Zhi-Jiang Zeng

The integrative analysis of microRNA and mRNA expression in Apis mellifera following maze-based visual pattern learning.

The honeybee (Apis mellifera) is a social insect with strong sensory capacity and diverse behavioral repertoire and is recognized as a good model organism for studying the neurobiological basis of learning and memory. In this study, we analyzed the changes in microRNA (miRNA) and messenger RNA (mRNA) following maze‐based visual learning using next‐generation small RNA sequencing and Solexa/lllumina Digital Gene Expression tag profiling (DGE). For small RNA sequencing, we obtained 13 367 770 and 13 132 655 clean tags from the maze and control groups, respectively. A total of 40 differentially expressed known miRNAs were detected between these two samples, and all of them were up‐regulated in the maze group compared to the control group. For DGE, 5 681 320 and 5 939 855 clean tags were detected from the maze and control groups, respectively. There were a total of 388 differentially expressed genes between these two samples, with 45 genes up‐regulated and 343 genes down‐regulated in the maze group, compared to the control group. Additionally, the expression levels of 10 differentially expressed genes were confirmed by quantitative reverse transcription polymerase chain reaction (qRT‐PCR) and the expression trends of eight of them were consistent with the DGE result, although the degree of change was lower in amplitude. The integrative analysis of miRNA and mRNA expression showed that, among the 40 differentially expressed known miRNAs and 388 differentially expressed genes, 60 pairs of miRNA/mRNA were identified as co‐expressed in our present study. These results suggest that both miRNA and mRNA may play a pivotal role in the process of learning and memory in honeybees. Our sequencing data provide comprehensive miRNA and gene expression information for maze‐based visual learning, which will facilitate understanding of the molecular mechanisms of honeybee learning and memory.

Comparison of learning and memory of Apis cerana and Apis mellifera.

The honeybee is an excellent model organism for research on learning and memory among invertebrates. Learning and memory in honeybees has intrigued neuroscientists and entomologists in the last few decades, but attention has focused almost solely on the Western honeybee, Apis mellifera. In contrast, there have been few studies on learning and memory in the Eastern honeybee, Apis cerana. Here we report comparative behavioral data of color and grating learning and memory for A. cerana and A. mellifera in China, gathered using a Y-maze apparatus. We show for the first time that the learning and memory performance of A. cerana is significantly better on both color and grating patterns than that of A. mellifera. This study provides the first evidence of a learning and memory difference between A. cerana and A. mellifera under controlled conditions, and it is an important basis for the further study of the mechanism of learning and memory in honeybees.

Nutrition affects longevity and gene expression in honey bee (Apis mellifera) workers

Nutrition is a major factor affecting animal health, resistance to disease, and survival. In honey bees (Apis mellifera), nectar or honey (carbohydrates) is the energy source, while pollen, which is the sole dietary source of protein, is essential for both larval and adult development. Royal jelly (RJ), a secretion from workers with high protein content, plays a critical role in which queens are fed throughout their lives, is responsible for switching the worker phenotype into the queen one. The role of RJ in extending the lifespan of caged workers is not clear. In this study, we determined longevity of caged workers fed with different diets (carbohydrate only, pollen, and pollen+ RJ) and also expression of six genes in these bees. We found that workers fed with pollen and royal jelly together (P+ RJ+) showed the best survival, followed by workers fed with pollen only (P + RJ−), and workers fed with neither pollen nor RJ (P− RJ−) had the shortest life. Pollen only (P + RJ−) and royal jelly together (P+ RJ+) significantly affected four of the six genes studied. While pollen and royal jelly together (P+ RJ+) only affected the vitellogenin gene compared to pollen only (P+ RJ−). These results demonstrate that pollen and RJ extended worker longevity, suggesting that they may improve the nutritional conditions of bees or contain health and longevity-promoting factors. Further analysis of the lifespan-extending genes may broaden our understanding of gene network involved in the regulation of longevity.

Gene expression analysis following olfactory learning in Apis mellifera

The honeybee has a strong learning and memory ability, and is recognized as the best model organism for studying the neurobiological basis of learning and memory. In this study, we analyzed the gene expression difference following proboscis extension response-based olfactory learning in the A. mellifera using a tag-based digital gene expression (DGE) method. We obtained about 5.71 and 5.65 million clean tags from the trained group and untrained group, respectively. A total of 259 differentially expressed genes were detected between these two samples, with 30 genes up-regulated and 229 genes down-regulated in trained group compared to the untrained group. These results suggest that bees tend to actively suppress some genes instead of activating previously silent genes after olfactory learning. Our DGE data provide comprehensive gene expression information for olfactory learning, which will facilitate our understanding of the molecular mechanism of honey bee learning and memory.

Differential protein expression analysis following olfactory learning in Apis cerana

Studies of olfactory learning in honeybees have helped to elucidate the neurobiological basis of learning and memory. In this study, protein expression changes following olfactory learning in Apis cerana were investigated using isobaric tags for relative and absolute quantification (iTRAQ) technology. A total of 2406 proteins were identified from the trained and untrained groups. Among these proteins, 147 were differentially expressed, with 87 up-regulated and 60 down-regulated in the trained group compared with the untrained group. These results suggest that the differentially expressed proteins may be involved in the regulation of olfactory learning and memory in A. cerana. The iTRAQ data can provide information on the global protein expression patterns associated with olfactory learning, which will facilitate our understanding of the molecular mechanisms of learning and memory of honeybees.

Transcriptome differences in the hypopharyngeal gland between Western Honeybees (Apis mellifera) and Eastern Honeybees (Apis cerana)

BackgroundApis mellifera and Apis cerana are two sibling species of Apidae. Apis cerana is adept at collecting sporadic nectar in mountain and forest region and exhibits stiffer hardiness and acarid resistance as a result of natural selection, whereas Apis mellifera has the advantage of producing royal jelly. To identify differentially expressed genes (DEGs) that affect the development of hypopharyngeal gland (HG) and/or the secretion of royal jelly between these two honeybee species, we performed a digital gene expression (DGE) analysis of the HGs of these two species at three developmental stages (newly emerged worker, nurse and forager).ResultsTwelve DGE-tag libraries were constructed and sequenced using the total RNA extracted from the HGs of newly emerged workers, nurses, and foragers of Apis mellifera and Apis cerana. Finally, a total of 1482 genes in Apis mellifera and 1313 in Apis cerana were found to exhibit an expression difference among the three developmental stages. A total of 1417 DEGs were identified between these two species. Of these, 623, 1072, and 462 genes showed an expression difference at the newly emerged worker, nurse, and forager stages, respectively. The nurse stage exhibited the highest number of DEGs between these two species and most of these were found to be up-regulated in Apis mellifera. These results suggest that the higher yield of royal jelly in Apis mellifera may be due to the higher expression level of these DEGs.ConclusionsIn this study, we investigated the DEGs between the HGs of two sibling honeybee species (Apis mellifera and Apis cerana). Our results indicated that the gene expression difference was associated with the difference in the royal jelly yield between these two species. These results provide an important clue for clarifying the mechanisms underlying hypopharyngeal gland development and the production of royal jelly.

RFID monitoring indicates honeybees work harder before a rainy day

Storms are usually accompanied by a drop in temperature, and an increase in wind and barometric pressure and rainfall, which have negative impacts on most activities, survival and reproduction in insects (Gillot, 2005). The majority of studies have mainly focused on how the flight activity of various flying insects such as honeybees, bumble bees, horse flies and leafminers were directly influenced by intraday weather changes (Burnett & Hays, 1974; Lundberg, 1980; Casas, 1989; Vicens & Bosch, 2000). However, accumulating evidences showed that animals can make behavioral changes before storms, which is enormously important for their survival in severe weather conditions. Before upcoming storms birds unusually chirp and bathe with sand; native frogs croak and hide their egg masses; spiders spin shorter and produce thicker webs and wasps hide their comb before rains (Galacgac & Balisacan, 2009; Acharya, 2011). In early 1893, honeybees were reported as more active before storms (Inwards, 1893). In this study, we compared the working habits of foragers on days that were followed by a sunny day and those that were followed by a rainy day using the Radio Frequency Identification (RFID), which was developed and manufactured by the Honeybee Research Institute of Jiangxi Agricultural University in collaboration with the Guangzhou Invengo Information Technology Co., Ltd., and we showed that honeybees worked harder before a rainy day. Three honeybee (Apis mellifera) colonies were maintained at the Honeybee Research Institute, Jiangxi Agricultural University, Nanchang, China. Each colony had four full frames, with approximately 6000 workers and a

Polymorphism analysis of csd gene in six Apis mellifera subspecies

The complementary sex determination (csd) gene is the primary gene determining the gender of honey bees (Apis spp). In this study we analyzed the polymorphism of csd gene in six Apis mellifera subspecies. The genomic region 3 of csd gene in these six A. mellifera was cloned, and identified. A total of 79 haplotypes were obtained from these six subspecies. Analysis showed that region 3 of csd gene has a high level of polymorphism in all the six A. mellifera subspecies. The A. m. anatolica subspecies has a slightly higher nucleotide diversity (π) than other subspecies, while the π values showed no significant difference among the other five subspecies. The phylogenetic tree showed that all the csd haplotypes from different A. mellifera subspecies are scattered throughout the tree, without forming six different clades. Population differentiation analysis showed that there are significant genetic differentiations among some of the subspecies. The NJ phylogenetic tree showed that the A. m. caucasica and A. m. carnica have the closest relationship, followed by A. m. ssp, A. m. ligustica, A. m. carpatica and A. m. anatolica that were gathered in the tree in turn.

csd alleles in the red dwarf honey bee (Apis florea, Hymenoptera: Apidae) show exceptionally high nucleotide diversity

Abstract  The single locus complementary sex determination (sl‐csd) gene is the primary gene determining the gender of honey bees (Apis spp.). While the csd gene has been well studied in the Western honey bee (Apis mellifera), and comparable data exist in both the Eastern honey bee (Apis cerana) and the giant honey bee (Apis dorsata), no studies have been conducted in the red dwarf honey bee, Apis florea. In this study we cloned the genomic region 3 of the A. florea csd gene from 60 workers, and identified 12 csd alleles. Analysis showed that similar to A. mellifera, region 3 of the csd gene contains a RS domain at the N terminal, a proline‐rich domain at the C terminal, and a hypervariable region in the middle. However, the A. florea csd gene possessed a much higher level of nucleotide diversity, compared to A. mellifera, A. cerana and Apis dorsata. We also show that similar to the other three Apis species, in A. florea, nonsynonymous mutations in the csd gene are selectively favored in young alleles.

Lateralization of gene expression in the honeybee brain during olfactory learning

In the last decade, it has been demonstrated that brain functional asymmetry occurs not only in vertebrates but also in invertebrates. However, the mechanisms underlying functional asymmetry remain unclear. In the present study, we trained honeybees of the same parentage and age, on the proboscis extension reflex (PER) paradigm with only one antenna in use. The comparisons of gene expression between the left and right hemispheres were carried out using high throughput sequencing. Our research revealed that gene expression in the honeybee brain is also asymmetric, with more genes having higher expression in the right hemisphere than the left hemisphere. Our studies show that during olfactory learning, the left hemisphere is more responsible for long term memory and the right hemisphere is more responsible for the learning and short term memory.