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.

Identification of genes related to high royal jelly production in the honey bee (Apis mellifera) using microarray analysis

Abstract China is the largest royal jelly producer and exporter in the world, and high royal jelly-yielding strains have been bred in the country for approximately three decades. However, information on the molecular mechanism underlying high royal jelly production is scarce. Here, a cDNA microarray was used to screen and identify differentially expressed genes (DEGs) to obtain an overview on the changes in gene expression levels between high and low royal jelly producing bees. We developed a honey bee gene chip that covered 11,689 genes, and this chip was hybridised with cDNA generated from RNA isolated from heads of nursing bees. A total of 369 DEGs were identified between high and low royal jelly producing bees. Amongst these DEGs, 201 (54.47%) genes were up-regulated, whereas 168 (45.53%) were down-regulated in high royal jelly-yielding bees. Gene ontology (GO) analyses showed that they are mainly involved in four key biological processes, and pathway analyses revealed that they belong to a total of 46 biological pathways. These results provide a genetic basis for further studies on the molecular mechanisms involved in high royal jelly production.

Identification of genes related to high royal jelly production in the honey bee (Apis mellifera) using microarray analysis.

China is the largest royal jelly producer and exporter in the world, and high royal jelly-yielding strains have been bred in the country for approximately three decades. However, information on the molecular mechanism underlying high royal jelly production is scarce. Here, a cDNA microarray was used to screen and identify differentially expressed genes (DEGs) to obtain an overview on the changes in gene expression levels between high and low royal jelly producing bees. We developed a honey bee gene chip that covered 11,689 genes, and this chip was hybridised with cDNA generated from RNA isolated from heads of nursing bees. A total of 369 DEGs were identified between high and low royal jelly producing bees. Amongst these DEGs, 201 (54.47%) genes were up-regulated, whereas 168 (45.53%) were down-regulated in high royal jelly-yielding bees. Gene ontology (GO) analyses showed that they are mainly involved in four key biological processes, and pathway analyses revealed that they belong to a total of 46 biological pathways. These results provide a genetic basis for further studies on the molecular mechanisms involved in high royal jelly production.

Adsorption equilibrium, kinetics and thermodynamics of dichloroacetic acid from aqueous solution using mesoporous carbon.

The presence of disinfection by-products, such as trihalomethanes and haloacetic acids in water, is believed to be harmful to human health. In this work, mesoporous carbon was synthesized with the evaporation-induced self-assembly method and employed to evaluate the effects of initial concentration, contact time, pH and temperature on the removal of dichloroacetic acid in batch experiments. Adsorption equilibrium was established in 480 min and the maximum adsorption (350mg/g) of dichloroacetic acid on the mesoporous carbon was observed to occur at 308 K and pH 3.0. Freundlich and Langmuir isotherms were used to analyse the equilibrium data at different temperatures; kinetic data were fitted to the pseudo-first-order and pseudo-second-order models and found that the adsorption capacity, mass transfer coefficient and diffusivity of dichloroacetic acid were directly affected by the physical and chemical parameters. In addition, the various thermodynamic parameters, such as Gibbs free energy (Delta G), enthalpy (Delta H = 54.35 kJmol-1) and entropy (Delta S = 258.36 Jmol-1 K-1) were calculated to analyse the adsorption process. The experimental results indicated that the mesoporous carbon was an excellent adsorbent for dichloroacetic acid removal from aqueous solutions.

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 4(th) to 6(th) 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.

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.

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.

Non-destructive genotyping and genetic variation of fanning in a honey bee colony.

The relationship between workers from different patrilines in a naturally mated queen honey bee colony is very complex due to queen polyandry, and still poorly characterized. Here, we report a means of determining the genotype of living workers in a natural honey bee colony by a new non-destructive method, which makes it possible to observe the relationship between behaviours and genotypes. DNA was extracted from the exuvia, found at the bottom of each brood cell, and confirmed to be identical to the DNA extracted from the thorax muscle of the bee emerging from that particular cell. The genotypes were thus determined using DNA from the exuviae without having to hurt or kill the organisms. The emerging workers were marked with coloured, numbered tags to enable behavioural observations over their entire life. Using this new method, we determined 20 patrilines in a naturally mated queen colony, and discovered that the patriline composition of bees exhibiting fanning behaviour was significantly different from the patriline composition of the whole colony. Our results confirm that the genetic structure of a natural insect society plays a fundamental role in the division of labour. The new non-destructive method reveals a novel avenue for the determination of relationships between the behaviours and genes of social insects.

[Isolation and identification of a newbacterial pathogen infecting larvae of honeybee (Apismellifera ) Perish].

A bacteroidal disease of honeybee (Apis mellifera ) larvae was found in some regions of Zhejiang Province, China , in early spring 2005. The diseased larvae lost its shine, became yellow and rotted when serious. This symptom was different to any bacteroidal disease of honeybee larval been reported. So, it is considered to be a new bacteroidal disease of honeybee larval. Five pure cultures of bacteria were separated from ten collections of diseased honeybee larvae, named as L1, L2, L3, IA and L5. Among these five pure cultures, only L2 could make the healthy honeybee larvae become diseased in both field and lab test. The symptom caused by L2 was similar to the natural-infection. From the diseased larvae caused by L2 could isolate bacteria the same as L2. Thus 12 was determined as the causing agent of this bacteroidal disease of honeybee larval. L2 was identified according to the characteristics of morphology, physiological biochemical characteristics and 16S rRNA gene sequence. As a result, the morphology and physiological biochemical characteristics of L2 were similar to E. faecium. And its 16S rRNA sequences highly matched to E. faecium, the similarities between them were higher than 99%. The overall similarity values between L2 and the published 16S rRNA sequences of 41 typical species of Enterococcus were 93.9% - 99.5% , the top value was between 12 and E. faecium. In the phylogenetic tree, L2 and E. faecium were assembled in the same ramification. So 12 was identified as E. faecium. Although Enterococcus faecium was known as pathogen to many post, account for 12% of all nosocomial infections, only second to E. coli, but there is no report about this bacteria infects honeybee up to now. So it is a new pathogen to honeybee. The isolation and identification of pathogen of this new bacteroidal larvae disease, afford a good feasibility for available prevention and cure to this new disease.

Microsatellite analysis of royal jelly producing traits of Italian honeybee (Apis mellifera Liguatica).

Genetic variations at 10 microsatellite loci were surveyed to determine the evolutionary relationships and molecular characteristics of three different honeybee (Apis mellifera L.) populations from Italy and China, i. e., native Italian (Ee), Chinese-Italian (Eb) and selected high royal jelly producing bees (Ea). A total of 96 alleles,an average of 9.6 alleles per locus,were scored in Ee,Eb and Ea bees at 10 loci. Out of which 48 (5%) were different. This indicated a high degree of polymorphism and ever, some genetic differentiation among the three populations due to artificial selection and geographical isolation. The polymorphic information contents (PIC) and heterozyosity of the three populations at 10 loci were 0.57, 0.50, 0.57, and 0.60, 0.57, 0.61, for Ee, Eb, Ea populations respectively, neither of which were different. This indicated same gene diversity within the three populations. The genetic distance was shorter between Ee and Eb bees as well as between Eb and Ea bees. Whereas that between Ee and Eb bees was longer. Further analysis indicated that the allele frequency of seven alleles at six loci (159 bp at A29,100 bp and 104 bp at A24; 110 bp at A7; 126 bp at A43, 221 bp at A14 and 221 bp at A113) increased going from Ee to Eb to Ea bees. Paired tests showed significant higher allele frequency between Ea and Eb bees,as well as between Ea and Eb bees. This indicates that these seven alleles are likely molecular markers of the high royal jelly producing bees. In addition,the allele frequency of four alleles at four loci (106 bp at A24,140 bp at A43;215 bp at A113 and 219 bp at A14) decreased going from Ea bees to Eb to Ee. Paired tests indicated significant lower allele frequency between Ea and Ee bees,as well as between Ea and Eb bees. Those four alleles may be the genetic markers for low royal jelly production.

[Cloning and sequence analysis of mrjp1 cDNA from Apis cerana cerana].

A cDNA library was constructed from eight-day-old worker heads of Apis cerana cerana. A probe derived from part of Apis cerana genomic mrjp3 segment was used to screen this library. A total of 120 positive clones of mrjps were screened out from the cDNA library with DIG-probe. The positive clones were amplified and sequenced with T3/T7 primer. 12 sequences were similar to Apis cerana india and Apis mellifera mrjp1 gene by BLAST analysis. The cDNA sequence analysis indicated that the homology was 93.78% between Apis mellifera mrjp1 and Apis cerana mrjp1 while the homology was 99.36% between Apis cerana cerana and Apis cerana india. This result confirmed in molecular level that Apis cerana cerana and Apis cerana india had a common ancestor since the relationship between Apis cerana and Apis mellifera was more far.

[Genetic analysis for developmental behavior of honeybee colony's royal jelly production traits in western honeybees].

Analyses of genotypic effects for colony's royal jelly (RJ) yields and RJ in each queen cell as well as acceptances of queen cell cups of three lines of Western honeybees (Apis melliffera Lingistica) were conducted by using a genotypic model for analyzing the genetic and non-genetic effects. Analytis approaches of conventional and conditional variance and correlation were employed to evaluate the developmental behavior of honeybee colony's RJ producing ability. The results indicated that significant variance due to genotypic effect was detected for colony's RJ yields and RJ in each queen cell cup at all stages, while the acceptance of queen cell cups was found variance significantly at 10 stages of its total 11. It meant that the three traits were dominated by genotypic effect. Significant conditional variances were found at some stages when no unconditional one being detected. Correlation analysis between same trait at different stages indicated that the significant coefficients always existed due to genotypic effects for RJ yields and RJ in each queen cell cup. Although the significant coefficients of the acceptance of queen cell cup at different stages were found at most of the stages, the coefficients were not found in come of the stages. These results indicated that the genotypic effects dominated in early stages of colony's RJ yield and RJ in each queen cell cup influencing its late stages in the same way, but the acceptance of queen cells cup was not. The analysis of correlation between different pair of traits showed that the coefficients between colony's RJ yields and RJ in each queen cell cup were significant at all stages, and while the coefficients between colony's RJ yields and the acceptance of queen cell cups were not always significant at all stages. It was because the genotypic effects dominated the former pair of traits had harmonious effects, while the later pair of traits were not.

[Research on morphological genetic marker of honeybee (Apis melli fera ligustica) in royal jelly production performance].

The lengths of hypopharyngeal glands (HG) from the left and right side were determined in 19 workers of honeybee(Apis mellifera ligustica). There were no significant differences (P<0.05) in length between the left and the right in one worker's hypopharyngeal gland. Three hundred and thirty workers were collected from eleven colonies of "ZND No.1" Italian honeybee(Apis mellifera ligustica) respectively. Head weight, body weight, ratio between head weight and body weight,bursa number and length of hypopharyngeal gland(HG)were tested in these samples. Royal jelly productions were determined during the flowing period of rape and Chinese milk retch from March 30 to April 26 in Chun'an County of Zhejiang Province in 2001. The correlation analysis between royal jelly production and head weight, ratio between head weight and body weight, bursa number of HG,and length of HG were conducted. The correlation coefficient between royal jelly production and length of HG was the largest. The correlation coefficient between royal jelly production and bursa number was the second. It was suggested that the length of HG could be used as one of genetic markers for the production performance of royal jelly.

[Research progress in malate dehydrogenase(MDH) of honeybees].

Malate dehydrogenase(MDH) is an important enzyme in glycometabolism. MDH of Apis mellifera showed three enzyme active zones, MDHI,MDHIIand MDHIII. MDHIand MDHIII maintained relative stability in different castes and developmental phases,but MDHIIwas polymorphic,and controlled by three alleles,a,b and c.MDH of Apis cerana was coded by S and F alleles,but some authors reported it is monomorphic.MDH was applied to the studies of A. mellifera, which included several aspects as follows: the number of queen matings,labor division in honeybee societies, the analysis of genetic constitution in honeybee populations and so on. The combination of both MDH and molecular biology will certainly promote honeybee studies.