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2-DE and CBB G-250 stained gels image showing differentially expressed protein spots profile obtained from different developmental stages of honeybee pupae heads. Each identified protein spot is specified by number and indicated by arrow, where the spot number with prefix ''u'' and ''d'' indicate up or down regulation. doi:10.1371/journal.pone.0020428.g003
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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 hea...
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... head weight between 13-17 days was significantly heavier than those at 19-20 days (Fig. 2). Subse- quently protein extracts from pupae head were separated using 2- DE and comparisons were done across 5 time points. As a result, the molecular weight (Mr) and pI of pupae head proteins ranged from 10.45 to 125.16 kDa and 3.7 to 9.56, respectively (Fig. 3). The 2-DE image analysis revealed nearly 400 spots on each gel, of which 85 spots showed differential expressions (.1.5 fold change, p,0.05) and 59 of them were successfully identified by MALDI-TOF MS (Table S2 and Fig. 3). The remaining unidentified proteins spots could be attributed to their lower abundance to produce enough spectra, ...
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... the molecular weight (Mr) and pI of pupae head proteins ranged from 10.45 to 125.16 kDa and 3.7 to 9.56, respectively (Fig. 3). The 2-DE image analysis revealed nearly 400 spots on each gel, of which 85 spots showed differential expressions (.1.5 fold change, p,0.05) and 59 of them were successfully identified by MALDI-TOF MS (Table S2 and Fig. 3). The remaining unidentified proteins spots could be attributed to their lower abundance to produce enough spectra, or because the databases search scores were not high enough (,95%) to yield unambig- uous results. In this study, some protein spots were identified as the same proteins, but appeared differently on 2-DE gels, most likely ...
Citations
... hypopharyngeal and mandibular glands) located in the heads [17][18][19]. Recently, a number of studies have focused on gaining insights on transcriptomic and proteomic changes in the honey bee heads in response to a stressor (nutrition or pesticides) [20][21][22][23][24]. Since honey bee heads house important brood food-producing glands, sensory structures (antennae), the brain and organs for visual acuity and taste, it is important to understand the impacts of dietary manipulations on the head regions of the nurse bees. ...
Phytosterols are important micronutrients that are precursors of important molting hormones and help maintain cellular membrane integrity in insects including bees. Previous research has shown that 24-methylenecholesterol is a key phytosterol that enhances honey bee longevity and improves nurse bee physiology. Nurse bees have the ability to selectively transfer this sterol to developing larvae through brood food. This study examines the physiological impacts of 24-methylenecholesterol on nurse bees, by analyzing the protein profiles of nurse bee heads upon dietary sterol manipulation. Dietary experimental groups consisting of newly emerged honey bees were provided with varying concentrations of 24-methylenecholesterol for three weeks. At the end of the study, honey bees were collected and proteomic analysis was performed on honey bee heads. A total of 1715 proteins were identified across experimental groups. The mean relative abundances of nutritional marker proteins (viz. major royal jelly proteins 1, 4, 5, 7) were higher in experimental groups supplemented with higher dietary sterol concentrations, when compared with the control dietary group. The mean relative abundances of important enzymatic proteins (aminopeptidase and calcium-transporting ATPase) were higher in control groups, whereas mean relative abundances of oxysterol-binding protein and fatty acid-binding protein were higher in higher dietary sterol groups.
... The odorant proteins are required for the adequate recognition of chemical stimuli by the insect olfactory system 69 , and decreased expression can impair the perception of stimuli by nurse bees and affect their functionality in the colony. The proteins 14-3-3 epsilon and 14-3-3 zeta that are involved in learning and memory in insects 70,71 were also downregulated in bees exposed to pesticides. These results suggest an impairment of these functions in bees due to exposure to fipronil, pyraclostrobin, and pyraclostrobin + fipronil. ...
Understanding the effect of pesticides on the survival of honeybee colonies is important because these pollinators are reportedly declining globally. In the present study, we examined the changes in the head proteome of nurse honeybees exposed to individual and combined pesticides (the fungicide pyraclostrobin and the insecticide fipronil) at field-relevant doses (850 and 2.5 ppb, respectively). The head proteomes of bees exposed to pesticides were compared with those of bees that were not exposed, and proteins with differences in expression were identified by mass spectrometry. The exposure of nurse bees to pesticides reduced the expression of four of the major royal jelly proteins (MRJP1, MRJP2, MRJP4, and MRJP5) and also several proteins associated with carbohydrate metabolism and energy synthesis, the antioxidant system, detoxification, biosynthesis, amino acid metabolism, transcription and translation, protein folding and binding, olfaction, and learning and memory. Overall, when pyraclostrobin and fipronil were combined, the changes in protein expression were exacerbated. Our results demonstrate that vital proteins and metabolic processes are impaired in nurse honeybees exposed to pesticides in doses close to those experienced by these insects in the field, increasing their susceptibility to stressors and affecting the nutrition and maintenance of both managed and natural colonies.
... A series of changes occur in the body structure and pigmentation of the compound eye during this period [75]. The earliest proteomic work on pupal heads using 2-DE found 58 differentially expressed proteins at five points in time (13,15,17,19 and 20 days), of which 36 proteins were involved in the organogenesis of the head occurring in early development [76]. However, 22 of these proteins were involved in regulating the development of pupal head neurons and glands that occurred later in the development of the pupae head [76]. ...
... The earliest proteomic work on pupal heads using 2-DE found 58 differentially expressed proteins at five points in time (13,15,17,19 and 20 days), of which 36 proteins were involved in the organogenesis of the head occurring in early development [76]. However, 22 of these proteins were involved in regulating the development of pupal head neurons and glands that occurred later in the development of the pupae head [76]. Furthermore, of the identified ∼129 proteins by 2-DE proteomics of worker red-eye pupae hemolymph [77], most of the proteins during non-feeding stage are related to physiological changes during the metamorphosis [77,78], and as red-eyes develop into a newly emerged bee, protein quality drops significantly [78]. ...
Advances in instrumentation and computational analysis in proteomics have opened new doors for honeybee biological research at the molecular and biochemical levels. Proteomics has greatly expanded the understanding of honeybee biology since its introduction in 2005, through which key signaling pathways and proteins that drive honeybee development and behavioral physiology have been identified. This is critical for downstream mechanistic investigation by knocking a gene down/out or overexpressing it and being able to attribute a specific phenotype/biochemical change to that gene. Here, we review how emerging proteome research has contributed to the new understanding of honeybee biology. A systematic and comprehensive analysis of global scientific progress in honeybee proteome research is essential for a better understanding of research topics and trends, and is potentially useful for future research directions.
... 2D-DIGE.Cardoen et al. (2012) found further overlap from 2D-DIGE against the gene screens ofGrozinger et al. (2007; n = 3 genes) andCardoen et al. (2011; n = 31 genes), strongly suggesting that several genelevel components of the regulatory networks are directly connected to the protein level above it. Additional studies on protein-level interactions that mediate sterility are necessary to identify hub proteins implicated in sterility(Duncan, Hyink, & Dearden, 2016;, caste differentiation(Begna, Han, Feng, Fang, & Li, 2012;Li et al., 2010), queen reproductive development(Pang et al., 2017), worker embryogenesis(Fang et al., 2014;Zheng et al., 2011), age-dependent division of labor(Huo et al., 2016), and other aspects of social foraging (the proboscis extension reflex; da SilvaMenegasso et al., 2017). Where available, information from these networks should serve to identify hub proteins and other topological features that, similarly to the gene networks, can be used to detail ideas on their origins and activity. ...
The study of social breeding systems is often gene focused, and the field of insect sociobiology has been successful at assimilating tools and techniques from molecular biology. One common output from sociogenomic studies is a gene list. Gene lists are readily generated from microarray, RNA sequencing, or other molecular screens that typically aim to prioritize genes based on the differences in their expression. Gene lists, however, are often unsatisfying because the information they provide is simply tabular and does not explain how genes interact with each other, or how genetic interactions change in real time under social or environmental circumstances. Here, we promote a view that is relatively common to molecular systems biology, where gene lists are converted into gene networks that better describe the functional connections that regulate behavioral traits. We present a narrative related to honeybee worker sterility to show how network analysis can be used to reprioritize candidate genes based on connectivity rather than their freestanding expression values. Networks can also reveal multigene modules, motifs, clusters or other system-wide properties that might not be apparent from an ab initio list. We argue that because network analyses are not restricted to "genes" as nodes, their implementation can potentially connect multiple levels of biological organization into a single, progressively complex study system.
... However, 22 of the proteins are involved in regulating the pupal head neuron and gland development that occur at a later stage of development of the pupae heads. 37 Furthermore, a 2-DE proteomic map of worker red-eye pupae hemolymph identified ∼129 proteins. 38 Most of the proteins identified during the non-feeding period are known to be involved in the mechanism of metamorphosis, 38,39 which results in the dramatic decrease of the overall protein quality as the red-eye pupa develops into a newly emerged bee. ...
The honeybee is one of the most valuable insect pollinators, playing a key role in pollinating wild vegetation and agricultural crops, with significant contribution to the world’s food production. Although honeybees have long been studied as model for social evolution, honeybee biology at the molecular level remained poorly understood until the year 2006. With the availability of the honeybee genome sequence and technological advancements in protein separation, mass spectrometry, and bioinformatics, aspects of honeybee biology such as developmental biology, physiology, behavior, neurobiology, and immunology have been explored to new depths at molecular and biochemical levels. This Review comprehensively summarizes the recent progress in honeybee biology using proteomics to study developmental physiology, task transition, and physiological changes in some of the organs, tissues, and cells based on achievements from the authors’ laboratory in this field. The research advances of honeybee proteomics provide new insights for understanding of honeybee biology and future research directions.
... The genome of A. mellifera predict 21 OBPs (OBP 1-21) [74,75], which are small water soluble molecules, binding reversibly to messenger molecules such as pheromones that are generally hydrophobic molecules with low solubility [76,77]. Although OBPs are commonly expressed in sensory organ of insects such as gustatory and olfactory sensilla [77], they have been also reported in ovary, brain, tegument [74,78], mandibular gland [79], hemolymph [37] and head salivary gland [80] of social bees. In the present work, OBP-13 was up-regulated in the Dufour's gland of younger nurse workers similarly to that previously reported to A. mellifera which OBP-13 expression is caste and age regulated, with high expression in younger workers [81]. ...
The colony of eusocial bee Apis mellifera has a reproductive queen and sterile workers performing tasks such as brood care and foraging. Chemical communication plays a crucial role in the maintenance of sociability in bees with many compounds released by the exocrine glands. The Dufour’s gland is a non-paired gland associated with the sting apparatus with important functions in the communication between members of the colony, releasing volatile chemicals that influence workers roles and tasks. However, the protein content in this gland is not well studied. This study identified differentially expressed proteins in the Dufour’s glands of nurse and forager workers of A. mellifera through 2D-gel electrophoresis and mass spectrometry. A total of 131 spots showed different expression between nurse and forager bees, and 28 proteins were identified. The identified proteins were categorized into different functions groups including protein, carbohydrate, energy and lipid metabolisms, cytoskeleton-associated proteins, detoxification, homeostasis, cell communication, constitutive and allergen. This study provides new insights of the protein content in the Dufour’s gland contributing to a more complete understanding of the biological functions of this gland in honeybees.
... Initially, differential proteomics was used to identify changes in protein expression between the normal status compared with a specified status such, as healthy and diseased; subsequently, its use is now widely reported for identifying toxicity biomarkers and induced immunity, discovering resistance mechanisms and providing new insights into complex mechanisms in many organisms (Kennedy, 2002;Sharma et al., 2004;Shi & Paskewitz, 2006;Nguyen et al., 2008). In several insect studies, proteomics has emerged as a powerful method for gaining insight into many physiological changes at the cellular level (Nyström, 2005;Seehuus et al., 2006;Zheng et al., 2011). The technique is reported to have successfully been used with many insects, particularly silkworm, mosquito, honeybee, fruit fly and cotton bollworm (Li et al., 2014). ...
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.
... Transcripts of hexamerin 110, 70a, and 70b have been detected in developing ovaries and testes; 110 is highly transcribed in the ovaries of egg-laying queens (Martins et al. 2010). An overexpression of six forms of 110 from the early to middle stages of the pupae head was observed (Zheng et al. 2011). The immunolocalization of hexamerin 70a revealed a tissue-specific role in developing and maturing ovarioles and testioles (Martins et al. 2011). ...
... During development, the Obps in the red-eye pupa hemolymph likely arises from Obp13, which is highly expressed in older larvae and throughout the pupal stages; from Obp14 and Obp15, which are found in larvae and are thought disappear after pupation; or from Obp10, which appears in pupae and reaches the highest level in the brain of newly emerged bees before declining in older bees (Foret and Maleszka 2006). We identified two of these Obps, Obp13 (NP_001035314) and Obp14 (NP_001035313), similarly to Zheng et al. (2011) who identified Obp13 and 14 expression during A. mellifera pupa head development (Zheng et al. 2011). Thus, our results demonstrated that Obp14 does not disappear after pupation and is a relatively abundant pupal protein. ...
... During development, the Obps in the red-eye pupa hemolymph likely arises from Obp13, which is highly expressed in older larvae and throughout the pupal stages; from Obp14 and Obp15, which are found in larvae and are thought disappear after pupation; or from Obp10, which appears in pupae and reaches the highest level in the brain of newly emerged bees before declining in older bees (Foret and Maleszka 2006). We identified two of these Obps, Obp13 (NP_001035314) and Obp14 (NP_001035313), similarly to Zheng et al. (2011) who identified Obp13 and 14 expression during A. mellifera pupa head development (Zheng et al. 2011). Thus, our results demonstrated that Obp14 does not disappear after pupation and is a relatively abundant pupal protein. ...
Apis mellifera Linnaeus is a holometabolous insect that undergoes complete metamorphosis in its nonfeeding pupal stage before transitioning to the adult stage. Its pupal stages are classifiable by the unique color pigmentation of its compound eyes and thorax; notably, there is a red-eye stage involving an unpigmented body that has a relatively short duration and is easy to recognize. The aim of the current study was to create a proteomic reference map of the worker red-eye pupa hemolymph. Hemolymph was collected from dorsal vessels using glass capillary tubes and was examined using pI 3–10 two-dimensional gel electrophoresis (2DE; 10 and 14 %) and matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF protein identification. This experimental approach allowed us to identify 129 different proteins organized into orthologous groups. Overall, the predominant category was post-translational modifications, protein turnover and chaperones (23.3 % of the identified proteins). In addition, we identified proteins in the non-orthologous groups of olfaction (2.3 % of the identified proteins) and storage hexamerins (3.1 % of the identified proteins). Quantitatively, the major protein isoforms that were accurately identified via 10 % 2DE were four forms of storage hexamerin: the 110, 70a, 70b, and 70c forms. The most abundant enzymes identified were short-chain dehydrogenases/reductases with pivotal developmental roles in ecdysteroidogenesis and a sigma class glutathione-S-transferase that most likely serves as a major protectant against the by-products of oxidative stress. Many of the identified proteins are known to be involved in the mechanisms of metamorphosis. All of the identified proteins are useful as markers for future comparative physiological and developmental studies.
... guani relationship. Energy production proteins play major roles in development (Zheng et al., 2011). In our view, the effects of parasitism on host energy metabolism are associated with increasing host energy reserves to benefit parasitoid development (Beckage et al., 1997;Nakamatsu and Tanaka, 2004;Nakamatsu et al., 2006). ...
Ectoparasitoid wasps deposit their eggs onto the surface and inject venom into their hosts. Venoms are chemically complex and they exert substantial impact on hosts, including permanent or temporary paralysis and developmental arrest. These visible venom effects are due to changes in expression of genes encoding physiologically relevant proteins. While the influence of parasitization on gene expression in several lepidopterans has been reported, the molecular details of parasitoid/beetle relationships remain mostly unknown. This shortcoming led us to pose the hypothesis that envenomation by the ectoparasitic ant-like bethylid wasp Scleroderma guani leads to changes in protein expression in the yellow mealworm beetle Tenebrio molitor. We tested our hypothesis by comparing the proteomes of non-parasitized and parasitized host pupae using iTRAQ-based proteomics. We identified 41 proteins that were differentially expressed (32↑- and 9↓-regulated) in parasitized pupae. We assigned these proteins to functional categories, including immunity, stress and detoxification, energy metabolism, development, cytoskeleton, signaling and others. We recorded parallel changes in mRNA levels and protein abundance in 14 selected proteins following parasitization. Our findings support our hypothesis by documenting changes in protein expression in parasitized hosts.
... In this study, a-glucosidase was detected on day 16, suggesting the upcoming task switch from nursing to foraging. DEGs related to metabolic enzymes and energy production provided substrates and complements to ensure the formation of HGs and other biological phenomenon [64]. ...
Transcriptome sequencing has become the main methodology for analyzing the relationship between genes and characteristics of interests, particularly those associated with diseases and economic traits. Because of its role of functional food for humans, commercial royal jelly (RJ) and its production are major research focuses in the field of apiculture. Multiple lines of evidence have demonstrated that many factors affect RJ output by activating or inhibiting various target genes and signaling pathways. Available coding sequences from the Honey Bee Genome Sequencing Consortium have permitted a pathway-based approach for investigating the development of the hypopharyngeal glands (HGs). In the present study, 3573941, 3562730, 3551541, 3524453, and 3615558 clean reads were obtained from the HGs of five full-sister honey bee samples using Solexa RNA sequencing technology. These reads were then assembled into 18378, 17785, 17065, 17105, and 17995 unigenes, respectively, and aligned to the DFCI Honey Bee Gene Index database. The differentially expressed genes (DEGs) data were also correlated with detailed morphological data for HGs acini.
