Project description:MRJP-3 is an RNA binding protein that binds both double- and single-stranded RNA in vitro. To test whether i) MRJP-3 binds RNA in its natural environment, the royal jelly (RJ) and ii) to characterize its RJ RNA partners; a pull down followed by RNA-seq system was developed. MRJP-3 bound RNA was compared to total RJ RNA extracted from the same hive. To establish the system, we first incubated biotinylated MRJP-3, or biotinylated BSA in RJ and pulled the proteins out with strepavidin coated magnetic beads followed by RNA extraction and bioanalyzer profiling. MRJP-3 pull out was enriched for RNA compare to the BSA and just beads controls. The RNA population pulled down with MRJP-3 has similar profile to the total RJ RNA, further demonstrating the MRJP-3 has no affinity to a certain RNA species.
Project description:Female larvae of the honeybee (Apis mellifera) develop into either queens or workers depending on nutrition during larval development. This nutritional stimulus triggers different developmental trajectories, resulting in adults that differ in physiology, behaviour and life-span. To understand how these developmental trajectories are established we have undertaken a comprehensive analysis of differential gene expression throughout larval development. Gene expression of honeybee queen and worker larval samples was analysed at seven time points during larval development (6 hr, 12 hr, 36 hr, 60 hr, 84 hr, 108 hr and 132 hr)
Project description:As a matter of fact, honeybees are vital for the pollination of more than 80 crops of agricultural interest. However, population decline has become an important global issue causing significant concerns among agricultural experts and the broader public. For this, parasites are known to be the major culprits responsible for the losses of millions of honeybee colonies so far. Among these parasites, Varroa destructor has been identified as a major cause for global losses in Western honeybee (Apis mellifera) colonies. Hygienic behavior (HB), on the other hand, is a collective response by adult honeybees to defend against parasites and diseases that is known to involve in resistance towards Varroosis. Even with the efforts made to elucidate the molecular mechanism underlying HB, it is still not understood. In our study, we have studied the proteomic correlates to HB using a honeybee line (selected for Varroa-specific HB for over a decade in Germany). We sampled individual worker bees from this line that showed HB after closer infrared video observations and compared the proteomes of their mushroom bodies and antennae with those of workers that came from the same set of colonies but didn't show the behaviour. Furthermore, we compared the pupal hemolymph for worker bees of the selected HB line and a control line using state-of-the art techniques of proteomics. We identified a total of 8609 proteins (covered >55% of the honeybee proteome) from these three honeybee tissues. This is the most comprehensive proteomic study of the honeybee HB to date, and the first to focus on individual bees expressing Varroa-specific HB. These results have significantly advanced our knowledge on the biology underlining HB to a new level. The uniquely found functional classes and pathways by the proteins identified in each tissue suggest that hygienic bees have shaped distinct proteome settings to underpin the HB. Moreover, during analysis of pupal hemolymph proteome, the HB-line has adapted a unique strategy to boost an individual and social immunity and drove pupal organogenesis via energy metabolism and protein biosynthesis. Moreover, in the mushroom bodies of different HB phenotypic worker bees, the hygienic bees have enhanced their neuronal sensitivity to promote the execution of HB by activation of synaptic vesicles and calcium channel activities. Moreover, in the antennae of two HB phenotypic worker bees, the hygienic bees have demonstrated strengthening of their sensitivity associated with olfactory senses and signal transmissions, which is important to input a strong signal to the mushroom bodies and initiate HB. In conclusion, our novel findings have significantly extended our understandings of the molecular mechanisms that underline the HB to combat Varroa infestation. Furthermore, we identified a wide array of novel markers that are useful for accelerating marker associated selection of HB to aid in the natural resistance to a parasite blamed for a global decline in honeybee health.
Project description:Social caste determination in the honey bee is assumed to be determined by the dietary status of the young larvae and translated into physiological and epigenetic changes through nutrient-sensing pathways. We have employed microRNA gene-microarray, and observed that both worker jelly and royal jelly showed dynamic changes in miRNA content during the 4th to 6th day of larval development . Adding specific miRNAs to royal jelly elicited significant changes in queen larval mRNA expression and in morphological characters of the emerging adult queen bee. We propose that miRNAs in the nurse bee secretions constitute an additional element in the regulatory control of caste determination in the honey bee. We collected worker and royal jelly of the Italian honeybee (ZND No.1, Apis mellifera ligustica) at 73~90 hours (4th-day larvae), 97~114 hours (5th-day larvae), and 121~138 hours (6th-day larvae) after hatching. After total RNA was extractedM-BM- and quantified , equal amounts of total RNAs from each of the three sampling days were analyzed on the LC Science miRNA-array to observe the expression variation of miRNAs between worker jelly and royal jelly along with the development time points (4th-day, 5th-day and 6th-day).
Project description:Social caste determination in the honey bee is assumed to be determined by the dietary status of the young larvae and translated into physiological and epigenetic changes through nutrient-sensing pathways. We have employed Illumina/Solexa sequencing to examine the small RNA content in the bee larval food source, and show that worker jelly is enriched in miRNA complexity and abundance relative to royal jelly. The miRNA levels in worker jelly were 7-215 fold higher than in royal jelly, and both jellies showed dynamic changes in miRNA content during the 4th to 6th day of larval development. Adding specific miRNAs to royal jelly elicited significant changes in queen larval mRNA expression and in morphological characters of the emerging adult queen bee. We propose that miRNAs in the nurse bee secretions constitute an additional element in the regulatory control of caste determination in the honey bee. We collected worker and royal jelly of the Italian honeybee (ZND No.1, Apis mellifera ligustica) at 73~90 hours (4th-day larvae), 97~114 hours (5th-day larvae), and 121~138 hours (6th-day larvae) after hatching. After total RNA was extractedM-BM- and quantified , relative equal amounts of total RNAs from each of the three sampling days were pooled into respectively worker and royal jelly samples, and the fraction of small RNAs less than 30nt long was retained and sequenced on the Illumina/Solexa high-throughput platform (HTP).
Project description:Honeybees are very important eusocial insects and are involved in the pollination of many plants. Queen bees and worker bees develop from the same fertilized eggs, and are thus genetically identical despite their substantial behavioural and physiological differences. The mechanism governing developmental differences between worker and queen bees has always attracted much interest. While there are several reports on mRNA expression related to caste differentiation, no systematic investigation of small RNAs has thus far been carried out. Results: Using deep sequencing we systematically profiled small RNA expression in 4th-6th day worker larvae and queen larvae (the critical stages at which the fates of workers and queens are determined), and found that 38 miRNAs were differentially expressed between worker and queen larvae. In addition, 639 mature miRNA candidates were identified in our work for the first time, of which, 526 were expressed only in workers (318) or queens (208). Conclusion: We present the first profile of honeybee small RNAs and explore the mechanism of caste differentiation between worker and queen bees. Caste-specific expression patterns and large discrepancies in small RNA profiles between worker and queen bees indicate that small RNAs may be related to the differential development of worker and queen bee larvae. Results presented here will make a valuable contribution to understanding the caste switch between worker and queen bees. Three healthy 10-frame colonies of ‘Zhenongda No.1’- a high-yielding royal jelly breed of Apis mellifera ligustica , were maintained at the Huajiachi campus of Zhejiang University. In each colony, the queen laid eggs over a period of 24 hours in one empty frame which was subsequently moved to an egg-free super-chamber. After 66 hours (less than 18 hours after hatching), we transferred 150 larvae into queen cups to rear queens in each colony and put the queen cup frames into their corresponding colonies. 40-60 worker larvae and queen larvae were collected from each colony after 4 days (73~90 h after hatching), 5 days (97~114 h after hatching) and 6 days (121~138 h after hatching). The larval samples were collected into 50 ml tubes, immediately frozen in liquid nitrogen and stored at -80C until being used for RNA extraction. After total RNA was extracted and quantified , relative equal amounts of total RNAs from each of the three sampling days were pooled into respectively worker and queen samples, and the fraction of small RNAs less than 30nt long was retained and sequenced on the Illumina/Solexa high-throughput platform (HTP).
Project description:This experiment was aimed at evaluating the presence or absence of transcripts in various body parts and at different developmental stages in the honeybee.
Project description:In this project, proteomic approaches were used to detect statistically significant changes in the honeybee proteome and to explore the mechanistic basis for the HG regulation that accompanies the seasonal changes.