Genome-wide characterization of long intergenic non-coding RNAs (lincRNAs) provides new insight into viral diseases in honey bees Apis cerana and Apis mellifera

Background

Long non-coding RNAs (lncRNAs) are a class of RNAs that do not encode proteins. Recently, lncRNAs have gained special attention for their roles in various biological process and diseases.

Results

In an attempt to identify long intergenic non-coding RNAs (lincRNAs) and their possible involvement in honey bee development and diseases, we analyzed RNA-seq datasets generated from Asian honey bee (Apis cerana) and western honey bee (Apis mellifera). We identified 2470 lincRNAs with an average length of 1011 bp from A. cerana and 1514 lincRNAs with an average length of 790 bp in A. mellifera. Comparative analysis revealed that 5 % of the total lincRNAs derived from both species are unique in each species. Our comparative digital gene expression analysis revealed a high degree of tissue-specific expression among the seven major tissues of honey bee, different from mRNA expression patterns. A total of 863 (57 %) and 464 (18 %) lincRNAs showed tissue-dependent expression in A. mellifera and A. cerana, respectively, most preferentially in ovary and fat body tissues. Importantly, we identified 11 lincRNAs that are specifically regulated upon viral infection in honey bees, and 10 of them appear to play roles during infection with various viruses.

Conclusions

This study provides the first comprehensive set of lincRNAs for honey bees and opens the door to discover lincRNAs associated with biological and hormone signaling pathways as well as various diseases of honey bee.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1868-7) contains supplementary material, which is available to authorized users.     

Hemolymph proteome changes during worker brood development match the biological divergences between western honey bees (Apis mellifera) and eastern honey bees (Apis cerana)

Background

Hemolymph plays key roles in honey bee molecule transport, immune defense, and in monitoring the physiological condition. There is a lack of knowledge regarding how the proteome achieves these biological missions for both the western and eastern honey bees (Apis mellifera and Apis cerana). A time-resolved proteome was compared using two-dimensional electrophoresis-based proteomics to reveal the mechanistic differences by analysis of hemolymph proteome changes between the worker bees of two bee species during the larval to pupal stages.

Results

The brood body weight of Apis mellifera was significantly heavier than that of Apis cerana at each developmental stage. Significantly, different protein expression patterns and metabolic pathways were observed in 74 proteins (166 spots) that were differentially abundant between the two bee species. The function of hemolymph in energy storage, odor communication, and antioxidation is of equal importance for the western and eastern bees, indicated by the enhanced expression of different protein species. However, stronger expression of protein folding, cytoskeletal and developmental proteins, and more highly activated energy producing pathways in western bees suggests that the different bee species have developed unique strategies to match their specific physiology using hemolymph to deliver nutrients and in immune defense.

Conclusions

Our disparate findings constitute a proof-of-concept of molecular details that the ecologically shaped different physiological conditions of different bee species match with the hemolymph proteome during the brood stage. This also provides a starting point for future research on the specific hemolymph proteins or pathways related to the differential phenotypes or physiology.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-563) contains supplementary material, which is available to authorized users.     

A SNP Based High-Density Linkage Map of Apis cerana Reveals a High Recombination Rate Similar to Apis mellifera

Background

The Eastern honey bee, Apis cerana Fabricius, is distributed in southern and eastern Asia, from India and China to Korea and Japan and southeast to the Moluccas. This species is also widely kept for honey production besides Apis mellifera. Apis cerana is also a model organism for studying social behavior, caste determination, mating biology, sexual selection, and host-parasite interactions. Few resources are available for molecular research in this species, and a linkage map was never constructed. A linkage map is a prerequisite for quantitative trait loci mapping and for analyzing genome structure. We used the Chinese honey bee, Apis cerana cerana to construct the first linkage map in the Eastern honey bee.

Results

F2 workers (N = 103) were genotyped for 126,990 single nucleotide polymorphisms (SNPs). After filtering low quality and those not passing the Mendel test, we obtained 3,000 SNPs, 1,535 of these were informative and used to construct a linkage map. The preliminary map contains 19 linkage groups, we then mapped the 19 linkage groups to 16 chromosomes by comparing the markers to the genome of A. mellfiera. The final map contains 16 linkage groups with a total of 1,535 markers. The total genetic distance is 3,942.7 centimorgans (cM) with the largest linkage group (180 loci) measuring 574.5 cM. Average marker interval for all markers across the 16 linkage groups is 2.6 cM.

Conclusion

We constructed a high density linkage map for A. c. cerana with 1,535 markers. Because the map is based on SNP markers, it will enable easier and faster genotyping assays than randomly amplified polymorphic DNA or microsatellite based maps used in A. mellifera.     

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

Background

Apis 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).

Results

Twelve 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.

Conclusions

In 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.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-744) contains supplementary material, which is available to authorized users.     

Comparative susceptibility and immune responses of Asian and European honey bees to the American foulbrood pathogen,Paenibacillus larvae

American foulbrood (AFB) disease is caused by Paenibacillus larvae. Currently, this pathogen is widespread in the European honey bee— Apis mellifera. However, little is known about infectivity and pathogenicity of P. lan'ae in the Asiatic cavity-nesting honey bees, Apis cerana. Moreover, comparative knowledge of P. larvae infectivity and pathogenicity between both honey bee species is scarce. In this study, we examined susceptibility, larval mortality, survival rate and expression of genes encoding antimicrobial peptides (AMPs) including defensin, apidaecin, abaecin, and hymenoptaecin in A. mellifera and A. cerana when infected with P. larvae. Our results showed similar effects of P. larvae on the survival rate and patterns of AMP gene expression in both honey bee species when bee larvae are infected with spores at the median lethal concentration (LC5 0 ) for A. mellifera. All AMPs of infected bee larvae showed significant upregulation compared with noninfected bee larvae in both honey bee species. However, larvae of A. cerana were more susceptible than A. mellifera when the same larval ages and spore concentration of P. larvae were used. It also appears that A. cerana showed higher levels of AMP expression than A. mellifera. This research provides the first evidence of survival rate, LC50 and immune response profiles of Asian honey bees, A. cerana, when infected by P. larvae in comparison with the European honey bee, A. mellifera.     

Chronic parasitization by Nosema microsporidia causes global expression changes in core nutritional,metabolic and behavioral pathways in honey bee workers (Apis mellifera)

Background

Chronic infections can profoundly affect the physiology, behavior, fitness and longevity of individuals, and may alter the organization and demography of social groups. Nosema apis and Nosema ceranae are two microsporidian parasites which chronically infect the digestive tract of honey bees (Apis mellifera). These parasites, in addition to other stressors, have been linked to increased mortality of individual workers and colony losses in this key pollinator species. Physiologically, Nosema infection damages midgut tissue, is energetically expensive and alters expression of immune genes in worker honey bees. Infection also accelerates worker transition from nursing to foraging behavior (termed behavioral maturation). Here, using microarrays, we characterized global gene expression patterns in adult worker honey bee midgut and fat body tissue in response to Nosema infection.

Results

Our results indicate that N. apis infection in young workers (1 and 2 days old) disrupts midgut development. At 2 and 7 days post-infection in the fat body tissue, N. apis drives metabolic changes consistent with energetic costs of infection. A final experiment characterizing gene expression in the fat bodies of 14 day old workers parasitized with N. apis and N. ceranae demonstrated that Nosema co-infection specifically alters conserved nutritional, metabolic and hormonal pathways, including the insulin signaling pathway, which is also linked to behavioral maturation in workers. Interestingly, in all experiments, Nosema infection did not appear to significantly regulate overall expression of canonical immune response genes, but infection did alter expression of acute immune response genes identified in a previous study. Comparative analyses suggest that changes in nutritional/metabolic processes precede changes in behavioral maturation and immune processes.

Conclusions

These genome-wide studies of expression patterns can help us disentangle the direct and indirect effects of chronic infection, and understand the molecular pathways that regulate disease symptoms.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-799) contains supplementary material, which is available to authorized users.     

Extensive intra-phylotype diversity in lactobacilli and bifidobacteria from the honeybee gut

Background

In the honeybee Apis mellifera, the bacterial gut community is consistently colonized by eight distinct phylotypes of bacteria. Managed bee colonies are of considerable economic interest and it is therefore important to elucidate the diversity and role of this microbiota in the honeybee. In this study, we have sequenced the genomes of eleven strains of lactobacilli and bifidobacteria isolated from the honey crop of the honeybee A. mellifera.

Results

Single gene phylogenies confirmed that the isolated strains represent the diversity of lactobacilli and bifidobacteria in the gut, as previously identified by 16S rRNA gene sequencing. Core genome phylogenies of the lactobacilli and bifidobacteria further indicated extensive divergence between strains classified as the same phylotype. Phylotype-specific protein families included unique surface proteins. Within phylotypes, we found a remarkably high level of gene content diversity. Carbohydrate metabolism and transport functions contributed up to 45% of the accessory genes, with some genomes having a higher content of genes encoding phosphotransferase systems for the uptake of carbohydrates than any previously sequenced genome. These genes were often located in highly variable genomic segments that also contained genes for enzymes involved in the degradation and modification of sugar residues. Strain-specific gene clusters for the biosynthesis of exopolysaccharides were identified in two phylotypes. The dynamics of these segments contrasted with low recombination frequencies and conserved gene order structures for the core genes. Hits for CRISPR spacers were almost exclusively found within phylotypes, suggesting that the phylotypes are associated with distinct phage populations.

Conclusions

The honeybee gut microbiota has been described as consisting of a modest number of phylotypes; however, the genomes sequenced in the current study demonstrated a very high level of gene content diversity within all three described phylotypes of lactobacilli and bifidobacteria, particularly in terms of metabolic functions and surface structures, where many features were strain-specific. Together, these results indicate niche differentiation within phylotypes, suggesting that the honeybee gut microbiota is more complex than previously thought.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1476-6) contains supplementary material, which is available to authorized users.     

Genome-wide analysis of signatures of selection in populations of African honey bees (Apis mellifera) using new web-based tools

Background

With the development of inexpensive, high-throughput sequencing technologies, it has become feasible to examine questions related to population genetics and molecular evolution of non-model species in their ecological contexts on a genome-wide scale. Here, we employed a newly developed suite of integrated, web-based programs to examine population dynamics and signatures of selection across the genome using several well-established tests, including F_ST, pN/pS, and McDonald-Kreitman. We applied these techniques to study populations of honey bees (Apis mellifera) in East Africa. In Kenya, there are several described A. mellifera subspecies, which are thought to be localized to distinct ecological regions.

Results

We performed whole genome sequencing of 11 worker honey bees from apiaries distributed throughout Kenya and identified 3.6 million putative single-nucleotide polymorphisms. The dense coverage allowed us to apply several computational procedures to study population structure and the evolutionary relationships among the populations, and to detect signs of adaptive evolution across the genome. While there is considerable gene flow among the sampled populations, there are clear distinctions between populations from the northern desert region and those from the temperate, savannah region. We identified several genes showing population genetic patterns consistent with positive selection within African bee populations, and between these populations and European A. mellifera or Asian Apis florea.

Conclusions

These results lay the groundwork for future studies of adaptive ecological evolution in honey bees, and demonstrate the use of new, freely available web-based tools and workflows (http://usegalaxy.org/r/kenyanbee) that can be applied to any model system with genomic information.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1712-0) contains supplementary material, which is available to authorized users.     

Differential gene expression of the honey bees Apis mellifera and A. cerana induced by Varroa destructor infection

Varroa destructor mite is currently the most serious threat to the world bee industry. Differences in mite tolerance are reported between two honey bee species Apis mellifera and Apis cerana. Differential gene expression of two honey bee species induced by V. destructor infection was investigated by constructing two suppression subtractive hybridization (SSH) libraries, as first steps toward elucidating molecular mechanisms of Varroa tolerance. From the SSH libraries, we obtained 289 high quality sequences which clustered into 132 unique sequences grouped in 26 contigs and 106 singlets where 49 consisted in A. cerana subtracted library and 83 in A. mellifera. Using BLAST, we found that 85% sequences had counterpart known genes whereas 15% were undescribed. A Gene Ontology analysis classified 51 unique sequences into different functional categories. Eight of these differentially expressed genes, representative of different regulation patterns, were confirmed by qRT-PCR. Upon the mite induction, the differentially expressed genes from both bee species were different, except hex 110 gene, which was up-regulated in A. cerana but down-regulated in A. mellifera, and Npy-r gene, which was down-regulated in both species. In general, most of the differential expression genes were involved in metabolic processes and nerve signaling. The results provide information on the molecular response of these two bee species to Varroa infection.     

Pathological effects of the microsporidium Nosema ceranae on honey bee queen physiology (Apis mellifera)

Nosema ceranae, a microsporidian parasite originally described in the Asian honey bee Apis cerana, has recently been found to be cross-infective and to also parasitize the European honey bee Apis mellifera. Since this discovery, many studies have attempted to characterize the impact of this parasite in A. mellifera honey bees. Nosema species can infect all colony members, workers, drones and queens, but the pathological effects of this microsporidium has been mainly investigated in workers, despite the prime importance of the queen, who monopolizes the reproduction and regulates the cohesion of the society via pheromones. We therefore analyzed the impact of N. ceranae on queen physiology. We found that infection by N. ceranae did not affect the fat body content (an indicator of energy stores) but did alter the vitellogenin titer (an indicator of fertility and longevity), the total antioxidant capacity and the queen mandibular pheromones, which surprisingly were all significantly increased in Nosema-infected queens. Thus, such physiological changes may impact queen health, leading to changes in pheromone production, that could explain Nosema-induced supersedure (queen replacement).     

A meta-analysis of effects of Bt crops on honey bees (Hymenoptera: Apidae)

Background

Honey bees (Apis mellifera L.) are the most important pollinators of many agricultural crops worldwide and are a key test species used in the tiered safety assessment of genetically engineered insect-resistant crops. There is concern that widespread planting of these transgenic crops could harm honey bee populations.

Methodology/Principal Findings

We conducted a meta-analysis of 25 studies that independently assessed potential effects of Bt Cry proteins on honey bee survival (or mortality). Our results show that Bt Cry proteins used in genetically modified crops commercialized for control of lepidopteran and coleopteran pests do not negatively affect the survival of either honey bee larvae or adults in laboratory settings.

Conclusions/Significance

Although the additional stresses that honey bees face in the field could, in principle, modify their susceptibility to Cry proteins or lead to indirect effects, our findings support safety assessments that have not detected any direct negative effects of Bt crops for this vital insect pollinator.     

Structural and functional differences in the antennal olfactory system of worker honey bees of Apis mellifera and Apis cerana

Olfactory cues are important sensory modalities on individual discrimination, perception, and efficient orientation to food sources in most insects. In honey bees, which are well known as eusocial insects, olfactory cues are mainly used to maintain a colony. Although much research has been reported on olfactory systems in honey bee olfaction, little is known about the differences between two major honey bee species, the European honey bee Apis mellifera and the Asian honey bee Apis cerana. In order to understand the differences of olfactory characteristics in the two species, we compared the distribution of sensory hairs on the antennae and antennal olfactory responses, using electron microscopy, electrophysiological recording and molecular expression level of odorant receptors. Our present study demonstrated that the antennae of A. cerana have more olfactory sensilla than A. mellifera, responding more strongly to various floral volatile compounds. At the molecular level, olfactory co-receptor (Orco), which makes heterodimers with other conventional olfactory receptors, is more abundantly expressed in the antenna of A. cerana than of A. mellifera. These findings extend our understanding of the olfactory systems and behavioral responses to various ecological and biological signals in two closely related honey bee species.     

Diet and cell size both affect queen-worker differentiation through DNA methylation in honey bees (Apis mellifera, Apidae)

Background

Young larvae of the honey bee (Apis mellifera) are totipotent; they can become either queens (reproductives) or workers (largely sterile helpers). DNA methylation has been shown to play an important role in this differentiation. In this study, we examine the contributions of diet and cell size to caste differentiation.

Methodology/Principal Findings

We measured the activity and gene expression of one key enzyme involved in methylation, Dnmt3; the rates of methylation in the gene dynactin p62; as well as morphological characteristics of adult bees developed either from larvae fed with worker jelly or royal jelly; and larvae raised in either queen or worker cells. We show that both diet type and cell size contributed to the queen-worker differentiation, and that the two factors affected different methylation sites inside the same gene dynactin p62.

Conclusions/Significance

We confirm previous findings that Dnmt3 plays a critical role in honey bee caste differentiation. Further, we show for the first time that cell size also plays a role in influencing larval development when diet is kept the same.     

Pyrosequencing analysis of the bacterial communities in the guts of honey bees Apis cerana and Apis mellifera in Korea

The bacterial communities in the guts of the adults and larvae of the Asian honey bee Apis cerana and the European honey bee Apis mellifera were surveyed by pyrosequencing the 16S rRNA genes. Most of the gut bacterial 16S rRNA gene sequences were highly similar to the known honey bee-specific ones and affiliated with Pasteurellaceae or lactic acid bacteria (LAB). The numbers of operational taxonomic units (OTUs, defined at 97% similarity) were lower in the larval guts (6 or 9) than in the adult guts (18 or 20), and the frequencies of Pasteurellaceae-related OTUs were higher in the larval guts while those of LAB-related OTUs in the adult guts. The frequencies of Lactococcus, Bartonella, Spiroplasma, Enterobacteriaceae, and Flavobacteriaceae-related OTUs were much higher in A. cerana guts while Bifidobacterium and Lachnospiraceae-related OTUs were more abundant in A. mellfera guts. The bacterial community structures in the midguts and hindguts of the adult honey bees were not different for A. cerana, but significantly different for A. mellifera. The above results substantiated the previous observation that honey bee guts are dominated by several specific bacterial groups, and also showed that the relative abundances of OTUs could be markedly changed depending on the developmental stage, the location within the gut, and the honey bee species. The possibility of using the gut bacterial community as an indicator of honey bee health was discussed.     

The resistance mechanism of the Asian honey bee,Apis cerana Fabr., to an ectoparasitic mite,Varroa jacobsoni Oudemans

A behavioral and physiological resistance mechanism of the Asian honey bee (Apis cerana) to an ectoparasitic mite, Varroa jacobsoni, which causes severe damage to the European honey bee (Apis mellifera) in the beekeeping industry worldwide, is reported here for the first time. Parasitism by the mite induced Asian worker bees to perform a series of cleaning behaviors that effectively removed the mites from the bodies of the adult host bees. The mites were subsequently killed and removed from the bee hives in a few seconds to a few minutes. The grooming behavior consists of self-cleaning, grooming dance, nestmate cleaning, and group cleaning. Worker bees can also rapidly and effectively remove the mites from the brood. The European bee showed cleaning behavior at low frequency and generally failed to remove the mites from both the adult bees and the brood.     

A Comparative Study of Relational Learning Capacity in Honeybees (Apis mellifera) and Stingless Bees (Melipona rufiventris)

Background

Learning of arbitrary relations is the capacity to acquire knowledge about associations between events or stimuli that do not share any similarities, and use this knowledge to make behavioural choices. This capacity is well documented in humans and vertebrates, and there is some evidence it exists in the honeybee (Apis mellifera). However, little is known about whether the ability for relational learning extends to other invertebrates, although many insects have been shown to possess excellent learning capacities in spite of their small brains.

Methodology/Principal Findings

Using a symbolic matching-to-sample procedure, we show that the honeybee Apis mellifera rapidly learns arbitrary relations between colours and patterns, reaching 68.2% correct choice for pattern-colour relations and 73.3% for colour-pattern relations. However, Apis mellifera does not transfer this knowledge to the symmetrical relations when the stimulus order is reversed. A second bee species, the stingless bee Melipona rufiventris from Brazil, seems unable to learn the same arbitrary relations between colours and patterns, although it exhibits excellent discrimination learning.

Conclusions/Significance

Our results confirm that the capacity for learning arbitrary relations is not limited to vertebrates, but even insects with small brains can perform this learning task. Interestingly, it seems to be a species-specific ability. The disparity in relational learning performance between the two bee species we tested may be linked to their specific foraging and recruitment strategies, which evolved in adaptation to different environments.     

Varroa destructor changes its cuticular hydrocarbons to mimic new hosts

Varroa destructor (Vd) is a honeybee ectoparasite. Its original host is the Asian honeybee, Apis cerana, but it has also become a severe, global threat to the European honeybee, Apis mellifera. Previous studies have shown that Varroa can mimic a host''s cuticular hydrocarbons (HC), enabling the parasite to escape the hygienic behaviour of the host honeybees. By transferring mites between the two honeybee species, we further demonstrate that Vd is able to mimic the cuticular HC of a novel host species when artificially transferred to this new host. Mites originally from A. cerana are more efficient than mites from A. mellifera in mimicking HC of both A. cerana and A. mellifera. This remarkable adaptability may explain their relatively recent host-shift from A. cerana to A. mellifera.     

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.