The transformation from embryo to first instar nymph is an essential process in the insect life cycle. In order to characterize protein expression in the Ericerus pela Chavannes (Hemiptera: Coccoidea) egg, high-throughput proteomics and bioinformatics methods were used. A total of 678 peptides were identified and assigned to 358 protein groups. The proteins exhibited a wide range of molecular weight (3.50–495.12 kDa) and isoelectric points (3.50–13.1). Gene Ontology annotation showed that the majority of proteins were associated with cellular processes, metabolic processes, and response to stimulus processes. The predominant molecular functions of E. pela egg proteins included binding, catalytic activity, transporter activity, and structural molecule activity. Kyoto Encyclopedia of Genes and Genomes annotations identified 137 pathways, and most proteins were assigned to metabolism events, including many enzymes participating in energy metabolism, protein folding, sorting, and degradation. The processes and functions of the identified proteins were closely related to the physiological status of egg and embryo development. We conclude that some identified proteins are related to important egg biological characteristics, and regard them as the target proteins for future study. Key words: Ericerus pela Chavannes, egg, protein profile Ericerus pela Chavannes (Hemiptera: Coccoidea) is one of the oldest cuticle and the male pupal stage (Yang et al. 2011, Yang and Chen economic insect. It has been used successfully in commercial wax 2014), and transcriptome analyses of adults and pupae (Yang production, and reared by humans for more than a thousand years. et al. 2012, 2015; Yu et al. 2016). The results of these aforemen- White wax is secreted only by the male E. pela, and has an increas- tioned studies, coupled with in-depth study of biology and ecology ingly wide utilization; examples include candle production, printing, of E. pela will lay the foundation for further proteomic study of medicine, food, cosmetic industries, and precision machinery (Chen E. pela eggs and understanding the roles of proteins at this stage. and Feng 2009). Proteomic analyses of other insect eggs or embryos have been well The oviposition of E. pela occurs about in March and April documented (Amenya et al. 2010; Li et al. 2010, 2011; Müller et al. each year, and incubation happens in April and May. Eggs are laid 2010; Gala et al. 2013), and can provide beneficial references for in the capsule of the female adult every day during the oviposition the present study. period; the maximum and minimum amount of eggs laid per adult The present study was performed to explore the protein expres- are 18,047 and 12,000, respectively (Wu and Zhong 1983). When the sion profile of E. pela eggs, generate hypotheses about the con- oviposition period is over, the ventral side of the female adult body is nections between certain proteins and biological characteristics of very close to its dorsal side, and the adult will soon die. The eggs, with E. pela eggs, and provide basic information for studying target pro- wax powder on their surface, will incubate in the closed capsule for teins in future. at least 29 d (Wu and Zhong 1983). After the newly hatched nymphs crawl out of the capsule, the male nymphs live in the shadow of the Materials and Methods host plant in a gregarious manner, and secret an amount of white wax Insect Culture to cover themselves until eclosion, while the female nymphs scatter E. pela were cultured on the branches of Ligustrum lucidum, in the on the host plant and do not secrete the white wax (Chen 2011). experimental field of Research Institute of Resources Insects of the Research on E. pela eggs at molecule level has not been reported Chinese Academy of Forestry in Kunming (longitude: 102°42ʹE; up to date. There are only proteomic analyses of the male adult © The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact email@example.com Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/6/4825055 by Ed 'DeepDyve' Gillespie user on 16 March 2018 2 Journal of Insect Science, 2018, Vol. 18, No. 1 latitude: 25°02ʹN). When the first nymphs crawled out of their cap- The raw data from LC–MS/MS was analyzed using maxquant sules in March, the eggs in these capsules were collected in tubes and 1.3, the parameters were set as: the maximum number of missed stored at -80℃. cleavages a peptide for 2, the trypsin digestion, carbamidomethyl (C) for fixed modification, oxidation (M), and acetyl (N-term) for vari- Protein Extraction able modifications, proteins false discovery rate ≤0.01, and as well as peptides, specifying the string for reverse and contaminant hits. In Before protein extraction, the eggs collected from different mother this study, an in-house database was used for proteomic data ana- scale insects were mixed and randomly divided into three groups, and lysis, constructed by combining the coding sequences from E. pela washed in phosphate buffer solution (pH 7.2) three times. Total pro- Illumina transcriptome sequencing databases (Yang et al. 2012) tein of each group was extracted on ice in 1 ml lysis buffer (contain- with insect sequence data downloaded from the National Center ing 10% glycerin, 2.5% SDS, 5% β-mercaptoethanol, and 62.5 mM for Biotechnology Information (NCBI) Nr (nonredundant) database Tris–HCl, pH 6.8) per mg of sample homogenate. The homogenate and the SwissProt protein database. extraction was kept for 10 min at room temperature and then sub- jected to five rounds of sonication treatment in an ice-bath, each time for 20 s with a 20 s interval. After centrifugation at 20,000 g, the Bioinformatic Analysis supernatant was aliquoted and stored at −20℃. Protein concentra- Functional analysis of the identified proteins was performed using tion was determined with a Bradford protein assay kit (Beyotime, UniProt Knowledgebase (Swiss-Prot + TrEMBL) (http://www. Shanghai, China), BSA (Sigma, USA) was taken as standard. uniprot.org), and proteins were grouped on the basis of their bio- logical process and molecular function of Gene Ontology terms SDS–PAGE Separation and In-gel Digestion (Gala et al. 2013). The GO annotation terms were obtained from Before being subjected to SDS–PAGE, the extracted total protein was Web Gene Ontology Annotation Plotting (http://wego.genomics. dissolved in SDS–PAGE loading buffer, boiled for 5 min, centrifuged at org.cn/). All the identified proteins were searched against the 20,000 g for 10 min, loaded on a 5% stacking gel and 12% separating Kyoto Encyclopedia of Genes and Genomes (KEGG) (http:// www. gel, and was run at 15 mA for 30 min and then 30 mA for 2 hr in a genome.jp/kegg) to identify the correlated pathways. The protein mini-vertical electrophoresis system (Bio-Rad, USA). After electrophor- enzyme commission numbers were obtained based on the best esis, the gel was stained overnight in a solution of 0.1% (w/v) Coomassie matches (E-value ≤e-15). Brilliant Blue G-250 (Sangon Biotech, Shanghai, China), 30% (v/v) methanol, and 10% (v/v) glacial acetic acid. After decolorization, the gel Results was analyzed for bands along with a molecular weight marker. Thereafter, the gel containing all bands was cut into 1 mm par- Molecular Weight and Isoelectric Point of the ticles for in-gel digestion: gel particles were washed three times in Identified Proteins deionized water and subsequently dehydrated with 100% aceto- In this study, about 678 unique peptides were identified, ascribed nitrile (ACN) for 10 min. The particles were incubated with 100 mM to 358 protein groups. These identified proteins exhibited a broad DTT for 30 min at 56℃. The resulting free thiol (–SH) groups were range of theoretical molecular weight (MW); one major group com- subsequently alkylated by incubating the samples with 200 mM prised proteins with MW between 10 and 30 kDa, and remarkably, iodoacetamide for 20 min in the dark. Gels were washed with there were eight proteins with MW exceeding 300 kDa. With regard 25 mM ammonium bicarbonate and dehydrated with 100% ACN to theoretical isoelectric point (pI), about 70% of the total proteins sequentially. Thereafter, 10 ng/μl trypsin (Promega, USA) was added had pI in the range of 4–8, the pI of three proteins was <4, and the and incubated for 20 hr at 37℃ for protein digestion. Supernatants pI of 15 proteins was >11 (Fig. 1 and Table 1). were transferred to fresh tubes for mass spectrometric analysis. Categorization of the Identified Proteins Protein Identification Using LC–MS/MS The identified proteins were categorized based on the biological pro- The resuspended extracts were separated and identified using HPLC cess and molecular function as predicted from associated GO terms (Easy nLC system, ThermoQuest, San Jose, CA) coupled with (Table 1). Most of the proteins were predicted to act on material Q-Exactive mass spectrometer (thermo Fisher, San Jose, CA). One conversion and transportation, and information modulation, and microliter of sample was loaded on a trapping column (Thermo sci- the proteins associated with metabolism of carbohydrates and en- entific EASY column (2 cm × 100 μm 5 μm-C )) each time. After ergy, amino acid metabolism, nucleotide metabolism, transcription flow-splitting, peptides were transferred to the analytical column and translation, and other categories are shown in Table 1. (Thermo scientific EASY column (75 μm × 100 mm 3 μm-C )) We found that a large number of proteins associated trans- for separation equilibrated with buffer A (0.1% methanoic acid in formation of matter and energy were expressed during egg devel- water) and buffer B (84% ACN, 0.1% methanoic acid in water), a opment. Many identified enzymes were related to the glycolytic 280 min linear gradient was set: buffer B started from 0 to 60% at pathway, pentose phosphate pathway and tricarboxylic acid cycle a flow rate of 250 nl/min, came to 100% subsequently, and then (Supp Table 1 [online only]). Pyruvate kinase (NP_001036906.1), maintained constantly at this flow rate. malate dehydrogenase (XP_001659012.1), phosphoglyceromu- The mass spectra of the peptides were recorded on a Q-Exactive tase (NP_001037540.1), aldehyde dehydrogenase 2 family (mito- mass spectrometer. The positive ions were adopted as the mode of chondrial) (NP_001087022.1), ATP synthase (NP_001040233.1), Scanning MS spectra, the MS analysis was performed with one full GI20614 (XP_002005703.1), and other enzymes were identified, MS scan (m/z 300–1800) with the resolution (R = 70,000) at m/z and assigned to different carbohydrate metabolism pathways and 200 and dynamic exclusion (40.0 s), followed by MS/MS scans on energy metabolism pathways, which suggested the carbohydrate and the 10 most intense ions from the MS spectrum. Collision-induced energy metabolism was vigorously performing in this stage. dissociation was conducted with normalized collision energy of 35% RNA-binding protein 8A (XP_001849141.1), ribosomal pro- and voltage of 27 eV. tein L28 (NP_001155658.1), and other 20 proteins took part Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/6/4825055 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 1 3 Fig. 1. Theoretical molecular weight (MW) and isoelectric point (pI) distribution of the identified proteins. The bar represents the number of proteins and the solid square represent the percentage of per group of proteins to the total identified proteins. (A) distribution of MW, (B) distribution of pI. in spliceosome pathway (ID: ko03040), ribosome pathway (ID: Biological Process and Molecular Functions of the ko03010), and other seven pathways (Supp Table 2 [online only]), Identified Proteins which suggested a number of transcription and translation programs Based on the biological process and molecular function according to were now in process, which is consistent with an embryo in a vigor- the GO terms, in total, 213 proteins were found to be involved in 22 ously developing stage. categories of biological processes (Fig. 2). Most proteins were related In the identified proteins, tubulin, actin, profilin, myosin, micro- to cellular process (84.04%) and metabolic process (73.71%), which tubule-associated proteins, other cytoskeleton proteins and some was consistent with the active cell division and vigorous metabolism chaperonin proteins, such as t-complex protein 1, were identified. during the course of embryo development. Proteins related to the GO These proteins involved in the phagosome pathway (ID: ko04145), term ‘response to stimulus’ showed high representation (25.82%), tight junction pathway (ID: ko04530), and gap junction pathway and proteins involved in ‘cell proliferation’, ‘multi-organism pro- (ID: ko04540). The organization of the cytoskeleton plays important cess’, and ‘growth’ made the lowest representation (0.47%) in our roles in cell morphogenesis, and the identification of these proteins protein profile. Molecular function terms associated with E. pela egg might be due to the various development programs acting during proteins revealed that most of the proteins were involved in binding embryogenesis (Supp Table 3 [online only]). (49.09%), followed by catalytic activity, transporter activity, and Another group of identified proteins were HSPs, among them, structural molecule activity. Proteins related to other functions were HSP 90 and HSP 70 were predominant (Supp Table 3 [online only]). represented as small groups (Fig. 3). In order to reveal the enzyme Many members of this group might perform chaperone function by classes in E. pela egg, proteins with catalytic feature were further stabilizing new proteins to ensure correct folding or by helping to classified (Fig. 4). The enzyme distribution illustrated that hydrolases refold proteins that were damaged by the cell stress. accounted for the largest proportion (49.28%), followed by oxidore- In addition, some proteins were protective for the embryo develop- ductases and transferases. ment. In this study, various proteasomes and ubiquitin proteins were identified (Supp Table 3 [online only]). The identified proteasomes KEGG Pathway Analysis were involved in the proteasome pathway (ID: ko03050) and antigen processing and presentation pathway (ID: ko04612). These might be When searched against KEGG reference pathway database, 129 necessary to regulate specific proteins and remove protein misfolding proteins were assigned to 137 KEGG pathways, which were during the progress of E. pela embryo development. Moreover, some ascribed to five categories: organismal systems, metabolism, genetic proteins associated with antioxidant system were identified (Supp information processing, environmental information processing, Table 3 [online only]), they might play important roles in removing and cellular processes (Fig. 5). In the metabolism term, there were harmful metabolites conducted in the process of embryo development. 56 pathways identified. In particular, 20, 24, and 12 proteins were, In the protein profile of the E. pela egg, some kinds of proteins respectively, found in connection with 14 carbohydrate metabo- were likely to be related to the specific differentiation and mor - lism pathways, five energy metabolism pathways, and nine amino phogenesis programs for various tissues and organs, as well as acid metabolism pathways. There were 18 identified biological early nymphal morphogenesis (Supp Table 4 [online only]). These pathways in genetic information processing, folding sorting and proteins included chitinase, cuticular protein analogous to peritro- degradation were so complex and active that six pathways were phins 3-B precursor, prophenoloxidase, similar to n-synaptobrevin associated with 32 proteins in this processing. Under the cellular CG17248-PA, muscular protein 20, transformer-2 sex-determining process category, 18 proteins were involved in four pathways and protein, and other proteins, which were likely to be the important linked with transport and catabolism. Environmental information proteins for cuticular, nerve tissue, and reproductive organ forma- processing included signal transduction (eight pathways) and sig- tion of the new nymph. From these results, it was presumed that the naling molecules and interaction term (three pathways), the pro- embryo development was in its later stage. The result was consistent teins involved in the former were more numerous than the proteins with what we would expect at a stage close to egg hatching. involved in the latter. In the organismal systems term category, 10 Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/6/4825055 by Ed 'DeepDyve' Gillespie user on 16 March 2018 4 Journal of Insect Science, 2018, Vol. 18, No. 1 Table 1. Categorization of the identified proteins of E. pela egg based on the GO analysis Protein description ACC Mol. Biological_process Molecular_function weight [kDa]/pI Carbohydrate and energy metabolism GF23287 XP_001964635.1 56/10 Acetyl-CoA biosynthetic pro- Pyruvate dehydrogenase (acetyl-transferring) cess from pyruvate activity AGAP011066-PA XP_309579.4 3.5/8.1 Oxidoreductase activity Isocitrate dehydrogenase XP_001971666.1 54/6.7 Isocitrate metabolic process NAD binding [NADP] MGC80785 protein NP_001087022.1 6.5/7.7 Oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor Putative uncharacterized XP_967960.2 56/7.2 Oxidoreductase activity, acting on the aldehyde protein or oxo group of donors, NAD or NADP as acceptor GJ19670 XP_002058888.1 80/7.6 Carbohydrate metabolic Hydrolase activity, hydrolyzing O-glycosyl process compounds AAEL004297-PA XP_001648848.1 123/7.2 Cellular carbohydrate meta- ATP binding bolic process AGAP009039-PA XP_319791.4 10/7.9 carbohydrate metabolic Carbohydrate binding process Glucose-6-phosphate isomeraseXP_002005703.1 62/7 Gluconeogenesis Glucose-6-phosphate isomerase activity phosphoglyceromutase NP_001037540.1 28/6.8 Glycolysis Phosphoglycerate mutase activity GJ15342 XP_002059015.1 37/9.2 ATP citrate synthase activity GI22035 XP_002001326.1 6.5/7.5 Catalytic activity pyruvate kinase NP_001036906.1 31/5.2 Glycolysis Pyruvate kinase activity GJ17558 XP_002052468.1 28/8.8 Porphyrin-containing com- Coproporphyrinogen oxidase activity pound biosynthetic process GE24063 XP_002098232.1 8.1/7.2 Holocytochrome-c synthase activity GH22974 XP_001995110.1 73/7.3 Tricarboxylic acid cycle Oxidoreductase activity, acting on the CH-CH group of donors AAEL008167-PB XP_001658987.1 52/8.6 Fumarate metabolic process Fumarate hydratase activity ATP synthase subunit alpha NP_001040233.1 13/9.6 ATP hydrolysis coupled proton ATP binding transport Oligomycin sensitivity-confer- XP_968733.1 7.2/10 ATP synthesis coupled proton Proton-transporting ATP synthase activity, rota- ring protein transport tional mechanism vacuolar ATP synthase cata- NP_001091829.1 45/5 ATP hydrolysis coupled proton ATP binding lytic subunit A transport V-type proton ATPase sub- P31402.1 7.4/9.5 ATP hydrolysis coupled proton Proton-transporting ATPase activity, rotational unit E transport mechanism GJ16665 XP_002051636.1 93/5.9 ATP hydrolysis coupled proton Hydrogen ion transmembrane transporter transport activity electron-transfer-flavoprotein NP_001040123.1 18/9 Electron carrier activity beta polypeptide Acyl carrier protein XP_311483.3 7.6/4.7 Fatty acid biosynthetic process malate dehydrogenase, putativeXP_002432539.1 36/9.4 Malate metabolic process l-Malate dehydrogenase activity malate dehydrogenase XP_001659012.1 14/9.3 Malate metabolic process l-Malate dehydrogenase activity luciferase-like protein BAI66602.1 60/8.8 Bioluminescence catalytic activity Amino acid metabolism peroxidase XP_001867956.1 9.6/6.6 Response to oxidative stress Peroxidase activity GE13192 XP_002090578.1 12/6.5 Response to oxidative stress Peroxidase activity AGAP000901-PA XP_316880.5 61/8.3 Biosynthetic process Catalytic activity phosphoserine XP_001849403.1 30/8.6 l-Serine biosynthetic process O-Phospho-l-serine:2-oxoglutarate aminotrans- aminotransferase ferase activity GF18621 XP_001955137.1 62/7.4 Proteolysis Aminopeptidase activity GI23169 XP_001999260.1 5.3/6.6 Proteolysis Aminopeptidase activity prophenoloxidase XP_001661190.1 6.4/6.7 Oxidation–reduction process Oxidoreductase activity Lipid metabolism AGAP011066-PA XP_309579.4 3.5/8.1 Oxidoreductase activity aldehyde dehydrogenase NP_001087022.1 6.5/7.3 Oxidoreductase activity (NAD+) fatty acid synthase XP_001845135.1 249/6 Biosynthetic process Hydrolase activity, acting on ester bonds S-acetyltransferase Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/6/4825055 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 1 5 Table 1. Continued Protein description ACC Mol. Biological_process Molecular_function weight [kDa]/pI Putative uncharacterized XP_971757.1 32/9.2 Catalytic activity protein GK10733 XP_002061054.1 11/9.6 Fatty-acyl-CoA reductase (alcohol-forming) activity Nucleotide metabolism MGC130953 protein NP_001090100.1 1/6.50 Purine nucleotide biosynthetic IMP cyclohydrolase activity process GE23527 XP_002099157.1 9.2/8 Purine nucleotide biosynthetic IMP cyclohydrolase activity process gualynate kinase-1 ACD69431.1 1/6.58 Purine nucleotide metabolic Guanylate kinase activity process Nudix (Nucleoside diphos- NP_001002323.1 7.3/5.9 Bis(5′-nucleosyl)-tetraphosphatase activity phate linked moiety X)-type motif 2 GMP synthase, putative XP_002427615.1 76/7.1 GMP biosynthetic process GMP synthase (glutamine-hydrolyzing) activity GK17059 XP_002061681.1 17/4.6 Transcription, DNA-dependent Ribonucleoside-diphosphate XP_001660977.1 30/7 DNA replication Ribonucleoside-diphosphate reductase activity, reductase thioredoxin disulfide as acceptor AAEL003193-PB XP_001656515.1 42/6.6 Phosphate-containing com- Inorganic diphosphatase activity pound metabolic process Inosine-5’-monophosphate XP_309514.2 14/8.3 GMP biosynthetic process IMP dehydrogenase activity dehydrogenase GD20335 XP_002103291.1 12/5.9 Flavin adenine dinucleotide binding transformer-2 sex-determining XP_002432020.1 15.6/10.5 Nucleic acid binding,nucleotide binding protein, putative Transcription and translation heterogeneous nuclear ribonu- NP_001093319.1 22/7.1 Nucleic acid binding cleoprotein A1 Putative uncharacterized pro- XP_973561.1 31/10 Nucleic acid binding tein GLEAN_07585 AAEL007239-PA XP_001658243.1 44/8.3 Nucleic acid binding GK13948 XP_002073099.1 43/12 Nucleotide binding GL19239 XP_002014541.1 86/7.9 Threonyl-tRNA Threonine-tRNA ligase activity aminoacylation leucyl-tRNA synthetase, XP_002422927.1 97/8.3 Leucyl-tRNA aminoacylation LEUCINE-tRNA ligase activity putative GL25810 XP_002018701.1 12/5.8 Alanyl-tRNA aminoacylation Alanine-tRNA ligase activity 60S ribosomal protein L10A, XP_002426587.1 25/11 Translation RNA binding putative ACYPI006342 protein NP_001155658.1 16/12 Translation Structural constituent of ribosome GH23036 XP_001995231.1 19/11 Translation RNA binding Putative uncharacterized XP_967571.1 24/9.8 Translation RNA binding protein eukaryotic translation initi- XP_001842254.1 1/4.68 RNA metabolic process Translation initiation factor activity ation factor 5 Lysine-tRNA ligase NP_572573.1 38/6.5 Lysyl-tRNA aminoacylation Lysine-tRNA ligase activity GTP-binding nuclear protein XP_002423913.1 50/9.2 Nucleocytoplasmic transport GTP binding RAN1, putative RNA-binding protein 8A XP_001849141.1 19/4.8 RNA processing RNA binding Folding, sorting, and degradation proteasome subunit alpha type, XP_002422679.1 4.7/5.8 Ubiquitin-dependent protein Threonine-type endopeptidase activity putative catabolic process Proteasome subunit alpha type NP_001040387.1 5.9/5.1 Ubiquitin-dependent protein Threonine-type endopeptidase activity catabolic process Proteasome subunit beta type XP_317882.3 25/6.9 Proteolysis involved in cellular Threonine-type endopeptidase activity protein catabolic process GK22666 XP_002072105.1 16/4.8 Regulation of catalytic activity Enzyme regulator activity heat shock 70 kDa protein XP_001850527.1 71/5.2 Response to stress ATP binding cognate 4 HSP 70 B2 XP_001861436.1 70/5.5 Response to stress ATP binding Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/6/4825055 by Ed 'DeepDyve' Gillespie user on 16 March 2018 6 Journal of Insect Science, 2018, Vol. 18, No. 1 Table 1. Continued Protein description ACC Mol. Biological_process Molecular_function weight [kDa]/pI 60 kDa HSP, mitochondrial XP_001850501.1 60/5.2 Protein refolding ATP binding GG25088 XP_001968832.1 61/6.1 Protein refolding ATP binding HSP 90 kDa alpha (cytosolic), NP_001025655.1 77/4.7 Protein refolding;response to stress class B member 1 HSP 83 XP_001865484.1 82/4.6 Response to stress ATP binding HSP 90 protein, putative XP_002432348.1 83/4.7 Protein refolding; response to ATP binding stress disulfide isomerase XP_001866126.1 11/6.2 Glycerol ether metabolic Isomerase activity process GG17350 XP_001980787.1 47/4.2 Protein folding Calcium ion binding Prkcsh-prov protein NP_001087124.1 5.5/4.1 N-glycan processing Calcium ion binding AGAP001424-PA XP_321706.5 91/4.6 Protein refolding ATP binding AAEL012827-PA XP_001662951.1 27/4.5 Protein refolding ATP binding 78 kDa glucose-regulated XP_001845218.1 72/4.8 ATP binding protein GH11975 XP_001991597.1 72/5 ATP binding Putative uncharacterized XP_971446.1 45/7.6 Response to heat ATP binding protein Thioredoxin domain-contain- ACO12744.1 26/4.6 Cell redox homeostasis ing protein 1 GJ22764 XP_002054516.1 55/9.1 Ubiquitin-protein ligase activity Transitional endoplasmic re- XP_966692.1 89/5.1 Nucleoside-triphosphatase activity ticulum ATPase TER94 Transport and catabolism Dnase2-prov protein NP_001086671.1 9.7/6.8 DNA metabolic process Deoxyribonuclease II activity GL12416 XP_002019473.1 136/7.4 ATP catabolic process ATPase activity, coupled to transmembrane movement of substances GG20906 XP_001974813.1 53/4.8 Microtubule-based process GTP binding Rab-protein 5 XP_001813105.1 24/8.6 Protein transport GTP binding GK13103 XP_002073518.1 21/4.6 Sphingolipid metabolic process RAC GTPase, putative XP_002429222.1 21/6.8 Small GTPase mediated signal GTP binding transduction Development and organism system GD25430 XP_002082004.1 13/6.5 Oxidation–reduction process Oxidoreductase activity AAEL012062-PC XP_001662217.1 45/5.2 ATP biosynthetic process Monovalent inorganic cation transmembrane transporter activity clathrin light chain XP_001868264.1 6.7/4.2 Intracellular protein transport Structural molecule activity GL25029 XP_002021066.1 10/9.5 Fatty-acyl-CoA binding GF20350 XP_001963345.1 10/8.9 Zinc ion binding AAEL003413-PA XP_001656777.1 28/6.9 Serine-type endopeptidase inhibitor activity AGAP007452-PA XP_001687921.1 299/7.1 Regulation of Rho protein Rho guanyl-nucleotide exchange factor activity signal transduction lumbrokinase-3(1) XP_001844812.1 5.4/6.7 Proteolysis Kinase activity GK12466 XP_002072489.1 9/4.7 Proteolysis Serine-type endopeptidase activity leukocyte elastase inhibitor NP_001089382.1 17/6 Serine-type endopeptidase inhibitor activity Alpha-2-antiplasmin NP_777095.1 13/5.5 Acute-phase response serine-type endopeptidase inhibitor activity Signaling AGAP007523-PB XP_308355.3 231/5.2 Motor activity GA13959 XP_001360276.2 5.4/5.2 Motor activity AAEL004141-PA XP_001648499.1 11/6.5 Transport GTP-binding protein alpha XP_001858618.1 41/5.1 G-protein coupled receptor Signal transducer activity subunit, gna signaling pathway GK23973 XP_002064648.1 4/7.8 Voltage-gated anion channel activity GK17256 XP_002061919.1 59/4.8 Regulation of cell adhesion Peptidyl-prolyl cis–trans XP_002134398.1 25/10 Protein peptidyl-prolyl Peptidyl-prolyl cis-trans isomerase activity isomerase isomerization Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/6/4825055 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 1 7 Fig. 2. Biological process components by proportion according to GO classification. Fig. 3. Molecular function components by proportion according to GO classification. pathways were included in the immune system, and the proteins Target Proteins Selection related to immune system were the most numerous, which would One aim of our study was to identify for future study proteins that be consistent with the embryo requiring active protection from may be closely connected with biological and ecological characteris- pathogens at this stage. tics of E. pela. These proteins were selected through comparison and analysis using the known functional information of the most similar protein in another insect as a justification, combining this informa- Similarity Distribution of the Identified Proteins tion with the biological and ecological characteristics of E. pela and The identified nonredundant proteins were analyzed for their sim- the known information on the molecular level about E. pela. The ilarity distribution in the database. A majority of the annotated protein named GK10733 (XP_002061054.1) has fatty-acyl-CoA re- proteins shared similarity with proteins from arthropods (Fig. 6). ductase (alcohol-forming) activity and is related to cutin, suberine, E. pela shared maximum similarity with Drosophila (26.19%), and wax biosynthesis pathway according to GO and KEGG ana- followed by different mosquito fauna, beetles, Pediculus humanus lysis (Table 1 and Supp Table 4 [online only]). We hypothesized that corporis, and Maconellicoccus hirsutus Green (Hemiptera: fatty-acyl-CoA reductase is relevant to wax secretion on the surface Pseudococcidae), etc. Out of the identified nonredundant proteins, of E. pela eggs. A number of HSPs with different molecular weight only about 6.09% proteins exhibited similarity with the known were identified (Table 1 and Supp Table 3 [online only]), and we proteins of scale insects. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/6/4825055 by Ed 'DeepDyve' Gillespie user on 16 March 2018 8 Journal of Insect Science, 2018, Vol. 18, No. 1 Fig. 4. Enzyme classes of nonredundant proteins according to GO classification. Fig. 5. Classification of pathways according to the definition in KEGG. The pathways were clustered into cellular processes (A), environmental information processing (B), genetic information processing (C), metabolism (D), and organismal systems (E). have previously studied some HSP genes of E. pela (Liu et al. 2013) study. The egg is light yellow in color, and has features commonly in an earlier study. We hypothesized that these HSPs are very likely found in an insect egg. E. pela embryonic development begins with tied to stress resistance to the environment. We analyzed the possible cleavage by karyokinesis, goes through the formation of blasto- relationship between the genes identified in this study and the typical derm and germ band, formation and disappearing of the amnion ecological and biological characteristics of the E. pela egg in part of and serosa, differentiation of the germinal layer, germ band section- our discussion, and will use this as a basis for further investigation. alization, the formation of appendage, as well as the formation of the alimentary canal, nerve tissue, dorsal blood vessel, and genera- tive cells (Zhao and Wu 1990). The external body is well developed Discussion on the 15th day after oviposition, and the nymph crawls out of the chorion on the 18th day after oviposition. However, the duration of E. pela is one of the most economically valuable insects, belonging the egg stage varies with temperature and other factors (Zhao and to the family Coccidae. There exists minimal research about E. pela Wu 1990). During the egg development process, protein expression embryo development. The protein component of the E. pela egg is active, and biosynthesis and catabolism programs are performed. during embryo development stage has not been reported until this Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/6/4825055 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 1 9 Fig. 6. Pie diagram showing similarity distribution of nonredundant proteins assigned by GO analysis (represented as the percentage of total similarity proteins). Embryo development is a sequential and complex process controlled out that the expression of FAR protein in the egg stage is possibly in by genes. Some proteins are constitutively expressed throughout the preparation for the secreting wax behavior of E. pela nymphs. development process—these are indispensable for egg development. The existence of proteins expressed in specific stages of the egg sug- HSPs in E. pela Egg gests that the different developmental stages need specific protein(s) HSPs are known to play a vital role in both normal cellular homeo- to proceed correctly (Fang and Li 2010). stasis and stress response, and are involved in many biological func- In this study, we obtained the protein profile of the E. pela egg tions such as cellular communication, immune response, protein at stages close to hatching. A large number of identified proteins transport, cell cycle regulation, apoptosis, gametogenesis, and aging were related to metabolism and organismal systems pathways, and (Sarkar et al. 2011). A report documented that Hsp70 and small the results were in accordance with the physiological development HSPs are probably the major players in midgut metamorphosis in features of E. pela egg. The identified enzymes in the E. Pela egg Spodoptera litura (Gu et al. 2012); this viewpoint provides valu- shared maximum similarity with proteins in Drosophila, and the able insight into the roles of the HSP superfamily in insect meta- fact that Drosophila proteins are generally very well characterized morphosis. Furthermore, HSPs are documented widely as defensive was helpful to predict the function of E. pela proteins. On the basis response proteins to stress factors including heat shock, cold shock, of KEGG pathway analysis and GO analysis, we discuss the possible and other abiotic stresses and biotic stresses in insects (Zhao and relationship among some identified proteins and the biological and Jones 2012). The functions of various HSP often overlap but can ecological characteristics of the E. pela egg. be different between different proteins (Zhang and Denlinger 2010, Benoit et al. 2011, Michaud et al. 2011, Xu et al. 2011). In the pres- FAR and Secreting Wax Behavior ent study, heat shock 70 kDa protein cognate 4 and HSP 70 B2 were According to the wax ester biosynthesis pathway in organism identified, and these were primarily involved in the spliceosome (Cheng and Russell 2004, Doan et al. 2009, Liénard et al. 2010, pathway, the protein processing in endoplasmic reticulum pathway, Teerawanichpan and Qiu 2010, Teerawanichpan et al. 2010), fatty- the MAPK signaling pathway, the endocytosis pathway and antigen acyl-CoA reductase (FAR) and wax synthase are the key enzymes. In processing and presentation pathway on the basis of KEGG ana- this study, some detected proteins were predicted to be involved in lysis, which showed these proteins were associated with multiple white wax synthesis. Among them, a protein named GK10733 had biological processes. Moreover, some higher molecular weight pro- fatty-acyl-CoA reductase (alcohol-forming) activity, and was found teins, HSP 90 kDa alpha (cytosolic) and HSP 83, were identified to be related to the cutin, suberin, and wax biosynthesis pathways for the plant–pathogen interaction pathway, the progesterone-me- according to KEGG analysis (pathway ID: ko00073). The mRNA diated oocyte maturation pathway, the antigen processing and pres- level of E. pela FAR gene in nymphs has previously been analyzed entation, the NOD-like receptor signaling pathway, the PI3K-Akt using qRT-PCR, and E. pela FAR was assumed the key enzyme to signaling pathway, and the protein processing in endoplasmic re- white wax biosynthesis (Yang et al. 2012). In each capsule, all eggs, ticulum pathway. In addition, other HSPs were identified, including without exception, are covered with some wax powder in natural HSP cognate 5, HSP beta-6-like isoform 1, HSP 68a, 60 kDa HSP conditions. Before this study, some researchers postulated that the (mitochondrial), and a few putative small HSPs. We hypothesized wax adhering to the surface of eggs was secreted by the mother (Wu that the identified HSPs likely exhibit very important role in aiding and Zhong 1983), but there was not enough evidence to support organogenesis by folding newly synthesized proteins, binding other this hypothesis. In the present study, we predict that FAR is likely non-native proteins, and assisting proteins in the correct folding and involved in the wax formation on the surface of E. pela eggs, though functional actualization. the quantity of wax is so small that this has not generally been a pri- Furthermore, synthesis of the relevant literature about E. pela, ority for study by researchers. On the other hand, we also cannot rule particularly as regards its ecological strategy, some HSPs likely Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/6/4825055 by Ed 'DeepDyve' Gillespie user on 16 March 2018 10 Journal of Insect Science, 2018, Vol. 18, No. 1 primarily function in response to very high heat and humidity stress. against toxic ROS, superoxide dismutases, catalases, peroxidases, We reasoned that, first, thousands of eggs are laid in relatively closed thioredoxin, glutathione peroxidase, and other enzymes (Wang ootheca, and they need to respond to intense heat, and likely hypoxia. et al. 2008). Superoxide dismutase (XP_002048532.1), peroxidase Secondly, temperature and humidity is very high in the source region (XP_001867956.1), manganese superoxide dismutase (AEL79287.1), of white wax during the period of E. pela hatching, thus the egg glutathione S-transferase theta (ACB36909.1), aldo-keto reductase likely responds to this abiotic stress by using the regulatory mecha- (XP_001844819.1), and other proteins were found to be expressed in nism of HSPs. The host plant is often infested by pathogens because the E. pela eggs stage. These proteins might participate in the protec- of the scale insect colonization, and there are many pollutants on the tive pathways in order to provide essential protection from harmful surface of egg capsule, but surprisingly impaired eggs were not found metabolites during embryo development of E. pela. in our investigation. For this reason, we hypothesized that there is some inhibitory mechanism, potentially HSPs, in effect to protect Conclusions the eggs from these harms. Therefore, considerable further work is needed to fully understand these mechanisms, and the HSPs will be E. pela is a model for scale insects. This study provided the first pro- target proteins in our future work. teomic analysis in the eggs of E. pela near hatching, which provided a basis to elucidate the mechanism underlying embryogenesis, and illuminated candidate proteins for deeper research. Some identified Amount of Metabolic Energy Required for proteins might be directly correlated to the biological characteris- Development tics of the eggs at the stage at which they were examined. Further A very radical morphological transformation is exhibited from egg research is needed to verify the functions of these important proteins. to nymphal stage, and large amount of carbohydrate metabolism and energy production is needed to undergo extensive organogenesis during this process. In this study, according to GO and KEGG clas- Supplementary Data sification, some important proteins were implicated in carbohydrate Supplementary data are available at Journal of Insect Science online. metabolism and energy production. In particular, the citrate cycle (TCA cycle), glycolysis/gluconeogenesis, pyruvate metabolism, pro- panoate metabolism, and pentose and glucuronate interconversions Acknowledgments were assigned 41 proteins (32%), and 24 proteins (19%) involved This work is supported by Research Funds for the Central Non-profit in energy metabolism. 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Journal of Insect Science – Oxford University Press
Published: Jan 1, 2018
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