Mao Feng

Proteomic analysis of honeybee worker (Apis mellifera) hypopharyngeal gland development

BackgroundHypopharyngeal glands (HG) of honeybee workers play an important role in honeybee nutrition and caste differentiation. Previous research mainly focused on age-dependent morphological, physiological, biochemical and genomic characters of the HG. Here proteomics and biochemical network analysis were used to follow protein changes during the HG development.ResultsA total of 87, 76, 85, 74, 71, and 55 proteins were unambiguously identified on day 1, 3, 6, 12, 15 and 20, respectively. These proteins were major royal jelly proteins (MRJPs), metabolism of carbohydrates, lipids and proteins, cytoskeleton, development regulation, antioxidant, molecule transporter, regulation of transcription/translation, proteins with folding functions. The most interesting is that MRJPs that have been detected in the HG of the newly emerged worker bees. The MRJPs expression is at peak level from 6-12 days, was validated by western blot analysis of MRJP1, 2 and 3. Moreover, 35 key node proteins were found in the biochemical networks of the HG.ConclusionsHG secretes RJ at peak level within 6-12 days, but the worker bee can secrete royal jelly (RJ) since birth, which is a new finding. Several key node proteins play an important role in the biochemical networks of the developing HG. This provides us some target proteins when genetically manipulating honeybees.

Proteomics Analysis of Major Royal Jelly Protein Changes under Different Storage Conditions

Protein changes in fresh royal jelly (RJ) were compared when stored at -20, 4 degrees C, and room temperature (RT) for 12 months. Protein was partially identified using combinations of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/MS), gel filtration chromatography, nanoLC MS/MS, and a protein engine identification tool applied to the honeybee genome. Significantly more protein spots were found in fresh (85 spots) and -20 degrees C (81 spots) stored RJ than in samples stored at 4 degrees C (73 spots) and at RT (70 spots) for 1 year. Most identified spots, 56, 57, 51, 46, corresponding to RJ sample of the fresh, -20 degrees C, 4 degrees C, and RT, were assigned to major royal jelly proteins (MRJPs). Marked differences were found in the heterogeneity of the MRJPs, in particular, MRJP3. The quantity of MRJP1 decreased significantly following the temperature trend in all images, but MRJP 2 and -3 did not increase or decrease following the temperature trend, thus, suggesting that MRJP 1-3 are sensitive to temperature. However, MRJP4, 5, glucose oxidase (GOD), peroxiredoxin (PRDX), and glutathione S-transferase (GST) S1 were clearly absent in all images in samples held at RT for 1 year. This indicates that they are the proteins most sensitive to storage temperature and protein markers for freshness of RJ. Combining chromatography and nanoLC MS/MS results, we tentatively conclude that MRJP5 is a reliable freshness marker and that the best way to maintain quality of RJ is under freezing conditions.

Proteome and phosphoproteome analysis of honeybee (Apis mellifera) venom collected from electrical stimulation and manual extraction of the venom gland

BackgroundHoneybee venom is a complicated defensive toxin that has a wide range of pharmacologically active compounds. Some of these compounds are useful for human therapeutics. There are two major forms of honeybee venom used in pharmacological applications: manually (or reservoir disrupting) extracted glandular venom (GV), and venom extracted through the use of electrical stimulation (ESV). A proteome comparison of these two venom forms and an understanding of the phosphorylation status of ESV, are still very limited. Here, the proteomes of GV and ESV were compared using both gel-based and gel-free proteomics approaches and the phosphoproteome of ESV was determined through the use of TiO2 enrichment.ResultsOf the 43 proteins identified in GV, < 40% were venom toxins, and > 60% of the proteins were non-toxic proteins resulting from contamination by gland tissue damage during extraction and bee death. Of the 17 proteins identified in ESV, 14 proteins (>80%) were venom toxic proteins and most of them were found in higher abundance than in GV. Moreover, two novel proteins (dehydrogenase/reductase SDR family member 11-like and histone H2B.3-like) and three novel phosphorylation sites (icarapin (S43), phospholipase A-2 (T145), and apamin (T23)) were identified.ConclusionsOur data demonstrate that venom extracted manually is different from venom extracted using ESV, and these differences may be important in their use as pharmacological agents. ESV may be more efficient than GV as a potential pharmacological source because of its higher venom protein content, production efficiency, and without the need to kill honeybee. The three newly identified phosphorylated venom proteins in ESV may elicit a different immune response through the specific recognition of antigenic determinants. The two novel venom proteins extend our proteome coverage of honeybee venom.

Differential Expressions of Nuclear Proteomes between Honeybee (Apis mellifera L.) Queen and Worker Larvae: A Deep Insight into Caste Pathway Decisions

Honeybees (Apis mellifera L.) possess individuals (castes) in their colonies, to which specific tasks are allocated. Owing to a difference in nutrition, the young female larvae develop into either a fertile queen or a sterile worker. Despite a series of investigations on the underlying mechanisms of honeybee caste polyphenism, information on proteins and enzymes involved in DNA and RNA regulation in the nucleus is still missing. The techniques of nuclear protein enrichment, two-dimensional electrophoresis, mass spectrometry and bioinformatics were applied to understand the nuclear proteome changes in response to changes in environmental settings (nutrition and time) during the early developmental stages at the third (72 h), fourth (96 h), and fifth (120 h) instars of the two caste intended larvae. A total of 120 differentially expressed nuclear proteins were identified in both caste intended larvae during these developmental stages. The third, fourth and fifth instars of queen prospective larvae expressed 69%, 84%, and 68% of the proteins that had altered expression, respectively. Particularly, the prospective queen larvae up-regulated most of the proteins with nuclear functions. In general, this changing nuclear proteome of the two caste intended larvae over the three developmental stages suggests variations in DNA and RNA regulating proteins and enzymes. These variations of proteins and enzymes involved in DNA and RNA regulation in response to differential nutrition between the two caste intended larvae lead the two caste larvae to pursue different developmental trajectories. Hence, this first data set of the nuclear proteome helps us to explore the innermost biological makings of queen and worker bee castes as early as before the 72 h (3rd instar). Also, it provides new insights into the honeybees polymorphism at nuclear proteome level and paves new ways to understand mechanisms of caste decision in other eusocial insects.

Novel royal jelly proteins identified by gel-based and gel-free proteomics.

Royal jelly (RJ) plays an important role in caste determination of the honeybee; the genetically same female egg develops into either a queen or worker bee depending on the time and amount of RJ fed to the larvae. RJ also has numerous health-promoting properties for humans. Gel-based and gel-free proteomics approaches and high-performance liquid chromatography-chip quadruple time-of-flight tandem mass spectrometry were applied to comprehensively investigate the protein components of RJ. Overall, 37 and 22 nonredundant proteins were identified by one-dimensional gel electrophoresis and gel-free analysis, respectively, and 19 new proteins were found by these two proteomics approaches. Major royal jelly proteins (MRJPs) were identified as the principal protein components of RJ, and proteins related to carbohydrate metabolism such as glucose oxidase, α-glucosidase precursor, and glucose dehydrogenase were also successfully identified. Importantly, the 19 newly identified proteins were mainly classified into three functional categories: oxidation-reduction (ergic53 CG6822-PA isoform A isoform 1, Sec61 CG9539-PA, and ADP/ATP translocase), protein binding (regucalcin and translationally controlled tumor protein CG4800-PA isoform 1), and lipid transport (apolipophorin-III-like protein). These new findings not only significantly increase the RJ proteome coverage but also help to provide new knowledge of RJ for honeybee biology and potential use for human health promotion.

Towards posttranslational modification proteome of royal jelly.

Royal jelly (RJ) is a secretory protein from the hypopharyngeal glands of nurse honeybee workers, which contains a variety of proteins of which major royal jelly proteins (MRJPs) are some of the most important. It plays important roles both for honeybee and human. Each family of MRJP 1-5 displays a string of modified protein spots in the RJ proteome profile, which may be caused by posttranslational modifications (PTMs) of MRJPs. However, information on the RJ PTMs is still limited. Therefore, the PTM status of RJ was identified by using complementary proteome strategies of two-dimensional gel electrophoresis (2-DE), shotgun analysis in combination with high performance liquid chromatography-chip/electrospray ionization quadrupole time-of-flight/tandem mass spectrometry and bioinformatics. Phosphorylation was characterized in MRJP 1, MRJP 2 and apolipophorin-III-like protein for the first time and a new site was localized in venom protein 2 precursor. Methylation and deamidation were also identified in most of the MRJPs. The results indicate that methylation is the most important PTM of MRJPs that triggers the polymorphism of MRJP 1-5 in the RJ proteome. Our data provide a comprehensive catalog of several important PTMs in RJ and add valuable information towards assessing both the biological roles of these PTMs and deciphering the mechanisms underlying the beneficial effects of RJ for human health.

An Integrated Proteomics Reveals Pathological Mechanism of Honeybee (Apis cerena) Sacbrood Disease

Viral diseases of honeybees are a major challenge for the global beekeeping industry. Chinese indigenous honeybee (Apis cerana cerana, Acc) is one of the major Asian honeybee species and has a dominant population with more than 3 million colonies. However, Acc is frequently threatened by a viral disease caused by Chinese sacbrood virus (CSBV), which leads to fatal infections and eventually loss of the entire colony. Nevertheless, knowledge on the pathological mechanism of this deadly disease is still unknown. Here, an integrated gel-based and label-free liquid chromatography-mass spectrometry (LC-MS) based proteomic strategy was employed to unravel the molecular event that triggers this disease, by analysis of proteomics and phosphoproteomics alterations between healthy and CSBV infected worker larvae. There were 180 proteins and 19 phosphoproteins which altered their expressions after the viral infection, of which 142 proteins and 12 phosphoproteins were down-regulated in the sick larvae, while only 38 proteins and 7 phosphoproteins were up-regulated. The infected worker larvae were significantly affected by the pathways of carbohydrate and energy metabolism, development, protein metabolism, cytoskeleton, and protein folding, which were important for supporting organ generation and tissue development. Because of abnormal metabolism of these pathways, the sick larvae fail to pupate and eventually death occurs. Our data, for the first time, comprehensively decipher the molecular underpinnings of the viral infection of the Acc and are potentially helpful for sacbrood disease diagnosis and medicinal development for the prevention of this deadly viral disease.

In-depth Proteomics Characterization of Embryogenesis of the Honey Bee Worker (Apis mellifera ligustica)

Identifying proteome changes of honey bee embryogenesis is of prime importance for unraveling the molecular mechanisms that they underlie. However, many proteomic changes during the embryonic period are not well characterized. We analyzed the proteomic alterations over the complete time course of honey bee worker embryogenesis at 24, 48, and 72 h of age, using mass spectrometry-based proteomics, label-free quantitation, and bioinformatics. Of the 1460 proteins identified the embryo of all three ages, the core proteome (proteins shared by the embryos of all three ages, accounting for 40%) was mainly involved in protein synthesis, metabolic energy, development, and molecular transporter, which indicates their centrality in driving embryogenesis. However, embryos at different developmental stages have their own specific proteome and pathway signatures to coordinate and modulate developmental events. The young embryos (<24 h) stronger expression of proteins related to nutrition storage and nucleic acid metabolism may correlate with the cell proliferation occurring at this stage. The middle aged embryos (24–48 h) enhanced expression of proteins associated with cell cycle control, transporters, antioxidant activity, and the cytoskeleton suggest their roles to support rudimentary organogenesis. Among these proteins, the biological pathways of aminoacyl-tRNA biosynthesis, β-alanine metabolism, and protein export are intensively activated in the embryos of middle age. The old embryos (48–72 h) elevated expression of proteins implicated in fatty acid metabolism and morphogenesis indicate their functionality for the formation and development of organs and dorsal closure, in which the biological pathways of fatty acid metabolism and RNA transport are highly activated. These findings add novel understanding to the molecular details of honey bee embryogenesis, in which the programmed activation of the proteome matches with the physiological transition observed during embryogenesis. The identified biological pathways and key node proteins allow for further functional analysis and genetic manipulation for both the honey bee embryos and other eusocial insects.

Proteome analysis of hemolymph changes during the larval to pupal development stages of honeybee workers (Apis mellifera ligustica).

Hemolymph is vital for the flow and transportation of nutrients, ions, and hormones in the honey bee and plays role in innate immune defense. The proteome of the hemolymph changes over the life of a honey bee, but many of these changes are not well characterized, including changes during the life cycle transition from the larval to pupal stages of workers. We used two-dimensional gel electrophoresis, mass spectrometry, bioinformatics, and Western blot to analyze the proteome changes of the honeybee hemolymph during the transition from newly hatched larvae to five-day-old pupae. Of the 49 nonredundant proteins that changed in abundance (identified by 80 protein spots), 29 (59.2%) and 20 (40.8%) were strongly expressed in the larvae and the pupae, respectively. The larval hemolymph had high expressions of major royal jelly proteins and proteins related to metabolism of carbohydrates and energy, folding activities, development, and the cytoskeleton and antioxidant systems. Proteins involved in food storage and the metabolism of fatty acids and amino acids were abundantly expressed during the late larval to pupal development stages. The proteins expressed by the young larvae are used to enhance their development process and as a temporal innate immune protection mechanism until they gain immunity with age development. The pupae use more energy storage related proteins as they prepare for their non-diet-driven pupation. Our data provide new evidence that changes in the hemolymph at the proteome level match the processes during life transitions in the honeybee.

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.