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An Integrated Proteomics Reveals Pathological Mechanism of Honeybee (Apis cerena) Sacbrood Disease Bin 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 of Agricultural Science, Beijing, China S Supporting Information *

ABSTRACT: Viral diseases of honeybees are a major challenge for the global beekeeping industry. Chinese indigenous honeybee (Apis cerana cerana, Acc) is one of the major Asian honeybee species and has a dominant population with more than 3 million colonies. However, Acc is frequently threatened by a viral disease caused by Chinese sacbrood virus (CSBV), which leads to fatal infections and eventually loss of the entire colony. Nevertheless, knowledge on the pathological mechanism of this deadly disease is still unknown. Here, an integrated gel-based and label-free liquid chromatography−mass spectrometry (LC−MS) based proteomic strategy was employed to unravel the molecular event that triggers this disease, by analysis of proteomics and phosphoproteomics alterations between healthy and CSBV infected worker larvae. There were 180 proteins and 19 phosphoproteins which altered their expressions after the viral infection, of which 142 proteins and 12 phosphoproteins were down-regulated in the sick larvae, while only 38 proteins and 7 phosphoproteins were up-regulated. The infected worker larvae were significantly affected by the pathways of carbohydrate and energy metabolism, development, protein metabolism, 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 the first time, comprehensively decipher the molecular underpinnings of the viral infection of the Acc and are potentially helpful for sacbrood 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 upon honeybee pollination services.1 The pollinator decline will have severe consequences for food security.2 China has over 8.7 million honeybee colonies, of which the Chinese indigenous honeybee (Apis cerana cerana, Acc) represents over 3 million.3 The Acc has a long apicultural history and has stronger biological 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 of plants and crops, as well as the maintenance of the vegetation cover and biodiversity.6,7 In addition, the Acc is a major honey producer and its honey is a popular bee-product in the Asian market. Unfortunately, Acc has been stricken by a fatal viral disease caused by Chinese sacbrood virus (CSBV), which results in severe and deadly infections of the colony and eventually losses of the entire colony.8 This has become a huge challenge for the Chinese and even for the Southeast Asian beekeeping industry. The CSBV is a small RNA virus (picorna-like virus) that has an icosahedral virion with a diameter of 26−30 nm, and its genome consists of a single positive-strand RNA molecule with 8.8 kb.9 Although the CSBV shows a close genetic relationship to its western counterpart, sacbrood virus (SBV), no cross infection has been reported yet.10 The CSBV primarily affects © 2013 American Chemical Society

honey bee worker larvae aged 1−3 days, but the apparent symptoms are exhibited at their prepupal stage. Once the colonies get infection, the nurse bees can distinguish and uncap the sealed sick larvae in the comb cells.11 The most obvious symptom of the CSBV infection is that diseased larvae fail to pupate and further take a sac-like appearance because the ecdysial fluid is accumulated between the prepupal and pupal skins.12 In addition, the color of the infected larvae changes from 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 also reduce 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 methods such as electron microscopy,16 enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR).17 Recently, attempts have been made to use RNA interference to treat this disease.18 However, there is still a scarcity of information regarding the pathological changes of this fatal honeybee disease at the molecular level. Similar to all other insects, the honeybees lack a classically adaptive immune system as in the case of mammals. To survive, they have evolved cellular and humoral immune responses Received: December 31, 2012 Published: February 19, 2013 1881

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

Journal of Proteome Research

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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 five honeybee colonies. Briefly, to ensure the exactly the same age of the larvae to be sampled, the egg laying queen bee was confined to a single wax comb frame containing worker cells for 5 h with a cage made of a queen excluder, through which workers but not the queen could pass. Subsequently, the queen was removed, and the fertilized eggs contained in the frame were maintained in the honeybee colony for further development. At the experimental time point, each 100 larvae of the CSBV infected and healthy larvae were sampled and frozen at −80 °C until use. Each 100 larvae were pooled as one biological replicate, and three independent biological replicates were produced.

constituted of the defense mechanisms to cope with microbial infections.19 Therefore, the in vitro reared honeybee larvae could respond with a prominent humoral reaction to aseptic injury, and Gram-negative bacteria challenged bee larvae can increase synthesis of antimicrobial peptides.20 Two-dimensional gel electrophoresis (2-DE) based proteomics technology permits quantitative and multicolor fluorescence detection of phosphoproteins and total proteins within a single gel electrophoresis experiment. This has been widely used in unraveling the pathological mechanism in human diseases.21 Meanwhile, the label-free LC−MS based proteomics strategy is able to precisely and reproducibly quantify protein expression directly without using any labeling, which has been broadly applied to the proteomic profiling, biomarker discovery and disease diagnostics.22−24 However, despite rapid development in proteomics technologies and their application in honeybee developmental biology,25−27 no such study has been conducted on protein expression and phosphorylation changes associated with the pathological mechanism in the honeybee disease. Therefore, the current study employed gel-based (2DE) and shotgun proteomic (label-free LC−MS based) strategies, which have complementary natures, to gain an indepth understanding of the pathological mechanism of the fatal viral disease of the Acc by comparison of the proteome-wide change of the healthy and sick worker larvae. This may be potentially helpful for early diagnosis and development of a more specific target medicine to treat this disease and facilitate the investigation of sacbrood disease at cellular level with the availability of immortalize honey bee cell lines.28

2.3. Protein Extraction and 2-DE

Larval protein extractions were carried out according to our previously described method with some modifications.31 Briefly, the larvae were homogenized with lysis buffer (LB, 8 M urea, 2 M thiourea, 4% CHAPS, 20 mM Tris-base, 30 mM dithiothreitol (DTT), and 2% Biolyte pH 3−10). The mixture was homogenized for 30 min on ice and sonicated 20 s per 5 min during this time, then centrifuged at 12 00g and 4 °C for 10 min, and further centrifuged at 15 000g and 4 °C for 10 min. Three volumes of ice-cold acetone was added to the collected supernatants, and then the mixture was kept on ice for 30 min for protein precipitation and desalting. Subsequently, the mixture was centrifuged twice at 15 000g and 4 °C for 10 min. The supernatant was discarded and the pellets were resolved in LB; then, the mixture was homogenized for 5 min on ice and sonicated for 2 min. Protein concentration was determined according to the Bradford method using BSA as the standard 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 according to manufacturer’s instructions (Protean IEF Cell, Bio-Rad). The equilibration of IPG strips and the second-dimension electrophoresis were carried out as previously described.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 pH gradient (IPG) strips that were from Bio-Rad (Hercules, CA). Modified sequencing grade trypsin was from Roche (Mississauga, ON, Canada). Chemicals used but not specified here are noted with their sources in the text. All reagents used were analytical grade or better. 2.2. Biological Samples

The honeybee larvae (Acc) infected by Chinese sacbrood virus with typical symptom (A shot brood pattern in the comb, the sick larvae found in the uncapped cells with raised mouth parts. When taking the infected larva out of cell it presents a sac-like appearance) (Supporting Information Figure 1) were collected from five bee colonies maintained in the apiary of the Institute of Apicultural Research, Chinese Academy of Agricultural Sciences in Beijing. The infection of the CSBV virus was confirmed by morphological observation under electronic microscopy and RT-PCR test according to Chen et al.29 and Yan et al.30 For purification of the virus, CSBV-infected larvae were homogenized in 5 mL of NT buffer (100 mM NaCl, 10 mM Tris, pH 7.4) and the mixture was centrifuged at 1000g for 10 min. The supernatant was extracted with an equal volume of 1,1,2-trichlorotrifluoroethane before the aqueous phase was layered 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 CsCl interface was harvested. The purified virus was added into sugar syrup, which was made of 1 part sugar to 1 part water by volume, and then the sugar syrup was fed to five honeybee

2.4. Image Acquisition and Statistical Analysis

After 2-DE, Pro-Q Diamond (Invitrogen, Eugene, OR) was used for phosphoprotein stain and Comassiee Blue Brilliant (CBB, G-250) was applied to detect total proteins. In brief, the 2-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-DE gels were incubated in Pro-Q Diamond solution in the dark for 3 h followed by destaining with three successive washes of destaining solution (20% acetonitrile (ACN) in 50 mM of sodium acetate, pH 4.0, (30 min per wash). After destaining, the gels were again washed two times in deionized water for 5 min per wash in order to reduce the possible corrosion on images due to destaining solution. Three independent 2-DE gel images from triplicate samples of the CSBV infected and healthy larvae were scanned for phosphoprotein spots using a Pharos FX plus system (Bio-Rad, Hercules, CA) at an 1882



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excitation of 532 nm with a 610 BP 30 band-pass emission filter. Subsequently, the total proteins were stained with CBB G-250, and the gels were digitized using an ImageScanner (GE Healthcare, 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 matched with one of the selected reference gel. The match analysis was performed in an automatic mode, and further manual editing was performed to correct the mismatched and unmatched spots. The expression level of a given protein spot was expressed in terms of the volume of the spot. The software was used to perform gel alignment, spot averaging and normalization. The differences of protein spots were considered to be statistically significant with p < 0.05 and at least 2-fold changes. The q-value which determines adjusted p-values for each test was calculated by the Samespot software to estimate false positive results.

Taxonomy, all entries; Enzyme, trypsin; Missed cleavages, 1; Precursor ion mass tolerance, ±50 ppm; Fragment ion mass tolerance, ±0.05 Da. When the identified peptides matched multiple members of a protein family or a protein appears under the same name and accession number, the match was considered in terms of a higher Mascot score, the putative function, and differential patterns of protein spots on 2-DE gels. Protein identification was accepted if they contained at least two identified peptides having both minimal cutoff Mascot score 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 was performed with three replicate injections of each sample on the Q Exactive mass spectrometer (Thermo Fisher Scientific, Germany). A 50 μL sample of protein was subjected to 200 μL of ice-cold acetone, and then the mixture was kept on ice for 30 min for protein precipitation and desalting. Subsequently, the mixture was centrifuged at 15 000g and 4 °C for 5 min and the resulting supernatants were discarded, and the pellets were dried. The dried pellets were dissolved in 100 mM NH4HCO3, and the proteins were reduced with 10 mM DTT and alkylated with 50 mM iodoacetamide. Proteins were digested using trypsin at a 1:50 enzyme/protein concentration at 37 °C for 14 h. After digestion, 1 μL of formic acid was added into the solution to stop the reaction, and then dried using a Speed-Vac system. Reverse phase chromatography was performed using the Thermo EASY-nLC 1000 with a binary buffer system consisting of 0.5% acetic acid (buffer A) and 80% ACN in 0.5% acetic acid (buffer B). The peptides were separated by a linear gradient of buffer B up to 40% in 120 min with a flow rate of 250 nL/min in the EASY-nLC 1000 system. The following gradient program was 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, from 70 to 90% B in 3 min and 90% B for 10 min. The LC was coupled to a Q Exactive mass spectrometer via the nanoelectrospray source (Proxeon Biosystems, now Thermo Fisher Scientific). The Q Exactive was operated in the data dependent mode with survey scans acquired at a resolution of 70 000 at m/ z 300. Up to the top 10 most abundant isotope patterns with charge ≥2 from the survey scan were selected with an isolation window of 1.6 Th and fragmented by HCD with normalized collision energies of 25. The maximum ion injection times for the survey scan and the MS/MS scans were 20 and 60 ms, respectively, and the ion target value for both scan modes was set to 1 × 106. Repeat sequencing of peptides was kept to a minimum by dynamic exclusion of the sequenced peptides for 30 s. The acquired MS data (profile mode) was processed with Progenesis LC−MS (v.2.6 Nonlinear Dynamics, U.K.) program according to developer’s guidelines. The quantify-then-identify approach taken by Progenesis LC−MS quantified all detected peaks and identifications retrieved later once they exhibited expression alteration (>2-fold change and p < 0.02) using oneway ANOVA that the q-value was used to estimate false positive results in the multiple test (Progenesis LC−MS). Proteins with differential expression were identified on the basis of tandem MS data using in-house Mascot search engine (v. 2.3 Matrixes Science, U.K.). Searching parameters were same as the 2-DE protein spot identification, expect a precursor ion mass tolerance was ±15 ppm and fragment ion mass tolerance was ±20 mmu. The decoy search was performed to estimate the

2.5. Trypsin Digestion and Protein Identification by Mass Spectrometry (MS)

The differentially expressed proteins spots were manually excised from the CBB-stained gels of healthy and CSBV infected larval samples and distained for 30 min using 100 mL of ACN (50%) and 25 mM NH4HCO3 (pH 8, 50%) until the gels were transparent. The gels were dehydrated for 10 min and dried for 30 min using a Speed-Vac system (RVC 2-18, Marin Christ, Germany). Then, 10 μL of trypsin solution (final concentration 10 ng/μL) was pipetted on each dried protein spot. Protein digestion and peptide extraction were done according to our previously established protocol.32 The digested protein spot was analyzed by LC−MS system equipped with a 1200 Series nanoflow HPLC (high performance liquid chromatography) system (Agilent Technologies, Santa Clara, CA) interfaced with a Chip-cube (G4240A, Agilent Technologies) to a 6520 Q-TOF (quadruple time-of-flight time-of-flight, Agilent Technologies). Peptides were separated by reversed phase chromatography using a microfluidic Chip comprised of an analytical column (75 μm i.d., 150 mm length with a 300 Å C18 stationary phase) and a 160 nL trap column (5 mm). All data were acquired in the positive ionization mode within mass to charge ratio (m/z) range of 300−2000. The mass spectrometry were operated in auto MS/MS acquisition mode and the top three most intense precursor ions were selected for MS/MS. The peptides were loaded in 0.1% formic acid at 4 μL/min and then resolved at 500 nL/min for 15 min. Elution from the analytical column was performed by a binary solvent mixture composed of water with 0.1% formic acid (solvent A) and ACN with 0.1% formic acid (solvent B). The following 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 database generated from protein sequences of Apis (downloaded May 2011) augmented with sequences from Drosophila melanogaster (downloaded May 2011), Sacharomyces cerevisiae (downloaded May 2011) and common repository of adventitious proteins (cRAP, from The Global Proteome Machine Organization, downloaded May 2011), totaling 72 672 entries. Search parameters: Carbamidomethyl (C) was selected fixed modification and Oxidation (M) was selected as variable modifications. The other parameters used were the following: 1883



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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 Comassiee Blue Brilliant (CBB, G-250). (B) The 2-DE gels stained with the phosphoprotein-specific fluorescent dye (Pro-Q Diamond). Proteins are separated on 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. Differentially expression protein spots of known identity are labeled with color codes, where red indicates up-regulation and blue indicates down-regulation at each developmental stage.

false discovery rate (FDR). For label-free LC−MS based protein quantitation, Mascot results were imported into Progenesis LC−MS. Similar proteins were grouped and only nonconflicting features were used for quantitation.

CSBV infected larvae from both 2-DE and label-free LC−MS analysis were further analyzed by the Interologous Interaction Database (I2D) v1.9I2D (http://ophid.utoronto.ca/i2d),33,34 which integrated known and predicted mammalian and eukaryotic PPI data sets from D. melonogaster sources and mapped them to fly protein orthologs. PPI networks were annotated, visualized and analyzed using NAViGaTOR v2.2.1 (http://ophid.utoronto.ca/navigator/). Only protein nodes with more than three interaction degrees were considered.

2.7. Bioinformatics Analysis

The identified proteins were annotated by searching against the Uniprot database (http://www.uniprot.org/) and Flybase (http://flybase.org/) and grouped on the basis of their biological process of GO terms. To enrich the identified proteins to specific GO functional terms, the protein list was analyzed by CluoGo (v. 1.4, a Cytoscape plugin) software applying to the Drosophila database downloaded from the GO database (release date, February 28, 2012). Ontology was selected as a biological process. Enrichment analysis was done by right-side hypergeometric statistical testing and the probability value was corrected by Bonferroni method. The results are represented visually in graphical form. For the protein−protein interaction network analysis, the protein list of the differential expression of the healthy and

2.8. Quantitative Real-Time PCR

Total RNA was extracted from the five respective healthy and CSBV infected larvae using TRIzol regent (Takara Bio, Kyoto, Japan). Each sample was analyzed individually and processed in triplicate. Nineteen differentially expressed proteins were examined to detect the corresponding mRNA levels by quantitative real-time PCR, based on the sequences in honeybee cDNA library. Gene names, accession number, forward and reverse primer sequence were listed and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the reference gene (Supporting Information Table 1). 1884



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

3. RESULTS

Reverse transcription was performed using a RNA PCR Kit (Takara Bio, Kyoto, Japan), according to the manufacturer’s instructions. Real-time PCR amplification was conducted on iQ5Multicolor Real-Time PCR Detection System (Bio-Rad, Hercules, CA) as previously described.35 Gene expression data was normalized by GAPDH. After verifying amplification efficiency of the selected genes and GAPDH in approximately equal levels, the differences in gene expression were calculated using 2−ΔΔCt method.36 The statistical analysis of gene expression was performed by one-way ANOVA (SPSS version 16.0, SPSS, Inc.) using Duncan’s multiple-range test. An error probability p < 0.05 was considered statistically significant.

3.1. 2-DE Analysis of Differential Proteome and Phosphoproteome

To investigate proteomics and phosphoproteomics alterations between healthy and CSBV infected Acc worker larvae under natural conditions, first a multifluorescent stain approach based 2-DE was used to detect proteins and phosphoproteins simultaneously. Figure 1A is a representative 2-DE gel image of total proteins stained with CBB. Overall, 421 and 409 protein spots were reproducibly detected in the healthy and CSBV infected larvae. By contrast, 137 and 129 phosphoprotein spots were visualized by Pro-Q Diamond dye in the healthy and CSBV infected larvae, respectively (Figure 1B). In general, approximately 32% of the total proteins were modified by phosphorylation, which generally agrees with the notion that about 1/3 of proteins are phosphorylated in a Eukaryotic organism.37 Quantitatively, 92 total protein spots and 32 phosphoprotein spots showed significant changes of expression (>2-fold and p < 0.05). Of these, 77 total protein spots and 20 phosphoprotein spots were successfully identified (Supporting Information Tables 2 and 3). Of these, 51 and 26 total protein spots, 13 and 7 phosphoprotein spots were up-regulated in the healthy and sick larvae, respectively. The remaining proteins and phosphoproteins were not identified either because of their abundance was too low to produce enough spectra or because the database search scores can not yield unambiguous results (>95%). Moreover, some of the highly sensitive fluorescent stained proteins were present in low abundance and under the detection limit of CBB stain during spot excision.

2.9. Western Blot

To further verify the variation tendency of differentially expressed proteins identified by the proteomic approaches, heat shock protein (Hsp)60, Hsp90, actin, and tubulin were selected for Western blot analysis by the method we described previously with some modifications.35 Briefly, equal amount of protein sample (12 μg/lane) were separated by stacking (4%) and separating (12%) SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels and then transferred to a nitrocellulose transfer membrane (0.2 μm pore size) (Invitrogen, Eugene, OR) using the iBlot apparatus (Invitrogen, Eugene, OR). After blocking, the membranes were incubated for 1.5 h at room temperature with primary rabbit polyclonal antibodies of anti-Hsp60, Hsp90, actin and tubulin antibodies (Abcam, Cambridge, MA) at a dilution of 1:1000. Following three washes, the membranes were further incubated with horseradish peroxidase-conjugated rabbit anti-goat secondary antibody at a dilution of 1:8000 for 1.5 h. Immunoreactive protein bands were detected using the ECL Western Blotting Substrate (Pierce, Rockford, IL) and quantified by densitometry using Quantity-one image analysis system (Bio-Rad, Hercules, CA). GAPDH was detected simultaneously as loading control of the analysis.

3.2. Label-Free LC−MS Analysis of Differential Proteome

For the purpose of a more comprehensive evaluation of larval proteome changes by CSBV infection, a label-free LC−MS based approach was accomplished as a verification and complement of the 2-DE results. The analysis of healthy and infected larvae was achieved by three replicate injections of 1885



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Figure 3. Qualitative comparison of the up-regulated protein numbers in healthy and infected worker larvae of ACC. A total of 180 nonredundant proteins 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 different protein functional groups. The percentage of each functional group is obtained based on the number of proteins under each of the functional group of the total number of identified phosphoproteins in Supporting Information Table 3.

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

both trypsin digested samples on the Q Exactive LC−MS/MS system followed by data processing using Progenesis LC−MS software. In the two-dimensional feature maps created by the software, a total of 120 480 features corresponding to more than 500 proteins (FDR < 1%, at least two unique peptides) were aligned within the retention time. Then, 28 058 features 1886



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

(20), lipid metabolism (21), antioxidant activities (6) and silk protein (4) (Figure 3). In particular, the healthy larvae overexpressed more proteins in almost all the functional categories expected for lipid metabolism. More interestingly, the proteins involved in cytoskeleton, antioxidation, and silk proteins were up-regulated only in the healthy larvae. Of the 20 differentially regulated phosphoproteins, those involved in cytoskeleton were the most represented (9, or 45%), followed by 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 and energy metabolism (Figure 4). For a better understanding of the biological significance of protein expression levels in physiological changes after a viral

Among these, 123 and 29 proteins were found up-regulated in the healthy and sick larvae, respectively. 3.3. Qualitative and Quantitative Comparisons of Differentially Expressed Proteins and Phosphoproteins

In total, of the 180 nonredundant proteins which were differentially expressed between two samples by both 2-DE and label-free LC−MS based techniques, 28 and 119 proteins were exclusively identified and 33 proteins were overlapped (Figure 2). Notably, proteins up-regulated in the healthy larvae represented 79% (142 proteins), meaning the diseased larvae down-regulated this number, and only 21% (38 proteins) were up-regulated in the sick larvae. They were mainly related to carbohydrate and energy metabolism (22), development (44), protein metabolism (43), cytoskeleton (20), protein folding 1887



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Figure 6. Functional enrichment analysis of the differentially expressed proteins. Single (*) or double (**) asterisk indicate significant enriched GO terms 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.

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

challenge, the expression level of each protein between the two samples was compared. In 2-DE analyses, 13 proteins showed >10-fold higher expression of the 51 total protein spots upregulated in the healthy larvae (Figure 5A). On the other hand, of the 26 protein spots up-regulated by infected larvae, four proteins showed >10-fold changes (Figure 5A). As for the phosphoproteins, 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 upregulation in the healthy larvae, while only five proteins were highly expressed (>10-fold) in the infected larvae (Figure 5C).

3.5. Protein−Protein Interaction (PPI) Analysis

In a living cell, proteins perform functions cooperatively through interactions by forming PPI networks, and the study of these synergies is fundamental to the understanding of biological events in a systemic way. Therefore, of all the identified proteins, 89 proteins were linked in the PPI network with variation of interaction degree measured by the number of edges (interaction between the corresponding proteins) adjacent to each protein node (Figure 7). Therefore, protein metabolism was the most abundant in the network (28, or 31.5%). Of these, 18 proteins were up-regulated in the healthy larvae, 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 upregulated in the healthy larvae and three were up-regulated in the sick larvae. On the other hand, of the 15 proteins (16.9%) networked in protein folding, eight and seven proteins were upregulated in the healthy and infected larvae, respectively. Likewise, from 13 (14.6%) proteins associated with carbohydrate and energy metabolism, 11 were up-regulated in the healthy larvae, whereas two were up-regulated in the CSBV infected larvae. Four (4.5%) proteins implicated with lipid metabolism were associated with equal numbers of proteins upregulated in the healthy and sick larvae. Particularly, all eight

3.4. GO (Gene Ontology) Functional Term Enrichment

To enrich the identified proteins to specific functional GO terms and elucidate the possible different biological events behind the proteomic data, the two protein lists, proteins upregulated in the healthy and CSBV infected larvae, were separately analyzed by ClueGo software. Accordingly, the proteins up-regulated in healthy larvae were significantly enriched into five major functional groups, i.e., carbohydrate and energy metabolism, development, protein metabolism, cytoskeleton and protein folding (Figure 6A). The functional leading term (with lowest statistical p value) in carbohydrate and energy metabolism was “generation of precursor metabolites and energy”. And the leading term in development was “cell redox homeostasis”. In the third functional group, cytoskeleton, “cytoskeletal organization” was the leading term. The leading term in protein metabolism was “protein polymerization”. Finally, “’de novo’ protein folding” was the leading term in protein folding. By comparison, the proteins up-regulated in the CSBV infected larvae were significantly enriched in three major functional groups, i.e., small molecule 1888



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Figure 7. Protein−protein interaction (PPI) network. PPI was predicted using I2D and Navigator software. PPI was predicted using I2D and Navigator 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 proteins up-regulated in infected larvae. Blue lines indicate interactions between proteins. The intensity of the interaction degree is indicated by color gradient as noted on the key bar on the down right side of the figure.

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

(8.9%) cytoskeletal proteins and three (3.4%) antioxidant proteins in the network were up-regulated in the healthy larvae. On the basis of the interaction degree, 89 proteins linked in the PPI network with 19 proteins had more than 100 degrees of interaction (Supporting Information Table 6), i.e., RPLP2, eIF5A, Rpn11, EF-2, EF-1-alpha, PSMD4 and RPS6 in protein metabolism; CCT-zeta, HSC70-5, HSC70-4, HSP90 and HSP60 in protein folding; TPS in carbohydrate and energy metabolism; SUMO-3 and 14-3-3zeta in development; tubulin, ADF and actin in cytoskeleton group; SOD involved in antioxidant group.

3.7. Western Blot Analysis

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

3.6. Test of Differentially Expressed Proteins by qRT-PCR

To test the tendency of protein expression between its encoding gene at the transcript level, 20 key node proteins with high degrees in the PPI network from six major functional groups (carbohydrate and energy metabolism, development, protein metabolism, protein folding, cytoskeleton and antioxidation) were selected for qRT-PCR analysis. The trend of mRNA 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 consistent with the protein expression. However, three genes, atpb, sumo-

4. DISCUSSION The viral diseases of honeybees have a detrimental influence on the development of the beekeeping industry, and cause serious economic losses worldwide both of bee-products and pollination services. So far, over 18 viruses have been reported to trouble the honeybee.38 As one of the major honeybee species in Asia and China, the Acc are the most susceptible to 1889



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Figure 8. Test of the 20 differentially expressed proteins at mRNA level by quantitative real time PCR analysis. The black and the gray bars represent fold changes (healthy to infected honeybee worker larvae) of protein and mRNA, respectively. The positive values indicate higher expression in the healthy 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 of honeybee worker were subjected to SDS-PAGE followed by Western blot analysis. HSP60, HSP90, actin, and tubulin were detected using corresponding antibodies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as reference control. (A) The Western blot images of Hsp60, Hsp90, actin, tubulin and GAPDH. (B) The relative fold change of Hsp60, Hsp90, actin, tubulin (normalized by GAPDH). The gray bars represent relative fold change of protein expression, positive values indicates higher expression in the healthy larvae, and negative values denote higher expression in the infected larvae. Error bar is standard deviation.

CSBV infections that have caused a deadly loss of honeybee colonies recently. Usually, the viral infection causes cell apoptosis, tissue damage and even functional disorder of the whole organism and all these changes can be further reflected in proteome alteration.39 To understand the underlying molecular pathological mechanism of the CSBV infection, gel-based and label-free LC−MS based proteomic strategies were conducted to analyzes the proteome and phosphoproteome alterations between the healthy and the CSBV infected Acc larvae in vivo. Accordingly, 180 nonredundant proteins were identified as being differentially expressed between the healthy and diseased larvae. Of these, 142 were up-regulated in the healthy larvae and only 38 proteins were up-regulated in the sick larvae (Figure 2). Proteins up-regulated in the healthy larvae were mainly enriched in carbohydrate and energy metabolism, development, protein metabolism, cytoskeleton and folding functions (Figure 6A). This is believed to coincide with the

large repertoire of protein demands of the healthy larvae to satisfy their normal development and metabolism.40 Whereas the normal metabolism of the honeybee larvae is terribly disturbed by the down-regulation of the above-mentioned pathways under infectious conditions, at same time, proteins related to small molecule catabolic process, cellular chemical homeostasis and folding activities have a stronger response to fight against the viral challengers (Figure 6B). As one of the most common post-translational modifications (PTMs), protein phosphorylation participates in almost all aspects of cell life.41 In this study, the identified differentially expressed phosphoproteins are mainly associated with cytoskeleton, protein metabolism and protein folding. By changing the phosphorylation status, they either play essential roles in metabolism, differentiation, cytoskeleton arrangement and cell cycle for the normal larva growth,42,43 or modulation of 1890



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both viral replication21,22 and interactions between viral and host proteins during infection.44 Our data indicates that the viral infection has triggered an essential proteome and phosphoproteome change of the larvae, which derail the larvae into a deadly developmental trajectory by collapse of several pathways such as energy supply, protein biosynthesis, hormonal regulation and antioxidant defenses. This gains a new molecular-level insight into the pathological mechanism of the CSBV diseases.

involved in a variety of cell functions besides their glycolytic activities, and play significant roles in disease processes and many others,56 such as growth control,57 cytoskeletal binding,58 temperature and salt stress tolerance.59 Aldolase, a glycolysis enzyme, has an abundant expression in the hemolymph of Drosophila larvae following both fungal exposure and bacterial infection60 as well as white spot syndrome virus (WSSV) invasion.61 Therefore, the escalated expression of aldolase and enolase might suggest their roles in modulation of the stress response by virus infection as heat shock proteins do.62 Meanwhile, since an insect virus could jeopardize oxygen delivery, the up-regulation of these enzymes is possibly responsible for enhancing anaerobic metabolism, and providing a special pathway for the larvae to get energy under pathological conditions.63

4.1. Viral Infection Disrupting Carbohydrate Metabolism and Energy Production

As a necessary nutrient substance, carbohydrates are paramount important energy fuels for the fast growth of honeybee larvae. Young worker larvae less than three days old are fed solely with royal jelly, which is a protein-rich glandular secretion with the sugar content (fructose and sucrose) of only 18%. During development, the old larvae are fed with a blend of honey and royal jelly based diet in which the sugar content increases up to 45% in the last two days before pupation.45 This programmed food change is the biological demand of the fast growing older larvae to satisfy their astounding energy requirement from the carbohydrates. However, the target of CSBV is the goblet cells of the midgut epithelium, and the virus multiplies in the cytoplasm of goblet cells as a prelude to systemic infection.46 Hence, the viral infection may affect nutritional assimilation in the midgut and influence the physiological metabolism of larvae. In this study, 18 proteins (8 with >10-fold decrease) related to carbohydrate and energy metabolism were downregulated in the CSBV infected larvae. For instance, 6phosphofructokinase is involved in glycolytic pathways; ATPcitrate synthase and NADP-dependent malic enzyme (NADPME) are important components in the TCA cycle, and NADPME also implicated in the glyoxylate cycle;47 ATP synthase catalyzes the synthesis of ATP that provides energy for the cell.48 All these enzymes coordinate in the network of catalyzing energy generation to gratify the demand for the larval development under normal physiological conditions. On the contrary, the viral infection probably severely hindered carbohydrate uptake in the sick larvae, and the carbohydrate metabolism enzymes mentioned above were down-regulated as a consequence. In addition, some other carbohydrate metabolism enzymes were still down-regulated in the infected larvae. For example, glucosamine-6-phosphate deaminase, catalyzing the formation of glucosamine-6-phosphate dependent upon fructose-6-phosphate,49 is an active precursor of chitin in arthropods. It plays a crucial role in construction of insect cuticle and peritrophic matrix that serves as a physical barrier to pathogens in the midgut.50 Trehalose-phosphate synthase is responsible for trehalose synthesis, and trehalose has been shown to enhance anoxia tolerance of fruit fly larvae.51 Arginine kinase, a crucial enzyme in promoting the efficient synthesis of ATP,52 could fulfill the long periods of energy demand in the midgut.53 Together, the down-regulation of these enzymes is thought to weaken the larvae’s defense capability to the viral challenge and facilitate further infection or cut down energy supply of the larvae, thus further damaging the regular development of the larvae. However, some enzymes related to metabolism of carbohydrates such as enolase and aldolase were up-regulated in the infected larvae. Enolase is a crucial enzyme in the glycolytic pathway catalyzing the dehydration of 2-phosphoglycerate to phosphoenolpyruvate.54,55 Eukaryotic enolases, however, are

4.2. Viral Infection Hampering Larval Development

During the larval−pupal metamorphosis of holometabolic (completely metamorphosing) insects, the larvae go through remarkable physiological changes to prepare themselves for pupation and metamorphosis, and most of the organs and tissues are remolded.64 Accordingly, the proteins related to development play vital roles in this moulting process. At the prepupal stage, although the larvae are still in their old form, radical changes are already in progress. For example, the cuticle of the young pupa has already formed, which is enveloped in the larval cuticle.64 However, the virus infection could inhibit pupation to facilitate its replication and transmission.65 To this end, the down-regulation of seven cuticle proteins in the sick larvae with four decreased >10-fold is believed to be the major reason that the CSBV infected larvae failed to pupate. Along with the formation of pupate cuticle, a part of the larval muscles should be disintegrated and then remodeled with the imaginal myoblasts,64 and this process is also disturbed by infection. In line with this, some proteins implicated in regulating muscle cell proliferation and muscle contraction had decreased expression, including myophilin, muscle-specific protein 20, calreticulin and calumenin.66−68 Larval ontogenesis and cell proliferation heavily rely on the mitosis; lamin Dm0, nuclear migration protein NudC and small ubiquitin-related modifier have been documented to have direct mitogenic activity.69−71 The decreased expression of these proteins is supposed to hamper the larval development in this regard. Also, IDGF4 is a growth factor that is produced by the fat body and transported through the insects’ hemolymph. Its down-regulation in the infected larvae suggests the weakened roles in stimulating proliferation and polarization of imaginal disc cells.72 The 14-33 protein family contributes to a wide variety of important signal transduction pathways that control cell cycle, apoptosis, and programmed gene expression.73 Its down-regulation in the sick larvae suggests the modulation roles in larval to pupal metamorphosis are suppressed. Notably, the phosphorylation level of 14-3-3 zeta was higher in the sick larvae. The phosphorylation in specific residues of 14-3-3 proteins has important regulatory roles,74 and cellular stress induced 14-3-3 zeta phosphorylation makes cells more susceptible to apoptotic signals.75 Hence, increased expression of this phosphorylated protein may directly catalyze the viral infection. On the other hand, three development related proteins were up-regulated in the CSBV infected larvae, i.e., transitional endoplasmic reticulum ATPase (TER94), translationally controlled tumor protein (TCTP) and cyclin-dependent kinase 6 (CDK6). TER94 (p97 or VCP in mammals), an ATPase 1891



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associated with various cellular activities, is required for exporting 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 that facilitates its function of transporting and releasing ubiquitinated proteins in ubiquitin/proteasome-dependent proteolytic pathways78 and determining its role in the repairing of DNA damage.79 TCTP, an evolutionarily conserved multifunctional protein, is implicated in the protection of cells against various stress conditions and apoptosis.80 In addition, it could protect the cell from death during the viral infection.81 CDK6 is an important cell-cycle regulator in the progression of a cell through G1 phase and the G1/S transition. And many viruses arrest the host cell division cycle to favor their own growth.82,83 Consequently, the stronger expressions of these proteins collectively suggest the virus infected larvae recruit them as a remedy for crisis.

The cofilin/actin-depolymerizing factor and myosin are members of the actin-binding protein family. Moreover, tubulin, a basic subunit of the microtubule, can provide structural support of cells and functions as a motional path of organelles.96 However, the functions of the cytoskeleton are seriously disturbed once the virus invades such that severe disruption of microtubule organization and centrosome function occurs.97 The destruction of cytoskeletal filaments or microtubules could further facilitate both virus replication98 and release of virus particles.99 Furthermore, the CSBV is thought to likely cause cell death by induction of apoptosis like other picornaviruses family members such as rhinovirus, poliovirus and foot-and-mouth disease virus,100,101 and apoptosis is always accompanied by a reduction in actin content and the dissolution of the cellular cytoskeleton.102,103 In addition, the function of tropomyosin in nonmuscle cells is to modulate the interaction between actin and actin-binding proteins to stabilize the stress fibers and protect them from disassembly.104 Therefore, reduction of tropomyosin may also lead to the disruption of both cytoskeletal and extracellular architecture.105 This is likely the reason that the sick larvae eventually take a sack like shape. Certainly, the up-regulation of these cytoskeletal proteins in the healthy larvae is believed to be important to accomplish their roles in supporting dramatic cell and tissue rebuilding at the period before pupation. In addition, the larger number and stronger expression of phosphorylated cytoskeletal proteins seems to be enhancing the metamorphosis processes. For instance, phosphoryaltion of myosin regulatory light chain (MRLC) controls its accumulation during cytokinesis.106 Also, phosphorylation regulates the function of cofilin/actindepolymerizing factor (ADF) that influences actin cytoskeletal dynamics.107 So, the increased phosphorylation level of these proteins indicates the significance of phosphorylation in the regulation of larval development in cellular processes such as signal transduction and cell differentiation.

4.3. Viral Infection Leading to Protein Degradation

The development of a multicellular organism has a stake in the protein anabolism and catabolism.84 The developing larvae require continuous protein synthesis, especially during metamorphosis; the viral invasion, however, interrupts regular protein metabolism.85 Ribosome is the organelle in charge of protein biosynthesis and many other translational factors function in this process.86 Consequently, the down-regulation of nine ribosomal proteins and six eukaryotic translation initiation factors in the sick larvae is likely projecting that the destroyed protein biosynthesis system cannot guarantee the normal growth of larvae. Specifically, as a source of amino acids for tissue reconstruction during pupal development, hexamerins are first synthesized by a larval fat body and released into the hemolymph where they accumulate to extraordinarily high concentrations. Then, before the initiation of metamorphosis, hexamerins are selectively sequestered by fat body cells via receptor-mediated endocytosis, and used to build adult structures.87 Nevertheless, the high level of hexamerins in the CSBV infected larvae until the prepupal stage suggests that the virus infection blocked the utilization of hexamerins. This is consistent with a higher stock of hexamerin 110 presented at starved larvae than nonstarved larvae, and the starved larvae cannot reach the cocoon-spinning stage.88 On the other hand, following the infection, the expression of some proteins involved in protein degradation was increased. As we know, the proteasome is a multisubunit enzyme complex that plays a central role in eliminating of ubiquitinated proteins in the cell.89 The ubiquitin−proteasome system mediated viral protein degradation constitutes a host defense process against some RNA viral infections.90 Moreover, proteasome activity and assembly are regulated by cAMP-dependent protein kinase A induced phosphorylation.91,92 Thereby, the up-regulation of two proteasome subunits (β2 and β7) and two phosphorylated subunits (α1 and α3) suggests the participation of proteasomes in the protein degradation that occurs during the viral infection.

4.5. Heat Shock Protein Plays Defensive Roles for Sick Larvae

Correct protein folding is essential for the protein to function properly, and many proteins called molecular chaperones participate in this process. The heat shock protein (Hsp) family is a highly conserved group of molecular chaperones existing in all organisms.108 In this study, a number of Hsps, such as Hsp90, Hsc70-3, Hsc70-4, Hsc70-5, Hsp60, and Hsp10, were significantly up-regulated (Hsp90 and Hsp60 > 10-fold change) in honeybee larvae challenged by the CSBV. In general, there is a close relationship between Hsps and viral infection. Several viruses, such as dengue virus,109 newcastle disease virus,110 simian virus 40 and polyoma virus111 could induce the expression of Hsps to protect infected cells against proteotoxic stresses by assisting protein correct fording, or by guiding damaged proteins to the proteasome for destruction.112 Therefore, the up-regualtion of Hsps suggests that the CSBV infected larvae have employed these stress proteins in response to the viral invasion as in the case of heat stress and bacterial infections in the honeybees.113−115 However, through interaction with viral proteins, Hsc70 and Hsp90 participate in the facilitation of viral production in host cells during the infection cycle.116,117 In insect cells, for example, Hsp90 plays a supportive role in both flock house virus and baculovirus RNA replication.116,118 Interestingly, Hsp90 and Hsp60 were phosphorylated and their expression escalated in the sick larvae.

4.4. Viral Infection Collapsing Cytoskeletal Structure

The cytoskeleton generates cell mitosis, powers the movements of motile cells and provides mechanical support for the cell.93 The actin assembles into long, fiber-like filaments, which play a dominant role in many cellular functions including cytokinesis, phagocytosis and muscle contraction.94 In response to intracellular and extracellular signals that stimulate cell division and differentiation, the actin protein is dynamically remodeled, and this reorganization is regulated by actin-binding proteins.95 1892



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generated as harm products of normal metabolism, and the high oxygen demand increases its accumulation.130 In the honeybee, it is reported that the antioxidant proteins are expressed in the development of the hypopharyngeal gland and larvae to protect cellular components from oxidative damages.35,40 On the other hand, the stronger expression of antioxidative proteins at the earlier stage of infection is an adaptive strategy for the survival of cells.131,132 However, the functionality of this system is weakened at the late stage of disease.131,133 Thus, the down-regulation of antioxidant enzymes in the sick larvae, such as superoxide dismutase (SOD), glutathione S-transferase (GST), phospholipid hydroperoxide glutathione peroxidase isoform 1 (PHGPx1), thioredoxin peroxidase (TPx) and peroxiredoxin, implies the increased levels of ROS caused by infection overwhelmed the cells’ antioxidant defenses. Moreover, the up-regulation of phosphorylated PHGPx1 and SOD in the healthy larvae suggests the enhanced protective capacity against oxidative stress during the larval metamorphosis.134,135 The proteins which participate in the life activities of the complex organism are involved in a variety of interactions rather than acting as separate entities. Proteins that are linked together in the context of networks through PPI, modifications and regulation of expressional relationships indicate their biological centrality. To recognize this fact, 89 key node proteins are supposed to play crucial roles in the CSBV infection, of which 65 proteins were down-regulated and 24 upregulated in the infected larvae. They were mainly related to protein metabolism, development, carbohydrate and energy metabolism and protein folding and cytoskeleton, which were the most networked groups. Coinciding with GO functional enrichment analysis, these five groups are considered to be the major pathways that are significantly influenced by viral infections of honeybee larvae. In addition, the tested results both at the protein and gene level provide important information of target hub proteins that can be potentially used for early diagnosis and medicinal development or selection of viral-resistant bees through gene manipulation.

There is evidence that phosphorylated Hsp90 plays an active role in reovirus infection.119 Likewise, the phosphorylated Hsp60 in the cell plasma membrane functions as a molecular chaperone that is appropriately regulated by phosphorylation.120,121 Consequently, we suggest that the increased expression and phosphorylation levels of Hsps after the CSBV challenge probably helps the larvae in suppressing the stress caused by infection, but also seems to support the productive replication cycle of CSBV. In contrast, as might be expected, the healthy larvae require the up-regulation of some of other molecular chaperones to facilitate normal tissue development as in the developing queen and worker larvae and embryos under normal physiological conditions.40,122 Chaperonin containing TCP-1 (CCT), an indispensable chaperone in the eukaryote, is required in folding of an essential subset of cytosolic proteins, localizes at sites of microfilament assembly, and functions in the facilitated folding of actins and tubulins.123 It is unable to fold sufficient cytoskeletal proteins without the assistance of other molecular chaperones, such as prefoldin (PFD). PFD plays a major role in de novo protein folding, and its absence functionally impairs the chaperonin pathway.124 In this study, three subunits of prefoldin (PFD2, PFD4 and PFD6) and four of eight subunits of CCT (beta, epsilon, zeta and eta) were increased in the healthy larvae. This suggests a key role of molecular chaperones in protecting accurate protein folding to guarantee normal growth of the cell and healthy larval development. 4.6. Viral Infection Causes Juvenile Hormone Disorder That Blocks Pupation

Lipid metabolism provides energy needed during extended nonfeeding periods that is essential for the larval development, particularly through metamorphosis.125 To this end, the upregulation of proteins involved in lipid metabolism in both larval samples implies the critical necessity of energy supply for the larvae. Intriguingly, two important enzymes involved in this functional category, juvenile hormone esterases (JHE) and juvenile hormone epoxide hydrolases (JHEH), are directly responsible for juvenile hormone (JH) degradation.126 In the life cycle of the eusocial insect, larval−pupal metamorphosis is precisely regulated by the cooperation of JH and ecdysone.127 During the larval development, the ecdysone initiates only larva-to-larva molts with enough JH present in the hemolymph, while pupation is triggered by ecdysone with lower amounts of JH. The JH titer of the honeybee drops to a minimal level both at the end of the larval feeding phase and just before pupation.128 Nevertheless, the opposite relationship between JHE expression levels and JH titer is to degrade JH and promote larval−pupal metamorphosis of honeybees.129 The high level of JHE (>10-fold) in the healthy larvae seems to assist in clearance of the JH and preparing old larvae for metamorphosis. However, the low abundance of JHE and JHEH in the diseased larvae suggests that they are not efficient in degrading the JH, thus, hindering the regular pupation of larvae. Therefore, these two enzymes are likely to be biomarkers for CSBV diagnosis. In addition, because the larvae should begin to spin cocoons before pupation, the downregulation of four silk proteins in the sick larvae is proof that pupation was blocked.

5. CONCLUSION The CSBV infection has triggered significant proteome and phosphoproteome variations of Acc larvae. There are 180 differentially expressed proteins and 19 phosphoproteins identified under CSBV infection in vivo using gel-based and label-free LC−MS based proteomic approaches. This complementary platform has offered an efficient avenue for molecularlevel mechanistic understanding of the honeybee pathology through proteome analysis. Our results demonstrate that the CSBV infection disrupted normal larvae development and hampered larval−pupal metamorphosis by disturbing several key metabolism pathways such as carbohydrate metabolism, protein synthesis, cytoskeletal structure and hormone regulation. On the other hand, the sick larvae have recruited some strategies to defend themselves using Hsps and antioxidant system. This comprehensive proteomics and phosphoproteomics analysis provides a new understanding of the pathological mechanism of CSBV disease. Some important node proteins have been identified and protein expressions were validated at the gene level, which have potential use as candidate targets of RNAi-based gene therapy, as well as breeding and prevention of this fatal viral disease through marker assisted selection.

4.7. Viral Infection Disables the Larval Normal Oxidative System

The antioxidant system is central for the reduction of reactive oxygen species (ROS)-induced oxidative damage. ROS are 1893



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ASSOCIATED CONTENT

S Supporting Information *

Pictures of healthy and Chinese sacbrood virus infected honeybee (Acc) worker larva, identification of differentially expressed proteins and phosphoproteins in healthy and Chinese sacbrood virus infected honeybee (Acc) worker larvae by 2-DE analysis, identification of differentially expressed proteins in healthy and Chinese sacbrood virus infected honeybee (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 analysis of the differentially expressed proteins, and information of each protein and peptide sequence identified. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86 10 6259 1449. E-mail: [email protected]. Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Katrina Klett from University of Minnesota, USA, for her help with the language of the manuscript. This work is supported by the earmarked fund for Modern Agro-industry Technology Research System (CARS-45), The National Natural Science Foundation of China (No. 30972148) and key projects of the national scientific supporting plan of the 12th Five-Year Development (2011-2015) (2011BAD33B04).

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ABBREVIATIONS CSBV, Chinese sacbrood virus; 2-DE, two-dimensional gel electrophoresis; SBV, sacbrood virus; ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcription-polymerase chain reaction; LC−MS, liquid chromatography−mass spectrometry; FDR, false discovery rate; PPI, protein−protein interaction; HSPs, heat shock proteins; PTMs, post-translational modifications.

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REFERENCES

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