Shotgun proteomic analysis of wing discs from the domesticated silkworm (Bombyx mori) during metamorphosis

Proteomic profiles from the wing discs of silkworms at the larval, pupal, and adult moth stages were determined using shotgun proteomics and MS sequencing. We identified 241, 218, and 223 proteins from the larval, pupal, and adult moth stages, respectively, of which 139 were shared by all three stages. In addition, there were 55, 37, and 43 specific proteins identified at the larval, pupal, and adult moth stages, respectively. More metabolic enzymes were identified among the specific proteins expressed in the wing disc of larvae compared with pupae and moths. The identification of FKBP45 and the chitinase-like protein EN03 as two proteins solely expressed at the larval stage indicate these two proteins may be involved in the immunological functions of larvae. The myosin heavy chain was identified in the pupal wing disc, suggesting its involvement in the formation of wing muscle. Some proteins, such as proteasome alpha 3 subunits and ribosomal proteins, specifically identified from the moth stage may be involved in the degradation of old cuticle proteins and new cuticle protein synthesis. Gene ontology analysis of proteins specific to each of these three stages enabled their association with cellular component, molecular function, and biological process categories. The analysis of similarities and differences in these identified proteins will greatly further our understanding of wing disc development in silkworm and other insects.     

Proteomic profiling of the silkworm skeletal muscle proteins during larval-pupal metamorphosis

The silkworm is a typical holometabolous insect going through drastic morphological changes upon metamorphosis from larvae to pupae. Comprehensive studies focusing on the changes help elucidate understanding of a biogenic mechanism. Here, we report the initial profile of the intersegmental muscle (ISM) proteins of the silkworm during larval-pupal metamorphosis. In total, 258 protein spots were resolved by two-dimensional gel electrophoresis (2-DE). Fifty-seven larval proteins were identified, where 3 proteins were exclusively detected in larval samples. Fifty-four other proteins were common in pupal samples. Of these, 12 proteins belonging to the contractile apparatus, metabolism, regulation, and signal transduction were altered in their contents during the metamorphosis from larvae to pupae. Three pupa-defective proteins were identified as isoforms of troponin I, followed by an immunoblotting validation. This data will be helpful in understanding the biochemistry of an insect ISM.     

Comparative proteomics analysis of salt response reveals sex-related photosynthetic inhibition by salinity in Populus cathayana cuttings

Male and female poplar ( Populus cathayana Rehd.) cuttings respond differently to salinity stress. To understand these differences better, comparative morphological, physiological, and proteomics analyses were performed. Treatments with different concentrations of NaCl applied to male and female poplar cuttings for 4 weeks showed that females reacted more negatively at the morphological and physiological levels than did males, visible as shriveled leaves, decreased growth, lowered photosynthetic capacities, and greater Na(+) accumulation. The proteome analysis identified 73 proteins from 82 sexually related salt-responsive spots. They were involved in photosynthesis, protein folding and assembly, synthesis and degradation, carbon, energy and steroid metabolism, plant stress and defense, redox homeostasis, signal transduction, and so forth. The sex-related changes of these proteins were consistent with the different morphological and physiological responses in males and females. In conclusion, the higher salt resistance of male P. cathayana cuttings is related to higher expression and lower degradation of proteins in the photosynthetic apparatus, more effective metabolic mechanism and protective system, and greater capacity of hydrogen peroxide scavenging. This research allows us to further understand the possible different management strategies of cellular activities in male and female Populus when confronted by salt stress.     

葱蝇非滞育、 冬滞育和夏滞育蛹发育和形态特征比较

昆虫非滞育、 冬滞育和夏滞育蛹具有不同的生理和发育过程。本研究以葱蝇Delia antiqua作为模式种, 以黑腹果蝇Drosophila melanogaster蛹的发育形态特征和命名为参照, 用解剖、 拍照、 长度测量和图像处理等方法系统地比较研究了非滞育、 冬滞育和夏滞育蛹的发育历期和形态变化, 重点在头外翻和滞育相关发育和形态特征, 目的在于弄清非滞育、 冬滞育和夏滞育蛹发育和形态特征差异, 为滞育发育阶段的识别、 滞育分子机理研究奠定形态学基础。冬滞育蛹的滞育前期、 滞育期和滞育后期分别为4, 85和27 d, 夏滞育蛹的滞育前期、 滞育期和滞育后期分别为2, 8和22 d。从化蛹至头外翻完成为滞育前期, 头外翻完成约10 h内复眼中央游离脂肪体可见。头外翻的完成是滞育发生的前提, 非滞育、 夏滞育和冬滞育蛹头外翻发生在化蛹后的48, 36和83 h, 在头外翻过程中发育形态没有差异。头外翻的过程为： 首先, 头囊和胸部附肢从胸腔外翻, 头部形态出现; 然后, 腹部肌肉继续收缩, 将血淋巴和脂肪体推进头部及胸部附肢。葱蝇蛹在完成蛹期有效积温约15%时进入冬滞育或夏滞育。在滞育期, 蛹的形态一直停留在复眼中央游离脂肪体可见这一形态时期, 且冬滞育和夏滞育的蛹在形态上没有区别。在体长、 体宽和体重上, 冬滞育蛹最大, 夏滞育蛹次之, 非滞育蛹最小。在滞育后期, 在黄色体出现期间, 非滞育蛹的马氏管清楚可见, 呈绿色, 而滞育蛹的马氏管几乎不可见。本研究为认知昆虫滞育生理、 从发育历期和形态上推断滞育发育进程提供了依据。     

Proteomic analyses of Oryza sativa mature pollen reveal novel proteins associated with pollen germination and tube growth

As a highly reduced organism, pollen performs specialized functions to generate and carry sperm into the ovule by its polarily growing pollen tube. Yet the molecular genetic basis of these functions is poorly understood. Here, we identified 322 unique proteins, most of which were not reported previously to be in pollen, from mature pollen of Oryza sativa L. ssp japonica using a proteomic approach, 23% of them having more than one isoform. Functional classification reveals that an overrepresentation of the proteins was related to signal transduction (10%), wall remodeling and metabolism (11%), and protein synthesis, assembly and degradation (14%), as well as carbohydrate and energy metabolism (25%). Further, 11% of the identified proteins are functionally unknown and do not contain any conserved domain associated with known activities. These analyses also identified 5 novel proteins by de novo sequencing and revealed several important proteins, mainly involved in signal transduction (such as protein kinases, receptor kinase-interacting proteins, guanosine 5'-diphosphate dissociation inhibitors, C2 domain-containing proteins, cyclophilins), protein synthesis, assembly and degradation (such as prohibitin, mitochondrial processing peptidase, putative UFD1, AAA+ ATPase), and wall remodeling and metabolism (such as reversibly glycosylated polypeptides, cellulose synthase-like OsCsLF7). The study is the first close investigation, to our knowledge, of protein complement in mature pollen, and presents useful molecular information at the protein level to further understand the mechanisms underlying pollen germination and tube growth.     

Proteomic analysis of pulmonary tissue in tail-suspended rats under simulated weightlessness

Weightlessness affects lung function and even causes certain damages to pulmonary tissue. This study used rat tail-suspension model to simulate the physiological effects of weightlessness and investigate the alterations of lung proteome, to reveal the mechanism of lung injury under weightlessness condition. Twenty male Sprague-Dawley rats were randomly divided into two groups: tail-suspended and control. Protein samples from pulmonary tissue of tail-suspended and control groups were separated by two-dimensional (2D) gel electrophoresis and analyzed with ImageMaster 2D elite software. Differentially expressed proteins were identified by high definition mass spectrometry (HDMS) in combination with database searching. Seventeen differentially expressed proteins were identified, among which 13 proteins were upregulated, and four proteins downregulated. The functions of these identified proteins can be classified into six classes related to: metabolism, oxidative stress, cellular functions, cytoskeletal proteins, signal tranduction, and protein degradation. They are mainly related to cellular energy metabolism, stress and inflammatory response, cell injury and repair, intracellular signal transduction, and other cellular functions, playing important roles in weightlessness-induced lung injury.     

小峰熊蜂蜂王蛹期发育蛋白质组分析

为了探明小峰熊蜂Bombus hypocrita蜂王蛹期发育蛋白质表达调控方面的特点,揭示其发育的分子机理。采用双向电泳法对小峰熊蜂蜂王蛹期发育进行蛋白质组研究,结果在小峰熊蜂蜂王蛹期的白眼期(A期)、褐眼期(B期)和黑眼期(C期)分别检测到81、80和75个蛋白点,特有蛋白质分别为8个、7个和2个,共有蛋白质为61个,A期到B期有4个蛋白质显著上调,5个显著下调,B期到C期有7个蛋白质显著上调,1个显著下调,A期到C期有10个蛋白质显著上调,有4个显著下调。此外,3个蛋白质是在A、B期表达C期关闭,6个蛋白质A、C期表达,B期关闭,5个蛋白质A期关闭,而B、C期表达。初步表明小峰熊蜂蜂王从蛹期发育到成蜂过程中,不仅需要一些保守蛋白质来调控,而且还需要一些特异蛋白质。     

Blood protein synthesis in pupae of the silkmoth, Hyalophora cecropia

The blood proteins of pupae of Hyalophora cecropia were studied by acrylamide gel electrophoresis. Analyses of fractions separated on Sephadex G-75 columns revealed that a series of low molecular weight proteins previously reported to be absent in diapausing pupae are a normal component of diapausing pupal blood. Incorporation of isotopically labelled leucine into blood protein bands of injured pupae, perfused pupae, and perfused isolated pupal abdomens (lacking a midgut) revealed that each of these three preparations could synthesize all of the low molecular weight protein bands, with maximal incorporation in the perfused whole pupae.     

Common and distinct roles of juvenile hormone signaling genes in metamorphosis of holometabolous and hemimetabolous insects

Insect larvae metamorphose to winged and reproductive adults either directly (hemimetaboly) or through an intermediary pupal stage (holometaboly). In either case juvenile hormone (JH) prevents metamorphosis until a larva has attained an appropriate phase of development. In holometabolous insects, JH acts through its putative receptor Methoprene-tolerant (Met) to regulate Krüppel-homolog 1 (Kr-h1) and Broad-Complex (BR-C) genes. While Met and Kr-h1 prevent precocious metamorphosis in pre-final larval instars, BR-C specifies the pupal stage. How JH signaling operates in hemimetabolous insects is poorly understood. Here, we compare the function of Met, Kr-h1 and BR-C genes in the two types of insects. Using systemic RNAi in the hemimetabolous true bug, Pyrrhocoris apterus, we show that Met conveys the JH signal to prevent premature metamorphosis by maintaining high expression of Kr-h1. Knockdown of either Met or Kr-h1 (but not of BR-C) in penultimate-instar Pyrrhocoris larvae causes precocious development of adult color pattern, wings and genitalia. A natural fall of Kr-h1 expression in the last larval instar normally permits adult development, and treatment with an exogenous JH mimic methoprene at this time requires both Met and Kr-h1 to block the adult program and induce an extra larval instar. Met and Kr-h1 therefore serve as JH-dependent repressors of deleterious precocious metamorphic changes in both hemimetabolous and holometabolous juveniles, whereas BR-C has been recruited for a new role in specifying the holometabolous pupa. These results show that despite considerable evolutionary distance, insects with diverse developmental strategies employ a common-core JH signaling pathway to commit to adult morphogenesis.     

葱蝇非滞育与夏滞育蛹蛋白的双向凝胶电泳比较分析

【目的】比较分析葱蝇Delia antiqua非滞育与夏滞育蛹的蛋白表达差异, 为进一步揭示昆虫滞育的分子调控机理和昆虫防治提供理论基础。【方法】以非滞育和夏滞育的蛹为材料, 提取总蛋白; 进行双向凝胶电泳和凝胶图像分析, 对并差异蛋白质进行MALDI-TOF MS质谱鉴定, 获得该点的质量指纹图谱; 利用MASCOT软件在NCBI和SWISS PORT蛋白质数据库中进行搜索鉴定。【结果】非滞育和夏滞育蛹的蛋白表达存在显著差异。通过质谱鉴定和生物信息学分析, 13个差异表达的蛋白质分别为胶原蛋白、 纺锤丝组装非正常蛋白6(SAS6)、 5,10-亚甲基四氢叶酸合成酶(MTHFS)、 Bnb蛋白(bangles and beads)及其他功能未知蛋白。【结论】葱蝇在夏滞育时期, 蛹体内的某些蛋白被上调或下调表达。本研究所鉴定的蛋白中, 部分可能是参与滞育相关的蛋白质网络中的成员, 它们可能在抗高温、 染色体分离、 叶酸代谢等生理过程中发挥重要作用。     

Proteomic analysis of salicylic acid induced resistance to Mungbean Yellow Mosaic India Virus in Vigna mungo

The role of salicylic acid (SA) in inducing resistance to MYMIV infection in Vigna mungo has been elucidated by proteomics. Twenty-nine proteins identified by MALDI-TOF/TOF, predicted to be involved in stress responses, metabolism, photosynthesis, transport and signal transduction, showed increased abundance upon SA treatment. Susceptible plants showed characteristic yellow mosaic symptoms upon MYMIV infection. A concentration dependent decrease in physiological symptoms associated with MYMIV was observed upon exogenous SA treatment prior to viral inoculation; and no visible symptom was observed at 100 μM SA. SA treatment stimulated SOD and GPX activity and inhibited CAT activity thus preventing ROS mediated damage. Significant increase in chlorophyll, protein, carbohydrate, phenolic content and H(2)O(2) were observed. Involvement of calmodulin for transmission of defense signal by SA is suggested. A metabolic reprogramming leading to enhanced synthesis of proteins involved in primary and secondary metabolisms is necessary for SA mediated resistance to MYMIV. Identification of proteins showing increased abundance, involved in photosynthetic process is a significant finding which restores virus-induced degradation of the photosynthetic apparatus and provides enhanced metabolites required for repartition of resources towards defense.     

Comparative proteomic analysis between fifth‐instar nymphs and adults of Asian citrus psyllid Diaphorina citri

Asian citrus psyllid Diaphorina citri Kuwayama is extremely problematic worldwide, particularly where Huanglongbing (HLB) disease, the most serious and devastating of citrus diseases, is found. The threat is a result of its ability to transmit the causal agent of HLB, Candidatus Liberibacter asiaticus (CLas) bacterium. Improvements in proteomics, mass spectrometry, bioinformatics tools and gene ontology annotation facilitate the mapping and large‐scale identification and quantification of proteins. To date, only a few comparative proteomic studies report the developmental proteomic changes of hemimetabolous and plant–disease vector insects. Two‐dimensional gel electrophoresis analysis of D. citri total protein is able to detect qualitative and quantitative developmental differences. Liquid chromatography‐tandem mass spectrometry identifies 89 protein spots. Most proteins are metabolism and bioenergetics‐related. Nineteen protein spots are found to be implicated in stress/defence/immunity; 7 in development regulation; 9 in nervous system functions; 4 in the reproductive system; 23 in cytoskeleton and muscle organization; and 4 in movement, flight and other processes. Significant increases in the level of proteins related to structural constitution of the skeleton, stress/defence/immunity, reproduction system, muscles, locomotion and flight are found in adults, consistent with the fact that D. citri is a hemimetabolous insect, whereas proteins involved in developmental regulation are higher in the nymphal stage. The identification of these variably expressed proteins between the nymph and adult stages, linked with the basis of their physiological roles, will lead to a better understanding of the factors influencing development in D. citri and the regulation of some crucial metabolic pathways. It may also help to identify targets for genetic manipulation using RNA interference or other techniques to disrupt Asian citrus psyllid development, lifespan or its ability to transmit CLas.     

Comparative proteomics analysis reveals an intimate protein network provoked by hydrogen peroxide stress in rice seedling leaves

Hydrogen peroxide (H2O2) plays a dual role in plants as the toxic by-product of normal cell metabolism and as a regulatory molecule in stress perception and signal transduction. However, a clear inventory as to how this dual function is regulated in plants is far from complete. In particular, how plants maintain survival under oxidative stress via adjustments of the intercellular metabolic network and antioxidative system is largely unknown. To investigate the responses of rice seedlings to H2O2 stress, changes in protein expression were analyzed using a comparative proteomics approach. Treatments with different concentrations of H2O2 for 6 h on 12-day-old rice seedlings resulted in several stressful phenotypes such as rolling leaves, decreased photosynthetic and photorespiratory rates, and elevated H2O2 accumulation. Analysis of approximately 2000 protein spots on each two-dimensional electrophoresis gel revealed 144 differentially expressed proteins. Of them, 65 protein spots were up-regulated, and 79 were down-regulated under at least one of the H2O2 treatment concentrations. Furthermore 129 differentially expressed protein spots were identified by mass spectrometry to match 89 diverse protein species. These identified proteins are involved in different cellular responses and metabolic processes with obvious functional tendencies toward cell defense, redox homeostasis, signal transduction, protein synthesis and degradation, photosynthesis and photorespiration, and carbohydrate/energy metabolism, indicating a good correlation between oxidative stress-responsive proteins and leaf physiological changes. The abundance changes of these proteins, together with their putative functions and participation in physiological reactions, produce an oxidative stress-responsive network at the protein level in H2O2-treated rice seedling leaves. Such a protein network allows us to further understand the possible management strategy of cellular activities occurring in the H2O2-treated rice seedling leaves and provides new insights into oxidative stress responses in plants.     

LARVAL X‐RAY IRRADIATION INFLUENCES PROTEIN EXPRESSION IN PUPAE OF THE ORIENTAL FRUIT FLY,BACTROCERA DORSALIS

The sterile insect technique (SIT) was developed to eradicate the new world screwworm from the southern United States and Mexico, and became a component of many area‐wide integrated pest management programs, particularly useful in managing tephritid fruit flies. SIT is based on the idea of rearing and sterilizing male pests, originally by ionizing radiation, and then releasing into field, where they compete for and mate with wild females. Mating with sterile males leads to reduced fecundity to lower pest populations. There are concerns with the use and distribution of radioisotopes for SIT programs, which have led to developing X‐ray irradiation protocols to sterilize insects. We considered the possibility that X‐ray irradiation exerts sublethal impacts aside form sterilizing insects. Such effects may not be directly observable, which led us to the hypothesis that X‐ray irradiation in one life stage creates alterations in biological fitness and protein expression in the subsequent stage. We tested our hypothesis by irradiating larvae of Bactrocera dorsalis. There are two major points. One, exposing larvae to X‐ray treatments led to reduced adult emergence, fecundity, fertility, and flight capacity from the corresponding pupae and emerged adults. Two, the X‐ray treatments led to substantial expression changes in 27 pupal proteins. We assorted the 67 spots representing these proteins into three groups, metabolism, development, and structure. Our interpretation is these X‐ray induced changes in biological performance and protein expression indicate their adult counterparts may be disabled in their abilities to successfully compete for and mate wild females in native habitats.     

Proteomic analysis of honeybee (Apis mellifera L.) pupae head development

The honeybee pupae development influences its future adult condition as well as honey and royal jelly productions. However, the molecular mechanism that regulates honeybee pupae head metamorphosis is still poorly understood. To further our understand of the associated molecular mechanism, we investigated the protein change of the honeybee pupae head at 5 time-points using 2-D electrophoresis, mass spectrometry, bioinformatics, quantitative real-time polymerase chain reaction and Western blot analysis. Accordingly, 58 protein spots altered their expression across the 5 time points (13-20 days), of which 36 proteins involved in the head organogenesis were upregulated during early stages (13-17 days). However, 22 proteins involved in regulating the pupae head neuron and gland development were upregulated at later developmental stages (19-20 days). Also, the functional enrichment analysis further suggests that proteins related to carbohydrate metabolism and energy production, development, cytoskeleton and protein folding were highly involved in the generation of organs and development of honeybee pupal head. Furthermore, the constructed protein interaction network predicted 33 proteins acting as key nodes of honeybee pupae head growth of which 9 and 4 proteins were validated at gene and protein levels, respectively. In this study, we uncovered potential protein species involved in the formation of honeybee pupae head development along with their specific temporal requirements. This first proteomic result allows deeper understanding of the proteome profile changes during honeybee pupae head development and provides important potential candidate proteins for future reverse genetic research on honeybee pupae head development to improve the performance of related organs.     

Differential proteomic analysis of Trichoplusia ni cells after continuous selection with activated Cry1Ac toxin

Development of insect resistance to Bacillus thuringiensis (Bt) toxins threatens the sustained successful application of Bt-based biological control tactics. Multi-mechanisms of resistance have been proposed, such as alteration of toxin-binding proteins, changes of proteases in midgut and so on. The other responses of the Cry1Ac-selected insects might also contribute to the evolution of resistance. Here, the Cry1Ac-selected Trichoplusia ni TnH5 cells with high resistance were subjected to analysis of proteome and the differentially expressed proteins were identified using mass spectrometry. The differential proteins included transporter, molecular chaperon, structural molecules and many other molecules involved in protein metabolism, signal transduction, nucleotide binding, lipid biosynthesis, carbohydrates metabolism and energy production, suggesting that a complex mechanisms involved in the development of insect resistance to Bt Cry1Ac toxins at cellular levels. The decrease of protein synthesis, changes of signal transduction, more rapid energy production, the enhanced lipid synthesis and the decline of possible Cry1Ac-binding proteins in cytoplasm and other events might contribute to the development of resistance in the selected cells. Our results provide some new cues for understanding the mechanism of Bt resistance.     

Prostaglandins A1 and E1 influence gene expression in an established insect cell line (BCIRL-HzAM1 cells)

Prostaglandins (PGs) and other eicosanoids exert important physiological actions in insects and other invertebrates, including influencing ion transport and mediating cellular immune defense functions. Although these actions are very well documented, we have no information on the mechanisms of PGs actions in insect cells. Here we report on the outcomes of experiments designed to test our hypothesis that PGs modulate gene expression in an insect cell line established from pupal ovarian tissue of the moth Helicoverpa zea (BCIRL-HzAM1 cells). We treated cells with either PGA(1) or PGE(1) for 12 or 24h then analyzed cell lysates by 2-D electrophoresis. Analysis of the gels by densitometry revealed substantial changes in protein expression in some of the protein spots we analyzed. These spots were processed for mass spectrometric analysis by MALDI TOF/TOF, which yielded in silico protein identities for all 34 spots. The apparent changes in three of the proteins were confirmed by semi-quantative PCR, showing that the changes in mRNA expression were reflected in changes in protein expression. The 34 proteins were sorted into six categories, protein actions, lipid metabolism, signal transduction, protection, cell functions and metabolism. The findings support the hypothesis that one mechanism of PG action in insect cells is the modulation of gene expression.