Shenglu Chen

Next-generation small RNA sequencing for microRNAs profiling in Apis mellifera: comparison between nurses and foragers.

MicroRNAs (miRNAs) are endogenous small non‐coding RNAs regulating gene expression in animals and plants. To find some differentially expressed miRNAs that may be associated with age‐dependent behavioural changes in honey bees (Apis mellifera), we applied next‐generation high‐throughput sequencing technology to detect small RNAs in nurses and foragers. Our results showed that both nurses and foragers had a complicated small RNA population, and the length of small RNAs varied, 22 nucleotides being the predominant length. Combining deep sequencing and bioinformatic analysis, we discovered that nine known miRNAs were significantly different between nurses and foragers (P < 0.01; absolute value of fold‐change ≥1). Some of their target genes were related to neural function. Moreover, 67 novel miRNAs were identified in nurses and foragers. Ame‐miR‐31a and ame‐miR‐13b were further validated using quantitative reverse‐transcription PCR assays. The present study provides new information on the miRNA abundance of honey bees, and enhances our understanding of miRNA function in the regulation of honey bee development.

Recipe for a Busy Bee: MicroRNAs in Honey Bee Caste Determination

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, 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 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.

East Learns from West: Asiatic Honeybees Can Understand Dance Language of European Honeybees

The honeybee waggle dance, through which foragers advertise the existence and location of a food source to their hive mates, is acknowledged as the only known form of symbolic communication in an invertebrate. However, the suggestion, that different species of honeybee might possess distinct ‘dialects’ of the waggle dance, remains controversial. Furthermore, it remains unclear whether different species of honeybee can learn from and communicate with each other. This study reports experiments using a mixed-species colony that is composed of the Asiatic bee Apis cerana cerana (Acc), and the European bee Apis mellifera ligustica (Aml). Using video recordings made at an observation hive, we first confirm that Acc and Aml have significantly different dance dialects, even when made to forage in identical environments. When reared in the same colony, these two species are able to communicate with each other: Acc foragers could decode the dances of Aml to successfully locate an indicated food source. We believe that this is the first report of successful symbolic communication between two honeybee species; our study hints at the possibility of social learning between the two honeybee species, and at the existence of a learning component in the honeybee dance language.

Viral infection affects sucrose responsiveness and homing ability of forager honey bees, Apis mellifera L.

Honey bee health is mainly affected by Varroa destructor, viruses, Nosema spp., pesticide residues and poor nutrition. Interactions between these proposed factors may be responsible for the colony losses reported worldwide in recent years. In the present study, the effects of a honey bee virus, Israeli acute paralysis virus (IAPV), on the foraging behaviors and homing ability of European honey bees (Apis mellifera L.) were investigated based on proboscis extension response (PER) assays and radio frequency identification (RFID) systems. The pollen forager honey bees originated from colonies that had no detectable level of honey bee viruses and were manually inoculated with IAPV to induce the viral infection. The results showed that IAPV-inoculated honey bees were more responsive to low sucrose solutions compared to that of non-infected foragers. After two days of infection, around 107 copies of IAPV were detected in the heads of these honey bees. The homing ability of IAPV-infected foragers was depressed significantly in comparison to the homing ability of uninfected foragers. The data provided evidence that IAPV infection in the heads may enable the virus to disorder foraging roles of honey bees and to interfere with brain functions that are responsible for learning, navigation, and orientation in the honey bees, thus, making honey bees have a lower response threshold to sucrose and lose their way back to the hive.

Molecular Cloning and Characterization of Juvenile Hormone Acid Methyltransferase in the Honey Bee, Apis mellifera, and Its Differential Expression during Caste Differentiation

Juvenile hormone acid methyltransferase (JHAMT) is an enzyme involved in one of the final steps of juvenile hormone biosynthesis in insects. It transfers a methyl group from S-adenosyl-L-methionine (SAM) to the carboxyl group of either farnesoic acid (FA) or JH acid (JHA). Several genes coding for JHAMT have been cloned and characterized from insects from different orders, and they have been shown to play critical roles in metamorphosis and reproduction. However, the significance of JHAMT in Hymenopteran insects is unknown. We used RACE amplification method to clone JHAMT cDNA from the honey bee, Apis mellifera (AmJHAMT). The full length cDNA of AmJHAMT that we cloned is 1253bp long and encodes a 278-aa protein that shares 32-36% identity with known JHAMTs. A SAM-binding motif, conserved in the SAM-dependent methyltransferase (SAM-MT) superfamily, is present in AmJHAMT. Its secondary structure also contains a typical SAM-MT fold. Most of the active sites bound with SAM and substrates (JHA or FA) are conserved in AmJHAMT as in other JHAMT orthologs. Phylogenetic analysis clustered AmJHAMT with the other orthologs from Hymenoptera to form a major clade in the phylogenetic tree. Purified recombinant AmJHAMT protein expressed in E. coli was used to produce polyclonal antibodies and to verify the identity of AmJHAMT by immunoblotting and mass spectrometry. Quantitative RT-PCR and immunoblotting analyses revealed that queen larvae contained significantly higher levels of AmJHAMT mRNA and protein than worker larvae during the periods of caste development. The temporal profiles of both AmJHAMT mRNA and protein in queens and workers showed a similar pattern as the JH biosynthesis. These results suggest that the gene that we cloned codes for a functional JHAMT that catalyzes the final reactions of JH biosynthesis in honey bees. In addition, AmJHAMT may play an important role in honey bee caste differentiation.

High-abundance mRNAs in Apis mellifera: Comparison between nurses and foragers

A large volume of honey bee (Apis mellifera) tag-seq was obtained to identify differential gene expression via Solexa/lllumina Digital Gene Expression tag profiling (DGE) based on next generation sequencing. In total, 4,286,250 (foragers) and 3,422,327 (nurses) clean tags were sequenced, 24,568 (foragers) and 13,134 (nurses) distinct clean tags could not be match to the reference database, and 7508 and 6875 mapped genes were detected in foragers and nurses respectively. 7045 genes were found differentially expressed between foragers and nurses. Of those genes, 1621 genes had significantly different expression, that is, they showed an expression ratio (foragers/nurses) of more than 2 and FDR (False Discovery Rate) of less than 0.001. We identified 101 genes that were uniquely expressed in foragers, and 9 genes that were only expressed in nurses. We performed the Gene Ontology (GO) category and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and found 415 genes with annotation terms linked to the GO cellular component category. 200 components of KEGG pathways were obtained, including 21 signaling pathways. The PPAR signaling pathway was the most highly enriched, with the lowest Q-value.

Expression of Apalbumin1 of Apis cerana cerana in the Larvae of Silkworm, Bombyx mori

Royal jelly (RJ) is a thick, milky material produced by both the hypopharyngeal and the mandibular glands of nurse honeybees. The main proteins of RJ, named apalbumins or major royal jelly proteins (MRJPs), have multiple biological functions. Apalbumin1 is the most abundant glycoprotein of RJ. In this study, Bacmid- apalbumin1 was constructed for Apis cerana cerana using the newly established Bac-to-Bac/BmNPV baculovirus expression system (BES). This procedure allowed us to obtain the recombinant A. cerana cerana ( Acc) apalbumin1 (r Accapalbumin1) from the hemolymph of silkworm larvae through the BmNPV bacmid system, 96 h postinfection. The r Accapalbumin1 was then purified by Ni-NTA spin columns and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. A 55 kDa protein with good solubility was then obtained. The peptide Ile-Phe was identified from trypsin production of r Accapalbumin1. Such a peptide has been reported to have an antihypertensive ability. Our results have therefore potential applications in biomedical research and open new perspectives for the study of apalbumins.

Differential expression of miRNAs related to caste differentiation in the honey bee, Apis mellifera

Honey bees are very important eusocial insects and are involved in the pollination of many plants. Queen bees and worker bees can develop from the same fertilized eggs and are thus genetically identical despite their substantial behavioral and physiological differences. The mechanism governing developmental differences between worker and queen bees has always attracted much interest. While there are several reports on messenger RNA (mRNA) expression related to caste differentiation or microRNA (miRNA) expression in one time point of caste differentiation, no systematic investigation of the dynamic expression of small RNAs along with these two caste development has, thus far, been carried out. In this study, we present the dynamic expression profiles of queen and worker bee small RNAs and show caste-specific miRNA expression patterns between them, indicating that miRNAs may be related to the differential development of worker and queen bee larvae. Results presented here will make a valuable contribution to understanding of the caste switch between worker and queen bees.

Differential transcriptome profiles of heads from foragers: comparison between Apis mellifera ligustica and Apis cerana cerana

Given the differences in ecology and biology between Apis cerana cerana and Apis mellifera ligustica, we first used the Illumina–Solexa deep sequencing technology to describe the differences in the heads of A. cerana cerana and A. mellifera ligustica foragers at the gene expression level. We obtained over 3.6 million clean tags per sample and found about 2,370 differentially expressed genes related to metabolism, development, and signal transduction between A. cerana cerana and A. mellifera ligustica. Also, the many antisense transcripts found in our study indicated that they may represent novel paths involving gene expression regulation in honeybees. Our results indicated that differences in head expression profiles relate to sets of genes, and there existed significant enrichment of 22 pathways in differentially expressed genes. We conclude that the deep sequencing method provides us a better insight into differences at the molecular level between species within the genus Apis.

Genomic and transcriptomic analysis of the Asian honeybee Apis cerana provides novel insights into honeybee biology.

The Asian honeybee Apis cerana is one of two bee species that have been commercially kept with immense economic value. Here we present the analysis of genomic sequence and transcriptomic exploration for A. cerana as well as the comparative genomic analysis of the Asian honeybee and the European honeybee A. mellifera. The genome and RNA-seq data yield new insights into the behavioral and physiological resistance to the parasitic mite Varroa the evolution of antimicrobial peptides, and the genetic basis for labor division in A. cerana. Comparison of genes between the two sister species revealed genes specific to A. cerana, 54.5% of which have no homology to any known proteins. The observation that A. cerana displayed significantly more vigilant grooming behaviors to the presence of Varroa than A. mellifera in conjunction with gene expression analysis suggests that parasite-defensive grooming in A. cerana is likely triggered not only by exogenous stimuli through visual and olfactory detection of the parasite, but also by genetically endogenous processes that periodically activates a bout of grooming to remove the ectoparasite. This information provides a valuable platform to facilitate the traits unique to A. cerana as well as those shared with other social bees for health improvement.