This study employed a systematic analysis of the whole time-range of the honeybee HG at the protein level, in parallel with metabolic pathway approaches. Some of the biochemical pathways were built up where 35 key node proteins were found in all the identified proteins. To our present knowledge, this is the first large scale and all around analysis of the development of HG in this model organism. The results provide a global perspective on the protein and the biochemical pathways involved in the HG development of the honeybee workers, helping us to better understand honeybee biology. Obviously, some different spots in gels were identified to be the same proteins in supplementary Table S (Additional File 1). They were isoforms and their different distributions in gels might be caused by some post-translational modifications (PTMs), such as phosphorylation and possibly alternative splicing that resulting in a shift of Mr and pI of these proteins.
The HG of honeybee workers goes through the undeveloped, developed and regressive phases accompanying the shifting of tasks from nursing to foraging within 20 days in a normal colony. In young workers, usually not more than 13 days past eclosion, the HG, which is fully developed and shows high rates of protein synthesis in nursing period, is already secreting RJ for brood breeding. Eighteen days later, when workers become foragers, the HG begins to regress and produce enzyme products to digest nectar into honey [2
]. In the present study, 6 time points were chosen in an attempt to distinguish proteins expressed at different time points in the whole range of the HG development. Based on the proteomic profile, we could clearly observe that 6 and 12 day-old HGs expressed more proteins than that of the others. The proteins that were identified in each time point varied greatly, and belong to a broad range of different classes and functional pathways (Figure ), implying a wide range of proteins are necessary for the HG development.
RJ, secreted from the HG and mandibular gland of the worker honeybee [15
], is the exclusive food for the queen honeybee and young larva [16
]. Theoretically, MRJPs, including MRJP1, 2, 3, 4 and 5, should be the main contents in the HG. The identified MRJPs in this study have validated these assumptions. Attributed to the potential glycosylation sites and extensive repetitive regions in the C-terminal of the proteins [17
], some isoforms of MRJP2 and MRJP3 were present in this study. Interestingly, MRJPs were identified in the 1-day-old HG and 3-day-old HG had a typical pattern of RJ (Figure ). This suggests that the newly emerged honeybee workers are likely have the ability to secrete RJ and start to produce RJ much earlier than expected. It was confirmed by western blot that MRJP1, 2 and 3 could be detected from day 1, and by day 3 express at significantly higher levels than those of day 1 (Figure ). Furthermore, the HG secreted RJ at peak level from 6 to 12 days, which could also be confirmed by western blot analysis (Figure ). These findings show that the period from 6 to 12 days is the peak time for the workers to secret RJ and nurse the brood. After that the output of RJ begins to decrease because of the shifting tasks and the shrinkage of the HG [9
Eighteen proteins were related to the metabolism of carbohydrate and energy production involved in glycolysis (spots 13, 32, 58, 63 and 87), citric acid circle (spot 78), and ATP generations (spots 1, 3, 19, 30, 31, 36, 40, 48, 57, 60, 65 and 66) (supplementary Table S in Additional File 1) in the present study. Seven of them were detected as key node proteins (CG6084, atpsyn-β, blw, ter94, eno, scp2 and CG5362) (Figure ). Both the higher number and most of the differentially expressed were in the early stages, suggesting that not only the development of the HG needs much metabolic energy for cell divisions and secretory synthesis in this early period, but also the honeybee has an evolutionary strategy to cope with the selective pressure on its extremely carbohydrate-rich diet [18
]. The down-regulated of these proteins in the latter period are coincided well with Drosophila
that gene encoding enzymes involved in energy metabolism are down-regulated during the late larval ecdysone pulse [20
]. Acyl-CoA dehydrogenase (spot 31) is involved in mitochondrial fatty acid β-oxidation, which fuels hepatic ketogenesis during prolonged fasting and periods of higher energy demands [21
]. The down-regulated here is probably due to the honeybee worker dropping its lipid storage dramatically prior to the onset of foraging. The main task for the forager bee is to collect nectar and divert it into honey by adding some enzymes, such as α-
amylase (spot 79) and α-glucosidase (spot 85). These transferases could digest polysaccharides, disaccharides into monosaccharide. The up-regulated of α-glucosidase (Figure ) coincides well with the task changes of the honeybee workers. The expression of glucose oxidase (GOD) in this study (spots 80, 81 and 86) is likely used for the biological production of gluconic acid and for the removal of either glucose or oxygen from foodstuffs in order to improve their storage capability [22
]. So the up-regulated of this acid is also related to the work of the workers.
Hsps function primarily as a molecular chaperone, facilitating the protein folding, preventing protein aggregation, or targeting improperly folded proteins in specific degradative pathways [23
]. The third largest group in this study was related to proteins with folding functions, of which Hsps were the most represented forms. Six of them, hsp83 (spot 83), hsc70-1 (spot49 and 50), hsp60 (spot 46 and 51), CG14207 (spot 10), hsc70-5 (spot 55) and hsc70-3 (spot 47), were the key node proteins for the HG development (Figure ). In honeybees, the enhanced expression of Hsps and cell death in the midgut of the bee's larvae infected with Paenibacillus larvae
or Bacillus larvae
], the larval salivary glands treated with acaricides [26
] and under heat stress conditions [27
] have been well documented, which could be a defense mechanism to prevent against stress tolerance. Hsps have widely been identified in honeybees, including worker larvae [3
], the embryos [19
], the head and brain of the workers [28
], the hemolymph [29
] and the venom gland [30
], respectively. The higher number of Hsps identified in the early stage (Figure ) and higher abundance expressed from day 6 to day 12 (Figure ) suggests that they probably act as molecular chaperones by assisting in the correct folding of nascent proteins, thus contributing to cell maintenance and protein (RJ) secretory activity of the HG since our experiment was carried out under normal physiological conditions.
The present identified antioxidant proteins involving jafrac1 (spot 8), thioredoxin reductase-1(spot 64), sod (spot 24), gsts1 (spot 7) and peroxiredoxin 2540 (spot 11), could also been found in the honeybee embryo [19
], the venom gland [30
] and the HG of wintering honeybee workers [31
]. Among them, gsts1, jafracl and sod played the key role for the antioxidant system in the developing HG (Figure ). Sod, thioredoxin reductase and peroxiredoxin 2540 are able to metabolize peroxides [32
]; and Jafracl, a superfamily of detoxication protein, can change xenobiotic to harmless products [34
]. The oxidative damage caused by the reactive oxygen species (ROS) is to be intensified with the increase of ROS due to a high demand for oxygen in fast growing organism [35
]. Therefore, the HG during the nursing period is fully developed and shows high secreting activity, which means the metabolism in this stage is vigorous; on the contrary, the metabolism is slowed down in the HG of foraging bees because of the regression and low activity of the HG [10
]. As a result, the up-regulated of gstsl in the early stage, followed by a down-regulated in the late stage, is responsible for the changes with the development of the HG in this study.
The cytoskeleton plays important roles in both intracellular transport and cellular division. Among the identified cytoskeletal proteins, β-tub60d (spot 34), F-actin capping protein α subunit (spot 16), tsr (spot 4 and 6), ptx1 (spot 29) were the key node proteins in the cytoskeletal system (Figure ). Among those key node proteins, tsr and ptx1 showed a significantly down-regulated trend (Figure ).
Actin plays an important role during dorsal closure throughout the embryonic development in Drosophila
], and tsr, which restricts the actin polymerization [37
], appears to have control of actin-based motility processes and enhances removal of ADP bound actin monomers from the pointed end of an actin filament [38
]. Down-regulated of them in the latter developmental phase coincides with the secretory activity and the acini size of the HG beginning to decrease after 15 days when workers becoming foraging bees [5
], cell death predominates in the latter foraging period [39
Obviously, the development of HG requires the presence of growth factors to ensure its development. Rack1 (spots 21 and 22) and l(2)37 Cc (spot 12), acting as key node proteins, were the growth factors in the HG development of the honeybee workers. In Drosophila
, rack1 is expressed at all developmental stages and in many tissues. It is essential at multiple steps of Drosophila
development, particularly in oogenesis [40
]. Lethal (2) 37 Cc is required for larval metabolism from larvae to pupae, and is expressed in early embryos, late embryos, late third instar larvae and adults of the Drosophila
]. Overall, the down-regulated of rack1 (Figure ) suggests that a decrease in the secretory activity and lower growth factor titers are demanded with the regression of the HG.
A group of proteins were identified as being responsible for the initiation of translation and accuracy of elongation, with 12 were the key node proteins (Figure ). PCNA (spot 15), a highly conserved protein, is an essential component in the DNA replication and DNA repair [42
]. It is also required for some repair pathways, including NER [43
] and mismatch repair [44
]. In Drosophila
, temperature shift studies reveal that the vital function of PCNA is required throughout virtually all stages of fly development [44
]. Eif-5a (spot 5) is involved in the first step of peptide bond formation in translation and is essential for cell proliferation and cell-cycle regulation [45
]. While ef-1α (spot 67) is a nuclear protein coding gene involved in the GTP-dependent binding of charged tRNAs to the acceptor site of the ribosome during translation. In Drosophila
, ef-1α is expressed at different times during development [46
]. At the same time, several ribosome proteins (Rps) (spots 28, 69, 70, 71, 72, 73 and 75) were identified involving in the cellular process of translation [47
]. In addition to their ubiquitous expression, these proteins are present simultaneously and in essentially fixed ratios in the ribosome and in the cell as a whole. It has shown that disrupting ribosome function can result in an array of fascinating dominant phenotypes in Drosophila
]. Rps3 (spot 71) is very crucial for translation as a component of the 40S ribosomal subunit, and also acts as a damage DNA endonuclease [49
]. Sop (spot 70) mainly participates in aminoacyl-transfer RNA binding to the ribosome, potentially affecting the fidelity of mRNA translation [50
]. Rplp0 (spot 28) plays an important role in polypeptide chain elongation during translation. Most of the Rps identified in this study that have been documented in ribosomal proteome of the Drosophila
]. Newly synthesized ribosomal proteins are found in association with ribosomal subunits as soon as 90 min after fertilization of Drosophila
]. The ribosome number is thought to control cellular growth [53
], in this regard, the fast growing cells need more Rps to insure their growth. The expression of Rps in this study did not increase within 6 to 12 days might attribute to the increased cell proliferation does not always correlate with the higher expression level of ribosomal [54
]. Some ribosomal proteins may have more specific roles in regulating proliferation than simply influencing the rate of protein biogenesis. As such, their increased expression may be a separate phenomenon from the general increase in the synthesis of ribosomal proteins in dividing cells [55
]. In general, the highest key node proteins in this group that altered their expression are likely to work together to regulate the process of transcription and translation for the regular development of the HG and RJ secretion.