such assignal transduction and cell differentiation.

4.5. Heat Shock Protein Plays Defensive Roles for SickLarvae

Correct protein folding is essential for the protein to functionproperly, and many proteins called molecular chaperonesparticipate in this process. The heat shock protein (Hsp) familyis a highly conserved group of molecular chaperones existing inall organisms.108 In this study, a number of Hsps, such asHsp90, Hsc70-3, Hsc70-4, Hsc70-5, Hsp60, and Hsp10, weresignificantly up-regulated (Hsp90 and Hsp60 > 10-fold change)in honeybee larvae challenged by the CSBV. In general, there isa close relationship between Hsps and viral infection. Severalviruses, such as dengue virus,109 newcastle disease virus,110

simian virus 40 and polyoma virus111 could induce theexpression of Hsps to protect infected cells against proteotoxicstresses by assisting protein correct fording, or by guidingdamaged proteins to the proteasome for destruction.112

Therefore, the up-regualtion of Hsps suggests that the CSBVinfected larvae have employed these stress proteins in responseto the viral invasion as in the case of heat stress and bacterialinfections in the honeybees.113−115 However, through inter-action with viral proteins, Hsc70 and Hsp90 participate in thefacilitation of viral production in host cells during the infectioncycle.116,117 In insect cells, for example, Hsp90 plays asupportive role in both flock house virus and baculovirusRNA replication.116,118 Interestingly, Hsp90 and Hsp60 werephosphorylated and their expression escalated in the sick larvae.

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There is evidence that phosphorylated Hsp90 plays an activerole in reovirus infection.119 Likewise, the phosphorylatedHsp60 in the cell plasma membrane functions as a molecularchaperone that is appropriately regulated by phosphoryla-tion.120,121 Consequently, we suggest that the increasedexpression and phosphorylation levels of Hsps after theCSBV challenge probably helps the larvae in suppressing thestress caused by infection, but also seems to support theproductive replication cycle of CSBV.In contrast, as might be expected, the healthy larvae require

the up-regulation of some of other molecular chaperones tofacilitate normal tissue development as in the developing queenand worker larvae and embryos under normal physiologicalconditions.40,122 Chaperonin containing TCP-1 (CCT), anindispensable chaperone in the eukaryote, is required in foldingof an essential subset of cytosolic proteins, localizes at sites ofmicrofilament assembly, and functions in the facilitated foldingof actins and tubulins.123 It is unable to fold sufficientcytoskeletal proteins without the assistance of other molecularchaperones, such as prefoldin (PFD). PFD plays a major role inde novo protein folding, and its absence functionally impairsthe chaperonin pathway.124 In this study, three subunits ofprefoldin (PFD2, PFD4 and PFD6) and four of eight subunitsof CCT (beta, epsilon, zeta and eta) were increased in thehealthy larvae. This suggests a key role of molecular chaperonesin protecting accurate protein folding to guarantee normalgrowth of the cell and healthy larval development.

4.6. Viral Infection Causes Juvenile Hormone Disorder ThatBlocks Pupation

Lipid metabolism provides energy needed during extendednonfeeding periods that is essential for the larval development,particularly through metamorphosis.125 To this end, the up-regulation of proteins involved in lipid metabolism in bothlarval samples implies the critical necessity of energy supply forthe larvae. Intriguingly, two important enzymes involved in thisfunctional category, juvenile hormone esterases (JHE) andjuvenile hormone epoxide hydrolases (JHEH), are directlyresponsible for juvenile hormone (JH) degradation.126 In thelife cycle of the eusocial insect, larval−pupal metamorphosis isprecisely regulated by the cooperation of JH and ecdysone.127

During the larval development, the ecdysone initiates onlylarva-to-larva molts with enough JH present in the hemolymph,while pupation is triggered by ecdysone with lower amounts ofJH. The JH titer of the honeybee drops to a minimal level bothat the end of the larval feeding phase and just beforepupation.128 Nevertheless, the opposite relationship betweenJHE expression levels and JH titer is to degrade JH andpromote larval−pupal metamorphosis of honeybees.129 Thehigh level of JHE (>10-fold) in the healthy larvae seems toassist in clearance of the JH and preparing old larvae formetamorphosis. However, the low abundance of JHE andJHEH in the diseased larvae suggests that they are not efficientin degrading the JH, thus, hindering the regular pupation oflarvae. Therefore, these two enzymes are likely to bebiomarkers for CSBV diagnosis. In addition, because the larvaeshould begin to spin cocoons before pupation, the down-regulation of four silk proteins in the sick larvae is proof thatpupation was blocked.

4.7. Viral Infection Disables the Larval Normal OxidativeSystem

The antioxidant system is central for the reduction of reactiveoxygen species (ROS)-induced oxidative damage. ROS are

generated as harm products of normal metabolism, and thehigh oxygen demand increases its accumulation.130 In thehoneybee, it is reported that the antioxidant proteins areexpressed in the development of the hypopharyngeal gland andlarvae to protect cellular components from oxidativedamages.35,40 On the other hand, the stronger expression ofantioxidative proteins at the earlier stage of infection is anadaptive strategy for the survival of cells.131,132 However, thefunctionality of this system is weakened at the late stage ofdisease.131,133 Thus, the down-regulation of antioxidantenzymes in the sick larvae, such as superoxide dismutase(SOD), glutathione S-transferase (GST), phospholipid hydro-peroxide glutathione peroxidase isoform 1 (PHGPx1),thioredoxin peroxidase (TPx) and peroxiredoxin, implies theincreased levels of ROS caused by infection overwhelmed thecells’ antioxidant defenses. Moreover, the up-regulation ofphosphorylated PHGPx1 and SOD in the healthy larvaesuggests the enhanced protective capacity against oxidativestress during the larval metamorphosis.134,135

The proteins which participate in the life activities of thecomplex organism are involved in a variety of interactionsrather than acting as separate entities. Proteins that are linkedtogether in the context of networks through PPI, modificationsand regulation of expressional relationships indicate theirbiological centrality. To recognize this fact, 89 key nodeproteins are supposed to play crucial roles in the CSBVinfection, of which 65 proteins were down-regulated and 24 up-regulated in the infected larvae. They were mainly related toprotein metabolism, development, carbohydrate and energymetabolism and protein folding and cytoskeleton, which werethe most networked groups. Coinciding with GO functionalenrichment analysis, these five groups are considered to be themajor pathways that are significantly influenced by viralinfections of honeybee larvae. In addition, the tested resultsboth at the protein and gene level provide importantinformation of target hub proteins that can be potentiallyused for early diagnosis and medicinal development or selectionof viral-resistant bees through gene manipulation.

5. CONCLUSION

The CSBV infection has triggered significant proteome andphosphoproteome variations of Acc larvae. There are 180differentially expressed proteins and 19 phosphoproteinsidentified under CSBV infection in vivo using gel-based andlabel-free LC−MS based proteomic approaches. This comple-mentary platform has offered an efficient avenue for molecular-level mechanistic understanding of the honeybee pathologythrough proteome analysis. Our results demonstrate that theCSBV infection disrupted normal larvae development andhampered larval−pupal metamorphosis by disturbing severalkey metabolism pathways such as carbohydrate metabolism,protein synthesis, cytoskeletal structure and hormone regu-lation. On the other hand, the sick larvae have recruited somestrategies to defend themselves using Hsps and antioxidantsystem. This comprehensive proteomics and phosphoproteo-mics analysis provides a new understanding of the pathologicalmechanism of CSBV disease. Some important node proteinshave been identified and protein expressions were validated atthe gene level, which have potential use as candidate targets ofRNAi-based gene therapy, as well as breeding and prevention ofthis fatal viral disease through marker assisted selection.

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■ ASSOCIATED CONTENT

*S Supporting Information

Pictures of healthy and Chinese sacbrood virus infectedhoneybee (Acc) worker larva, identification of differentiallyexpressed proteins and phosphoproteins in healthy andChinese sacbrood virus infected honeybee (Acc) worker larvaeby 2-DE analysis, identification of differentially expressedproteins in healthy and Chinese sacbrood virus infectedhoneybee (Acc) worker larvae by label-free LC−MS analysis,protein degrees in protein−protein interaction (PPI) network,primer sequences used for quantitative real-time PCR analysisof the differentially expressed proteins, and information of eachprotein and peptide sequence identified. This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86 10 6259 1449. E-mail: apislijk@126.com.

Author Contributions§These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

We thank Katrina Klett from University of Minnesota, USA, forher help with the language of the manuscript. This work issupported by the earmarked fund for Modern Agro-industryTechnology Research System (CARS-45), The NationalNatural Science Foundation of China (No. 30972148) andkey projects of the national scientific supporting plan of the12th Five-Year Development (2011-2015) (2011BAD33B04).

■ ABBREVIATIONS

CSBV, Chinese sacbrood virus; 2-DE, two-dimensional gelelectrophoresis; SBV, sacbrood virus; ELISA, enzyme-linkedimmunosorbent assay; RT-PCR, reverse transcription-polymer-ase chain reaction; LC−MS, liquid chromatography−massspectrometry; FDR, false discovery rate; PPI, protein−proteininteraction; HSPs, heat shock proteins; PTMs, post-transla-tional modifications.

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Journal of Proteome Research Article

dx.doi.org/10.1021/pr301226d | J. Proteome Res. 2013, 12, 1881−18971897