Integrative Proteomics and Metabolomics Analysis of Insect Larva

Article pubs.acs.org/jpr

Integrative Proteomics and Metabolomics Analysis of Insect Larva Brain: Novel Insights into the Molecular Mechanism of Insect Wandering Behavior Yi Li,† Xin Wang,† Yong Hou,† Xiaoying Zhou,† Quanmei Chen,‡ Chao Guo,† Qingyou Xia,† Yan Zhang,† and Ping Zhao*,† †

State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China Department of Biochemistry & Molecular Biology, Chongqing Medical University, Chongqing 400016, China

‡

S Supporting Information *

ABSTRACT: Before metamorphosis, most holometabolous insects, such as the silkworm studied here, undergo a special phase called the wandering stage. Insects in this stage often display enhanced locomotor activity (ELA). ELA is vital because it ensures that the insect finds a safe and suitable place to live through the pupal stage. The physiological mechanisms of wandering behavior are still unclear. Here, we integrated proteomics and metabolomics approaches to analyze the brain of the lepidopteran insect, silkworm, at the feeding and wandering stages. Using LC−MS/MS and GC−MS, in all we identified 3004 proteins and 37 metabolites at these two stages. Among them, 465 proteins and 22 metabolites were changed. Neural signal transduction proteins and metabolites, such as neurofilament, dopaminergic synapse related proteins, and glutamic acid, were significantly altered, which suggested that active neural conduction occurred in the brain at the wandering stage. We also found decreased dopamine degradation at the wandering stage. The proposed changes in active neural conduction and increased dopamine concentration might induce ELA. In addition, proteins involved in the ubiquitin proteasome system and lysosome pathway were upregulated, revealing that the brain experiences morphological remodeling during metamorphosis. These findings yielded novel insights into the molecular mechanism underlying insect wandering behavior. KEYWORDS: Wandering behavior, brain, proteomics, metabolomics, silkworm

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INTRODUCTION The brain is the central organ for connecting and coordinating body function. In insects, the brain regulates the activity of endocrine glands to control most biological activity, such as growth, development, reproduction, and diapause. The insect brain can be divided into the protocerebrum, deutocerebrum, and tritocerebrum.1 On both sides of the protocerebrum, a protuberant optic lobe is joined directly to the compound eyes. Behind the protocerebrum, there are three ocellar pedicels that connect to the dorsal ocellus and ocellae. Four kinds of structures (mushroom body,2−5 protocerebral bridge, central body,6 and accessory lobe7,8), which consist of neural cell globes and nerve fiber tracts in the nerve myelin of the protocerebrum, make up the controlling center of the insect brain. The deutocerebrum includes two intumescent antennal lobes to control the antennal action.9 The tritocerebrum is specialized by having a pair of ganglia in the first body segment; its main function is to connect with the frontal ganglion and labrum ganglion.10,11 Many insects experience an important stage called wandering stage at the end of the larval period. The function of wandering for holometabolous insects is to find an appropriate place for © 2015 American Chemical Society

metamorphosis. Studies in Manduca sexta show that wandering behavior is controlled by the larval nervous system and can be triggered by 20-hydroxyecdysone (20E).12−15 20E is secreted by prothoracic glands (PGs) under the stimulation of the prothoracicotropic hormone (PTTH), which is synthesized in the brain to initiate metamorphosis.16,17 The silkworm, Bombyx mori, which has been an important economic insect for over 5000 years, has become a model for lepidopteran insects. Generally, the silkworm undergoes five larval instars ending with the wandering stage. Early in wandering, the silkworm integument begins to become transparent and the silkworm displays enhanced locomotor activity (ELA). Studies of this process also showed that 20E induces wandering behavior under the regulation of PTTH.18,19 However, the detailed molecular mechanism of ELA at the wandering stage is still unclear. With the completion of the silkworm genome, silkworm research entered the postgenomic era.20 Over the past few years, techniques have been successfully used to elucidate Received: August 7, 2015 Published: December 8, 2015 193

DOI: 10.1021/acs.jproteome.5b00736 J. Proteome Res. 2016, 15, 193−204

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Journal of Proteome Research

buffer and 50 mmol/L NH4HCO3. 150 μL of 50 mmol/L NH4HCO3 containing 6 μg of trypsin (Sigma) was added to each sample, which was then incubated at 37 °C for 24 h.31 The digested peptides were collected by centrifugation, dried for 4 h in a vacuum concentrator, and redissolved in 100 μL of 0.1% (v/v) trifluoroacetic acid. Dissolved peptides were desalted with a Ziptip C18 pipette tip (Millipore) and eluted in 50% (v/v) acetonitrile with 0.1% (v/v) trifluoroacetic acid. Desalted peptides were dried for 3 h in a vacuum concentrator and finally dissolved in 0.1% (v/v) formic acid.

changes in the silkworm proteome in processes such as reproduction,21 development,22 and spinning,23 and research on silkworm metabolomics has been reported.24,25 As another model organism of Lepidoptera, metabolomics studies on the hemolymph of M. sexta have shown that changes in metabolites are involved in insect development.26,27 Among published reports, only a few studies have focused on brain-controlled behavior in insects. Using two-dimensional gel electrophoresis and MALDI-TOF-MS, Biron et al. identified changes in the brain proteome of the drumming katydid, Meconema thalassinum, infected by the nematomorph hairworm, Spinochordodes tellinii.28 In research on ant brain, de Bekker et al. identified differential changes in metabolites after fungal infection using LC−MS/MS.29 However, neither proteomics nor metabolomics techniques have been used in studies of insect wandering behavior. In this study, we analyzed silkworm brains from the feeding stage (third day of the fifth instar larva, 3d5I) and wandering stage by integrative proteomics and metabolomics techniques and found increased dopamine (DA) concentration in the brain of wandering stage larvae. These data provide novel insights into the molecular mechanism of insect wandering behavior.

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Protein Identification by LC−MS/MS and Database Searches

10 μL of each sample was first separated with a ThermoFisher Scientific EASY-nLC 1000 system equipped with an EASYSpray column (C18, 2 μm, 100 Å, 75 μm × 50 cm) using a 2− 100% (v/v) acetonitrile gradient in 0.1% (v/v) formic acid over 180 min at a flow rate of 250 nL/min. The separated peptides were analyzed using a Thermo Fisher Scientific Q-Exactive mass spectrometer operating in data-dependent mode. The temperature of the ion transfer capillary was set at 250 °C. The full mass scan range was 300 to 1800 m/z with a resolution 70 000 at m/z 200, and up to 20 of the most abundant ions with charge ≥2 from full mass scan were automatically selected by higher energy collisional dissociation for fragmentation with normalized collision energies of 27%. The maximum ion injection times for the full mass scan and the MS/MS scans were, respectively, 20 and 60 ms, with dynamic exclusion for previously sequenced ions set to 10 s. Raw MS data were converted into Mascot generic peak lists by MaxQuant, version 1.5.2.8.32,33 Proteins were identified by searching against a silkworm proteome database containing 35 859 protein sequences from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) and silkDB (http://silkworm.genomics.org.cn/). Search parameters included variable modifications of methionine oxidation and Nterminal acetylation and a fixed modification of carbamidomethyl cysteine. Precursor mass and fragment mass tolerances were set to 6 and 20 ppm, respectively. The minimal peptide length was six amino acids, and up to two missed cleavage sites were allowed. A strict 1% false discovery rate threshold was set to filter both the peptide and protein. After searching the database, protein data were first filtered to remove contaminants and identified proteins from the reverse database. In addition, at least one unique peptide was considered to be accurately identified. A label-free quantification (LFQ) algorithm was used to compare the protein abundances between different samples.34 On the basis of analyses of three biological replicates, only proteins with a log2 fold change (wandering stage/feeding stage LFQ intensity, log2FC) ≥ 1 or ≤ −1 (p-value < 0.05) were considered to be significantly altered.

MATERIALS AND METHODS

Insect Rearing and Sample Collection

Silkworms, strain P50 (Dazao), provided by the State Key Laboratory of Silkworm Genome Biology (Southwest University, Chongqing, China), were reared on fresh mulberry leaves at 27 ± 1 °C at a humidity of 75 ± 5%. Larval brains from feeding stage and wandering stage silkworms were dissected on ice. Locomotion Assay

Locomotion assays were performed as previously described30 with some modifications. Larvae from feeding stage and wandering stage (separated into 3 groups of 10 larvae each) were placed in the center of a rectangular grid (350 × 500 mm2) marked at 1 mm intervals. Photographs were taken by a digital camera at 1, 5, and 10 min after release. The coordinates of each larva at the midpoint of the sixth and seventh body segments were determined at each time point after release using Adobe Photoshop CC 14.0 software. The distances moved at 1, 5, and 10 min were measured for the locomotion assay. Sample Preparation for LC−MS/MS Proteomics

Fifty larval brains were used per sample, with three biological replicates. Larval brains were homogenized in lysis buffer (containing 8 mol/L urea, 100 mmol/L dithiothreitol (DTT)) using tissue grinding pestles, followed by centrifugation at 14 000g for 30 min at 10 °C. The supernatant was moved to a new centrifuge tube, and protein concentrations were measured by the Bradford method. A total of 300 μg of proteins was placed into an ultrafiltration device with 3 kDa interception (Millipore) and centrifuged at 11 000g for 15 min at room temperature. After centrifugation, 200 μL of UA buffer containing 8 mol/L urea and 100 mmol/L Tris-base (pH 8.0) was added to the ultrafiltration device, which was then centrifuged again three times. The resulting proteins were reduced by 10 mmol/L DTT for 150 min at 37 °C and then alkylated with 50 mmol/L iodoacetamide for 60 min at room temperature in the dark. The ultrafiltration device was centrifuged again and then washed 3× separately with UA

Protein Annotation, Gene Ontology (GO) Categories, and Pathway Analysis

Protein sequences were searched against Europe, ES: PublicDB (version b2g_sep 14) using Blast2GO 3.0 software to identify annotation and GO numbers.35 The GO number outputs were subjected to GO categories using the web gene ontology annotation plot (WEGO).36 The GO enrichment analysis was operated by Blast2GO using Fisher’s exact test.37 The protein sequences were searched against KEGG (Kyoto encyclopedia of genes and genomes) genes using the BLASTP program with a BLOSUM62 scoring matrix for pathway analysis.38 194


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°C. Samples were centrifuged at 18 000g for 20 min at 4 °C. The supernatant was moved to a clean centrifuge tube, dried for 3 h in a vacuum concentrator, and then dissolved in 200 μL of 0.2% (v/v) formic acid. QC samples were prepared by pooling 10 feeding stage larval brains and 10 wandering stage larval brains. 10 μL of each sample was injected into a ThermoFisher Scientific Ultimate 3000 system equipped with a Pursuit 3 PFP column (3 μm, 2 × 150 mm, Agilent). The flow phases used were (A) aqueous 0.2% (v/v) formic acid and (B) methanol. The gradient used was 2% B for 0.5 min, 2−60% B for 1 min, 60% B for 2.5 min, and re-equilibrate for 3 min.43 The flow rate was 0.3 mL/min, and the column oven temperature was kept at 40 °C. Targeted selected ion monitoring (SIM)/ddMS2 mode was used in the Q-Exactive mass spectrometer with an electrospray ion source. The precursor ion used to monitor DA in the brain samples is 154.086 [M + H]1+, and the product ion of MS/MS was confirmed by Mass Frontier 7.0 software. Quantifier SIM peak areas were compared to the calibration curve of external standard peak areas to determine the DA concentration.43

Quantitative Real-Time PCR (qRT-PCR) Analysis

Total RNA from each sample was extracted using TRIzol reagent according to the manufacturer’s guidelines (ThermoFisher Scientific)39 and then reverse-transcribed into cDNA with an oligo (dT)15-primer and GoScript reverse transcription system (Promega). Gene-specific primers were designed with Primer Premier 5.0 software and are listed in Table S1. qRTPCR was performed in 20 μL reactions with 100 ng of cDNA, 400 nmol/L each of the forward and reverse primers, and SYBR Green Mix (Takara). The cDNA was amplified in an ABI 7500 fast real-time PCR system (ThermoFisher Scientific) according to the following program: initial denaturation at 95 °C for 30 s and 40 cycles of 95 °C for 3 s and 60 °C for 30 s. Three replications of each RNA sample were performed for qRTPCR. Quantification of the amplified transcripts was determined by the Ct value of each transcript.40 Sample Extraction and Derivatization for Metabolomics

Each sample containing 100 brains (with five biological replicates) was homogenized in 500 μL of a water− methanol−chloroform mixture (1:2:2) and sonicated for 15 min (safety considerations: this work should be performed in a fume hood), followed by incubating on ice for 30 min. The samples were then centrifuged at 18 000g for 30 min at 4 °C. The supernatant was moved to a clean centrifuge tube and dried for 4 h in a vacuum concentrator, dissolved in 80 μL of pyridine containing 20 mg/mL methoxylamine hydrochloride, and then incubated at 37 °C for 1.5 h. Samples were trimethylsilylated with the addition of 65 μL of N-methyl-N(trimethylsilyl) trifluoroacetamide containing 1% (v/v) chlorotrimethylsilane for 1 h at 37 °C. The reaction was stopped by the addition of 10 μL of hexane. The quality control (QC) samples were prepared by pooling 50 larval brains from feeding stage and 50 larval brains from wandering stage with the same processing method described above.

20-Hydroxyecdysone Treatment

Each larva on the first day of the fifth instar was injected with 10 μL of solution containing 2.5 μg of 20E or deionized water (control). At 24 h after injection, the brains were dissected, and the concentration of DA was measured as described above. Each group has 20 larvae with three replicates.

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RESULTS

ELA Occurred in Wandering Stage Larvae

During the last larval instar, silkworms eat a large number of mulberry leaves. This period is called the feeding stage. At 3d5I (a typical feeding stage), many genes associated with growth, development, and signal transduction begin to express.44 At wandering stage, silkworms display ELA. To test the differential locomotion activity between feeding stage and wandering stage larvae, we performed a locomotion assay involving the horizontal climbing distance of silkworms on a rectangular grid, and the results are shown in Figures 1 and S1. At 1 min

Metabolites Identification by GC−MS

The derivative sample (1 μL) was injected at a 1:10 split ratio into a 7890B/5977A GC−MS (Agilent) equipped with a HP5MS column (length 30 m, i.d. 25 mm; Agilent), and the injection temperature was held at 280 °C. The oven temperature program was set as follows: initiate at 60 °C and hold for 3 min, elevate to 170 °C at a rate of 5 °C/min, to 234 °C at 4 °C/min, to 250 °C at 10 °C/min, ramp to 300 °C at a rate of 20 °C/min, and hold for 5 min. Mass spectra were recorded from 45 to 500 m/z. The samples were randomized for analysis. The raw data were converted into AIA format and sent to Xcms online (https://xcmsonline.scripps.edu/index. php) for data analysis.41 Peaks were identified by spectra matching against the National Institute of Standards and Technology; a relative score over 700 was considered to be a good match. On the basis of analyzing five biological replicates, only metabolites that changed ≥1.5-fold in relative ratios (pvalue < 0.05) were considered to be significantly altered. To verify separation trends between different samples, principle component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were performed using SIMCA-P 11.0 software. All identified metabolites were sent to the KEGG pathway database to identify the KEGG ID and perform pathway analysis.

Figure 1. Locomotion assay of silkworm larvae in feeding stage (3d5I) and wandering stage (WS). The blue column represents the average locomotion distance of feeding stage (3d5I) larvae, and the orange column represents the average locomotion distance of wandering stage (WS) larvae. ***, p-value < 0.001. Error bars, SEM.

after release, the locomotion distances of wandering stage larvae (4.184 ± 0.539 cm, mean ± SEM) significantly exceeded those of feeding stage larvae (1.817 ± 0.162 cm). As time went on, the locomotion distances of wandering stage larvae reached 10.819 ± 1.330 cm (5 min after release) and 16.530 ± 2.209 cm (10 min after release), but those of feeding stage larvae were only 1.975 ± 0.189 cm (5 min after release) and 2.109 ± 0.207

Measurement of the Concentration of DA by LC−MS/MS

Twenty larval brains (with three replicates) were collected in 200 μL of 0.2 M perchloric acid42 and vortexed for 30 min at 4 195


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proteolysis, chitin metabolic process, iron ion transport, tyrosine metabolic process, and hormone catabolic process were significantly enriched (Figure 3A). Furthermore, under molecular function and cellular component, terms for structure molecular activity, enzyme regulator activity, endopeptidase regulator activity, ribosome, proteasome complex, actin filament, and proteasome regulator particle were remarkably enriched (Figure 3B,C).

cm (10 min after release). These data confirmed that silkworm larvae displayed ELA at wandering stage. Global Analysis of the Feeding Stage and Wandering Stage Larval Brain Proteins

A total of 23 906 peptides were identified by a MaxQuant search (Table S2) and assembled into 3004 proteins (Table S3). Among them, 2595 proteins were identified in feeding stage larval brain, 2715 proteins were identified in wandering stage larval brain, and 2318 proteins were identified in both feeding stage and wandering stage larval brain (Figure S2). The annotated MS/MS spectra of proteins identified by a single peptide are shown in Figure S3. The isoelectric points (pI) of these proteins ranged from 3.7 to 12.6, and the molecular weights (Mw) ranged from 2.1 to 1539.8 kDa.

Neural Signal Transduction Related Proteins

Neural signal transduction is the physiological basis of braincontrolled behavior. Generally, neural signal transduction requires neurotransmitters, synapses, neurofilaments, ion channels, receptors, and specialized enzymes.45 In insects, neurotransmitters exist in many forms, such as acetylcholine, γaminobutyric acid (GABA), glycine, glutamic acid, 5-hydroxytryptophane (5-HT), DA, and neuropeptides.46 In this study, a total of 64 neural signal transduction related proteins were identified (Table S6); those with significant differences (p-value < 0.05) are listed in Table 1. Twenty neurotransmitter related proteins were identified in our data, which included acetylcholine synthesis and degradation enzymes, GABA transporter and receptor, glycine or glutamic acid tRNA ligase, DA synthetase and degradation enzymes, and transporter. The most remarkable protein was the DA degradation enzyme, ebony protein, which was significantly downregulated at wandering stage (p-value = 0.019, log2FC = −1.89). The expression of another DA metabolic enzyme, DA N-acetyl transferase, was also decreased slightly (p-value = 0.019, log2FC = −0.51). Synapse-associated protein and neurofilament heavy polypeptide (NF-H) were also identified. Further analysis showed that NF-H was significantly upregulated at wandering stage (pvalue = 0.029, log2FC = 1.46). Twenty-one ion channel proteins were identified, including sodium, potassium, chloride, calcium, and other cation or anion transporting proteins. Among these ion channel proteins, no differences in expression were evident between the two stages except for three of them (gi|512926870, gi|512928856, BGIBMGA011016). Neuropeptides are small protein-like molecules that function in insect signal transduction. Twenty-one neuropeptides were found in the brain proteome, including PTTHs (one 22 KPTTH and ten bombyxins), prothoracicostatic peptide precursor, paralytic peptide precursor, and diapause hormone. None of them were changed at the protein level in the wandering stage brain except for one bombyxin.

Differentially Expressed Proteins and GO Analysis

A total of 465 differentially expressed proteins were found in the wandering stage brain, including 390 upregulated proteins and 75 downregulated proteins (Figure S4 and Table S4). These proteins were classified into three major categories (cellular component, molecular function, and biological process) according to GO analysis (Figure 2). In the cellular

Figure 2. Gene ontology terms for differentially expressed proteins in silkworm brain. Proteins were classified into cellular component, molecular function, and biological process by WEGO according to GO terms. The black columns represent the GO terms of upregulated proteins, and the gray columns represent the GO terms of downregulated proteins.

Brain Remodeling Related Proteins

component category, the GO terms cell, cell part, and organelle were the most abundant. The term membrane-enclosed lumen was found only in the upregulated group. The number of proteins classified as ribonucleoprotein complex and ribosome was higher in the upregulated group than in the downregulated group. In the molecular function category, four terms (methyltransferase, molecular transducer, signal transducer, and translation regulator) were found only in the upregulated group. In the biological process category, many proteins were found for amine metabolic process, anatomical structure formation, death, locomotion, and signal transduction. To further understand the function of differentially expressed proteins, we performed a GO enrichment analysis. According to Fisher’s exact test, 107 GO terms were enriched (p-value < 0.05; Table S5). Under the biological process category, protein metabolic process, macromolecular biosynthetic process,

During insect metamorphosis, some larval tissues undergo programmed cell death and remodeling to the adult tissues. It has been reported that the larval brain experiences dissociation and remodeling during metamorphosis.47 Along with tissue dissociation, many proteins are degraded. Two main processes operate to degrade proteins in vivo: the lysosome pathway and the ubiquitin proteasome system (UPS). Cathepsins are a class of lysosome enzyme that play key roles in insect tissue dissociation. We identified seven cathepsins in silkworm brain, which belonged to four types of cathepsins (cathepsin B, D, F, and L) (Table S7). Three of them were significantly upregulated at wandering stage (Table 2). UPS related proteins were abundant in the brain. A total of 55 proteins were identified (Table S7), all of which were upregulated (Table 2). These upregulated proteins further confirmed the role of 196


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Figure 3. Functional enrichment of differentially expressed proteins in silkworm brain. A, B, and C represent biological process, molecular function, and cellular component, respectively. The test set in the blue columns represents the GO terms of differentially expressed proteins, and the reference set in the orange columns represents the GO terms of all proteins (background) identified in silkworm brain. The x axis stands for the percentage of annotated proteins in each group.

dissociation and remodeling in the silkworm brain during metamorphosis.

a high correlation between qRT-PCR results and proteome data (Figure 4).

Verification of the Differentially Expressed Proteins by qRT-PCR

Differential Metabolites Identified by GC−MS

In all, 37 metabolites were identified in the brain of feeding and wandering stage silkworms (Table S8). Response values of these identified metabolites were sent to PCA and PLS-DA. The results revealed a large separated trend between the two samples (Figure S6). By differential analysis, we found 22 metabolites that were changed at the wandering stage, including 9 amino acids, 7 sugars and alcohols, and 6 acids and metabolic intermediates (Table 3). Among them, L-threonine, β-alanine, and L-alanine were significantly increased at wandering stage with a relative

To verify the accuracy of the proteome data, 10 proteins were selected for qRT-PCR analysis to confirm their expression at the transcription level. They were dopa decarboxylase, DA transporter, ebony protein, DA N-acetyl transferase, NF-H, PTTH, proteasome subunit alpha type-1, proteasome 26S nonATPase subunit 4, cathepsin L precursor, and organic cation transporter protein. The muscle-type A1 actin (BmActin, BGIBMGA013945) was used as an internal control. There was 197


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Journal of Proteome Research Pathway Analysis

Table 1. Neural Signal Transduction Related Proteins protein ID

seq description

log2FC

gi|512921767 BGIBMGA003199 BGIBMGA004570 gi|220983689 gi|160333514 BGIBMGA013705 gi|512886672 gi|512933094 gi|255661412 gi|512905918

Tryptophan 5-hydroxylase Dopa decarboxylase Dopamine transporter Ebony protein Dopamine N-acetyl transferase Glycine N-methyl transferase Glutamate/proline-tRNA ligase Neurofilament heavy polypeptide Sarco/endoplasmic reticulum Ca2+ ATPase Plasma membrane Ca2+-transporting ATPase Ca2+-binding mitochondrial carrier protein Ca2+-transporting ATPase Organic cation transporter protein Bombyxin

−12.37 0.49 0.66 −1.89 −0.51 3.89 0.96 1.46 0.31 0.36

gi|512926870 gi|512928856 BGIBMGA011016 gi|115186

All of the altered proteins and metabolites were sent to the KEGG database to identify the pathways involved. After searching against the database, 75 and 98 pathways were identified by use of proteome data and metabolome data, respectively (Table S9). The most abundant 35 pathways are shown in Figure S7. It can be easily seen that the differentially expressed proteins were involved in ribosome, proteasome, and lysosome pathways and in amino acid, sugar, and fatty acid metabolism (Figure S7A). Furthermore, metabolites were abundant in amino acid, sugar, and fatty acid metabolism and ABC transporters (Figure S7B). These results were consistent with the GO enrichment analysis shown in Figure 3. Measurement of DA in Brain Tissues

1.03 −13.25 1.73 −3.01

The calibration curves were linear over the DA concentration range of 1−10000 pg/μL. The correlation coefficients (r2) for the calibration curves were higher than 0.999. The within-day repeatability of the method was studied at concentration levels of 100 and 5000 pg. The relative standard deviations were below 10%. The concentrations of DA in feeding and wandering stage larval brains are shown in Figure 5. As the ELA occurred at wandering stage, the concentration of DA was significantly increased to 9.113 ± 0.346 ng/tissue (mean ± SD), which is higher than that in feeding stage brains (1.143 ± 0.048 ng/ tissue). Furthermore, we found that the concentration of DA in larval brains also increased 2.62-fold after 20E treatment (pvalue = 0.0013; Figure S8). This result indicated that 20E can elevate DA concentration.

Table 2. Brain Remodeling Related Proteins protein ID BGIBMGA011581 BGIBMGA011803 gi|112983984 BGIBMGA002559 gi|114051245 gi|512891246 BGIBMGA013898 BGIBMGA003335 BGIBMGA004376 gi|512914947 gi|114052605 gi|114051770 BGIBMGA008595 BGIBMGA005994 BGIBMGA005560 BGIBMGA003212 gi|512914524 gi|512931070 BGIBMGA002953 BGIBMGA009562 gi|512909998 BGIBMGA005014 gi|112983576 BGIBMGA007061 BGIBMGA011342

seq description Ubiquitin-like protein Ubiquitin-conjugating enzyme E2 Polyubiquitin Ubiquitin conjugating enzyme E2 Proteasome alpha 3 subunit Proteasome subunit alpha type-1 Proteasome zeta subunit Proteasome subunit beta type-6 Proteasome subunit beta type-5 Proteasome subunit beta type-2 26S proteasome regulatory ATPase subunit 10B 26S proteasome non-ATPase regulatory subunit 13 26S proteasome non-ATPase regulatory subunit 8 Proteasome 26S non-ATPase subunit 7 Proteasome 26S non-ATPase subunit 4 26S proteasome non-ATPase regulatory subunit 6 26S proteasome non-ATPase regulatory subunit 1 26S proteasome non-ATPase regulatory subunit 2 Probable 26S proteasome non-ATPase regulatory subunit 3 Proteasome 25 kDa subunit 26S proteasome non-ATPase regulatory subunit 10 Proteasome activator complex subunit 4 Cathepsin D precursor Cathepsin B precursor Cathepsin L precursor

log2FC 1.45 0.56 1.13 0.91 0.84 1.12 1.16 1.19 0.96 0.98 0.89

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DISCUSSION Locomotion, which is controlled by the central nervous system (CNS) in living organisms, is the foundation of sustaining life. For holometabolous insects such as the silkworm, ELA in the wandering stage is vital because the wandering behavior ensures that a safe and suitable place can be found for the silkworm to live through the pupal stage, during which the pupa cannot move. Wandering behavior in a defined metamorphic stage is a general phenomenon in holometabolous insects. However, the molecular mechanism of wandering behavior has not been determined yet. We are interested in what proteins or metabolites are used by the brain and how they work to induce wandering behavior. To address this question, we took advantage of integrated proteomics and metabolomics techniques to explain the molecular mechanism of silkworm wandering behavior.

0.99 0.76 0.91 2.01 0.79 1.15 1.46 1.35 3.25 2.78

Dopamine Is a Key Molecule for Insect Wandering Behavior

2.81 1.44 1.51 1.74

DA is the most abundant monoamine, mainly expressed in the insect brain, and plays a role as a neurotransmitter. The insect brain includes several DA systems, some of which are involved in hormone release and body movement.48 In insects, DA is synthesized from L-dopa under the catalysis of dopa decarboxylase (DD), and L-dopa is synthesized from tyrosine by tyrosine hydroxylase (TH).49 Meanwhile, there are two main pathways for DA degradation: DA N-acetyl transferase (DAT) converts DA into N-acetyl dopamine and N-β-alanyl dopamine synthetase (ebony) converts DA conjugated with β-alanine into N-β-alanyl dopamine (NBAD).49 In Drosophila, the function of the ebony protein is to regulate the concentration of DA, which is located in glial cells,50 and is closely related to locomotor activity. Reducing

ratio > 1.8. Other amino acids like L-asparagine, serine, Lproline, and L-valine were remarkably decreased with a relative ratio < −1.8. Interestingly, glutamic acid was detected only at feeding stage. All of the sugars and alcohols were significantly decreased at wandering stage. 9,12-Octadecadienoic acid (linoleic acid) was decreased with a relative ratio of −1.87. As an important energy supplier for insects, trehalose and linoleic acid were decreased in the wandering stage brain, which demonstrated that the wandering stage brain needs more energy than it does at the feeding stage. 198


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Figure 4. Verification of differentially expressed proteins by qRT-PCR. (A) Dopa decarboxylase; (B) dopamine transporter; (C) ebony protein; (D) dopamine N-acetyl transferase; (E) neurofilament heavy polypeptide; (F) prothoracicotropic hormone preproprotein; (G) proteasome subunit alpha type-1; (H) proteasome 26S non-ATPase subunit 4; (I) cathepsin L precursor; (J) organic cation transporter protein. 3d5I, feeding stage; WS, wandering stage. *, p-value < 0.05. **, p-value < 0.01. ***, p-value < 0.001. Error bars, SD.

Table 3. Differential Metabolites in Silkworm Brain metabolite name L-Threonine

β-Alanine L-Alanine N-Formylglycine L-Asparagine Serine L-Proline L-Valine Glutamic acid D-(−)-Lactic acid Pentanedioic acid Propanoic acid

KEGG ID Amino Acids C00188 C00099 C00041 C00152 C00065 C00148 C00183 C00025 C00256 C00489 C00163

relative ratioa

metabolite name

2.41 D-(−)-Fructose 1.96 Myo-Inositol 1.94 D-Allose −2.81 Glucitol −2.78 D-(+)-Trehalose −2.05 Xylitol −1.96 Cyclohexanol −1.89 3d5I specific Acids and Metabolic Intermediates 2.41 9,12-Octadecadienoic acid 1.81 Butanoic acid 1.74 Aconitic acid

KEGG ID

relative ratioa

Sugars and Alcohols C00095 C00137 C01487 C00794 C01083 C00379 C00854

−5.65 −3.32 −2.65 −2.12 −1.80 −1.74 −1.67

C06426 C00246 C00417

−1.87 −1.59 −2.97

a

The relative ratio was calculated by the mean value of response value for each metabolite. Positive values represent upregulated metabolites, and negative values represent downregulated metabolites.

the expression level of ebony protein causes an elevation in DA concentration, which finally leads to abnormal locomotor activity.51,52 In our study, four proteins related to insect DA synthesis and degradation pathways, dopa decarboxylase, ebony protein, DA N-acetyl transferase, and tyrosine hydroxylase, were identified (Table S6). Differential analysis showed that dopa decarboxylase increased at wandering stage, whereas ebony protein and

DA N-acetyl transferase decreased. The increase of DA synthetase and the reduction of DA degradation enzymes elevated the concentration of DA. As a result, more DA transporter would be needed to maintain the regular function of dopaminergic synapses (Table 1 and Figure 4B).53 β-Alanine is another substrate of ebony protein.54 Thus, the content of βalanine should increase after downregulation of ebony protein, which coincided with our metabolome data (Table 3; 1.96-fold 199


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when levels of glutamic acid are low, DA is overactive and results in the expression of schizophrenic symptoms.55 Excess glutamic acid injection into rat brain also causes a reduction of DA concentration.56 Our data identified only glutamic acid at feeding stage; this striking change in glutamic acid concentration may cause the rise in DA concentration in the silkworm brain at wandering stage. 20E is another molecule that can regulate the concentration of DA.57 Numerous studies have revealed that 20E titer increases at wandering stage.58−61 In our data, after 20E injection, the concentration of DA in silkworm brain also increased significantly (Figure S8). The increased 20E titers at wandering stage may elevate the DA concentration in the brain. In vertebrates, the contribution of DA to locomotor control is generally attributed to ascending dopaminergic projections. Injections of DA in the mesencephalic locomotor region increased the locomotor activity in lamprey.62 Similarly,

Figure 5. Concentration of dopamine in silkworm brain. 3d5I, feeding stage; WS, wandering stage. ***, p-value < 0.001. Error bars, SD.

higher). Tyrosine hydroxylase was not changed at wandering stage. Small molecules also play key roles in regulating the concentration of DA. Glutamic acid imbalances appear to cause abnormal functioning in DA metabolism. For example,

Figure 6. Hypothetic model of changes in the wandering silkworm brain. Red font stands for an upregulated protein or metabolite, and green font stands for a downregulated protein or metabolite. (A) Changes in axons: NF-H is upregulated at wandering stage. (B) Changes in dopaminergic synapse: DD, DT, and β-alanine are upregulated at wandering stage, whereas DAT and ebony are downregulated at wandering stage. (C) Summary flowchart displaying the physiological changes in the wandering stage silkworm. Red arrows represent the stimulatory effect; green arrows represent the inhibitory effect. NF-H, neurofilament heavy polypeptide; DD, dopa decarboxylase; Glu, glutamate; DA, dopamine; DAT, N-acetyl transferase; ebony, N-β-alanyl dopamine synthetase; DT, dopamine transporter; PTTH, prothoracicotropic hormone; 20E, 20-hydroxyecdysone; UPS, ubiquitinproteasome system. 200


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the remodeling of fat body,74 gut,75 and silk gland.76 In this study, seven cathepsins were identified; three of them were significantly upregulated at wandering stage, which suggested that those enzymes may participate in brain tissue dissociation. Ubiquitin is a small regulatory protein that has been found in almost all eukaryotic tissues. Its main function is to mark proteins that need to undergo proteolysis. In Drosophila, the dendrites from mature neurons experience large-scale morphological remodeling in response to changes in the environment; 20E and UPS are cell-intrinsic signals that initiate dendrite dissociation.77 Also, UPS plays an essential role in proteolysis during Trypanosoma cruzi remodeling.78 A large number of UPS-related proteins were identified in the present study, which might take part in the protein degradation process during brain remodeling. Thus, we propose that PTTH induces 20E synthesis and that 20E may initiate the morphological remodeling of the brain by the lysosomal pathway and UPS during insect metamorphosis.

significantly increased locomotor activity of invertebrates due to a higher DA concentration is found in Drosophila melanogaster.52 Research on the dysb′ mutant in Drosophila shows that its abnormal locomotor activity is related to the concentration of DA and ebony protein.63 When silkworms are infected by baculovirus, the ebony gene is downregulated in infected brain and infected silkworms display abnormal locomotor activity.64 Altogether, we found that the increase in DA synthetase and the decrease in DA degradation enzymes and glutamic acid concentration increased the DA concentration in the wandering stage brain, and this higher DA concentration could be a major factor driving wandering behavior. Neurofilaments Lay the Basis of Accelerated Signal Transductions in the Wandering Stage Brain

The CNS consists of billions of neurons, and its basic function is signal conduction. Axons originate from the neuron cell body and can extend from several micrometers to meters. Neurofilament proteins are a major component of the neuronal cytoskeleton and are believed to function primarily to provide structural support for the axon.65 The expression level of the neurofilament gene directly regulates the axonal diameter, and the latter, in turn, controls how fast signals travel down the axon.65 We discovered that NF-H was significantly increased at both the mRNA and protein levels (Figure 4E and Table 1). This suggested that the speed and frequency of neuronal signals become faster in the wandering stage brain. Hence, the accelerated signal transduction may lead to wandering behavior.

Molecular Mechanism of Wandering Behavior

On the basis of integrated proteomics and metabolomics data for the silkworm brain, a hypothetical model for elucidating the molecular mechanism of insect wandering behavior is displayed in Figure 6. The protein expression level of NF-H significantly increased at wandering stage, which made the axons wider and accelerated the signal transduction (Figure 6A). The accelerated signal transduction guaranteed that the larvae can actively accept internal and external stimuli and rapidly respond to them during metamorphosis. Accordingly, the accelerated signal transduction laid the physiological foundation for the wandering behavior. In addition, DA synthetase (dopa decarboxylase) was upregulated at wandering stage, whereas the degradation enzymes (ebony and DA N-acetyl transferase) were downregulated (Figure 6B). The glutamic acid concentration in the brain significantly decreased at wandering stage. These changes elevated the DA concentration in the brain to a level that finally induced wandering behavior. On the basis of previous research and our results, the hypothetical molecular events occurring in the wandering stage brain are shown in Figure 6C. PTTH secreted from the brain at wandering stage stimulates the PGs and elevates the 20E concentration in the hemolymph. On one hand, 20E regulates the levels of lysosomal enzymes and UPS proteins by transcription factors73 to initiate the brain remodeling process. On the other hand, ebony and other proteins are regulated by 20E by unknown pathways79 to elevate the DA concentration in the brain and induce wandering behavior. Previous studies about DA and ebony in locomotion have focused on general locomotor activity like crawling in Drosophila and swinging in lamprey. None of them has connected DA and ebony with insect wandering behavior. Our study is the first report using multiomics technology to claim that DA induces wandering behavior.

PTTH Induces 20E to Initiate Morphological Remodeling of the Brain

Neuropeptides are a kind of bioactive substance that exist in nervous tissue and play key roles in regulating the function of the nervous system. It has been reported that 51 neuropeptides are expressed in the silkworm brain, such as neuropeptide F1, short neuropeptide F, bombyxin, PTTH, corazonin, and so on.66 PTTH is a homodimeric glycoprotein; our qRT-PCR results showed that its coding gene was highly expressed at wandering stage (Figure 4F), which was consistent with previous results.66 At the protein level, we identified PTTH in both feeding stage and wandering stage, and there was no significant change between these two stages. Secreted by the brain, PTTH is transported to the PGs and activates a receptor tyrosine kinase to synthesize and release 20E.16 Thus, the expression levels of PTTH between the two stages showed no difference at the protein level, probably because PTTH had already been transported outside the brain. 20E regulates insect development in combination with juvenile hormone. The titer of 20E increases significantly at wandering stage to induce metamorphosis,58−61 when many insect organs undergo dissociation and remodeling.67 The brain and neurons also undergo dissociation and remodeling processes.66,68 Along with the remodeling process, postembryonic neurogenesis61 and programmed cell death69 occur. It is believed that cathepsins and UPS participate in this process under the regulation of 20E.70−72 Cathepsins are a class of important hydrolytic enzymes in lysosomes that play key roles in tissue dissociation. In Helicoverpa armigera, the fat body is dissociated at the prepupal stage by the action of cathepsin L under the regulation of 20E.73 In Sarcophage, cathepsin L is suggested to play the important role in the degradation of the brain’s basement membrane, which makes brain remodeling easier during metamorphosis.72 In silkworm, cathepsin is also involved in

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CONCLUSIONS In this study, we integrated comparative proteomics and metabolomics to analyze the brains from feeding stage and wandering stage silkworms, which enabled us to further understand the molecular mechanism of insect wandering behavior. Our data showed that both DA related proteins and metabolites were significantly changed at wandering stage. The 201


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KEGG, Kyoto encyclopedia of genes and genomes; LC−MS/ MS, liquid chromatography with tandem mass spectrometry; LFQ, label-free quantification; MALDI-TOF-MS, matrixassisted laser desorption with ionization time-of-flight mass spectrometry; Mw, molecular weight; NBAD, N-β-alanyl dopamine; NF-H, neurofilament heavy polypeptide; PCA, principle component analysis; PGs, prothoracic glands; pI, isoelectric point; PLS-DA, partial least-squares discriminant analysis; PTTH, prothoracicotropic hormone; qRT-PCR, quantitative real-time PCR; QC, quality control; SIM, selected ion monitoring; TH, tyrosine hydroxylase; UPS, ubiquitin proteasome system; WEGO, web gene ontology annotation plot

concentration of DA increased in the wandering stage brain, which we propose induced the wandering behavior at wandering stage. On the basis of our results, the molecular mechanism of insect wandering behavior is becoming clearer. Therefore, our findings can have far-reaching consequences for our understanding of insect ethology.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.5b00736. Photographs of locomotion assay (Figure S1); number of identified proteins in each sample (Figure S2); annotated MS/MS spectra for a single peptide identified proteins in silkworm brain (Figure S3); number of differentially expressed proteins at wandering stage (Figure S4); total ion chromatogram from GC−MS in two samples (Figure S5); PCA and PLS-DA of identified metabolites by GC− MS (Figure S6); pathway analysis of altered proteins and metabolites in silkworm brain (Figure S7); concentration of dopamine in silkworm brain after injection with 20E (Figure S8); primers used in qRT-PCR (Table S1); identified peptides from the two stages of B. mori brain (Table S2); identified proteins from the two stages of B. mori brain (Table S3); differentially expressed proteins from the two stages of B. mori brain (Table S4); functional enrichment of the differentially expressed proteins in brain (Table S5); neural signal transduction related proteins from the two stages of B. mori brain (Table S6); brain remodeling related proteins from the two stages of B. mori brain (Table S7); identified metabolites from the two stages of B. mori brain (Table S8): pathway analysis of the differentially expressed proteins and the differential metabolites in brain (Table S9) (ZIP)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 23 68250885. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Ying Xiong for technical help with the DA measurements. This study was supported by the National Basic Research Program of China (no. 2012CB114600), National High Technology Research and Development Program of China (no. 2011AA100306), National Natural Science Foundation of China (no. 31201853), and Basic and Advanced Research Project of Chongqing (no. cstc2015jcyjA0469).

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ABBREVIATIONS 3d5I, third day of the fifth instar larva; 5-HT, 5hydroxytryptophane; 20E, 20-hydroxyecdysone; CNS, central nervous system; DA, dopamine; DAT, dopamine N-acetyl transferase; DD, dopa decarboxylase; DT, dopamine transporter; DTT, dithiothreitol; ELA, enhanced locomotor activity; FC, fold change; GABA, γ-aminobutyric acid; GC−MS, gas chromatography mass spectrometry; GO, gene ontology; 202


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