A scientific note about spectroscopic analysis of honey bee brood comb cappings in hygienic and non-hygienic honey bee colonies

Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)

The four hexamerin genes in the honey bee: structure, molecular evolution and function deduced from expression patterns in queens, workers and drones

Background: Hexamerins are hemocyanin-derived proteins that have lost the ability to bind copper ions and transport oxygen; instead, they became storage proteins. The current study aimed to broaden our knowledge on the hexamerin genes found in the honey bee genome by exploring their structural characteristics, expression profiles, evolution, and functions in the life cycle of workers, drones and queens. Results: The hexamerin genes of the honey bee (hex 70a, hex 70b, hex 70c and hex 110) diverge considerably in structure, so that the overall amino acid identity shared among their deduced protein subunits varies from 30 to 42%. Bioinformatics search for motifs in the respective upstream control regions (UCRs) revealed six overrepresented motifs including a potential binding site for Ultraspiracle (Usp), a target of juvenile hormone (JH). The expression of these genes was induced by topical application of JH on worker larvae. The four genes are highly transcribed by the larval fat body, although with significant differences in transcript levels, but only hex 110 and hex 70a are re-induced in the adult fat body in a caste-and sex-specific fashion, workers showing the highest expression. Transcripts for hex 110, hex 70a and hex70b were detected in developing ovaries and testes, and hex 110 was highly transcribed in the ovaries of egg-laying queens. A phylogenetic analysis revealed that HEX 110 is located at the most basal position among the holometabola hexamerins, and like HEX 70a and HEX 70c, it shares potential orthology relationship with hexamerins from other hymenopteran species. Conclusions: Striking differences were found in the structure and developmental expression of the four hexamerin genes in the honey bee. The presence of a potential binding site for Usp in the respective 5' UCRs, and the results of experiments on JH level manipulation in vivo support the hypothesis of regulation by JH. Transcript levels and patterns in the fat body and gonads suggest that, in addition to their primary role in supplying amino acids for metamorphosis, hexamerins serve as storage proteins for gonad development, egg production, and to support foraging activity. A phylogenetic analysis including the four deduced hexamerins and related proteins revealed a complex pattern of evolution, with independent radiation in insect orders.

Pollination of tomatoes by the stingless bee Melipona quadrifasciata and the honey bee Apis mellifera (Hymenoptera, Apidae)

The pollination effectiveness of the stingless bee Melipona quadrifasciata and the honey bee Apis mellifera was tested in tomato plots. The experiment was conducted in four greenhouses as well as in an external open plot in Ribeirao Preto, SP, Brazil. The tomato plants were exposed to visits by M. quadrifasciata in one greenhouse and to A. mellifera in another; two greenhouses were maintained without bees (controls) and an open field plot was exposed to pollinators in an area where both honey bee and stingless bee colonies are abundant. We counted the number of tomatoes produced in each plot. Two hundred tomatoes from each plot were weighed, their vertical and transversal circumferences were measured, and the seeds were counted. We collected 253 Chrysomelidae, 17 Halictidae, one Paratrigona sp, and one honey bee from the flowers of the tomato plants in the open area. The largest number of fruits (1414 tomatoes), the heaviest and largest tomatoes, and the ones with the most seed were collected from the greenhouse with stingless bees. Fruits cultivated in the greenhouse with honey bees had the same weight and size as those produced in one of the control greenhouses. The stingless bee, M. quadrifasciata, was significantly more efficient than honey bees in pollinating greenhouse tomatoes.

Changes in integument structure during the imaginal molt of the honey bee

The changing pattern of developing cuticle and associated epidermis is described during the imaginal molt in the honey bee. Observations began immediately after the pupal molt, and included histological analyses of the integument during apolysis and the subsequent deposition and differentiation of the adult cuticle. Apolysis coincides with a marked increase in the thickness and reorganization of the epidermal layer, reflecting changes in cell structure. The epidermis remains thickened during the period of cuticle deposition, suggesting intense biosynthetic activity, but turns into a very thin layer during cuticle differentiation, clearly indicating that secretory activity for cuticle formation is terminating. The thoracic cuticle differentiates earlier and becomes thicker than the abdominal. The observed changes in integument structure provide insights that permit an improved physiological characterization for staging pupal and pharate adult development.

Validation of reference genes for gene expression studies in the honey bee, Apis mellifera, by quantitative real-time RT-PCR

For obtaining accurate and reliable gene expression results it is essential that quantitative real-time RT-PCR (qRT-PCR) data are normalized with appropriate reference genes. The current exponential increase in postgenomic studies on the honey bee, Apis mellifera, makes the standardization of qRT-PCR results an important task for ongoing community efforts. For this aim we selected four candidate reference genes (actin, ribosomal protein 49, elongation factor 1-alpha, tbp-association factor) and used three software-based approaches (geNorm, BestKeeper and NormFinder) to evaluate the suitability of these genes as endogenous controls. Their expression was examined during honey bee development, in different tissues, and after juvenile hormone exposure. Furthermore, the importance of choosing an appropriate reference gene was investigated for two developmentally regulated target genes. The results led us to consider all four candidate genes as suitable genes for normalization in A. mellifera. However, each condition evaluated in this study revealed a specific set of genes as the most appropriated ones.

Ecdysteroid-Dependent Expression of the Tweedle and Peroxidase Genes during Adult Cuticle Formation in the Honey Bee, Apis mellifera

Cuticle renewal is a complex biological process that depends on the cross talk between hormone levels and gene expression. This study characterized the expression of two genes encoding cuticle proteins sharing the four conserved amino acid blocks of the Tweedle family, AmelTwdl1 and AmelTwdl2, and a gene encoding a cuticle peroxidase containing the Animal haem peroxidase domain, Ampxd, in the honey bee. Gene sequencing and annotation validated the formerly predicted tweedle genes, and revealed a novel gene, Ampxd, in the honey bee genome. Expression of these genes was studied in the context of the ecdysteroid-coordinated pupal-to-adult molt, and in different tissues. Higher transcript levels were detected in the integument after the ecdysteroid peak that induces apolysis, coinciding with the synthesis and deposition of the adult exoskeleton and its early differentiation. The effect of this hormone was confirmed in vivo by tying a ligature between the thorax and abdomen of early pupae to prevent the abdominal integument from coming in contact with ecdysteroids released from the prothoracic gland. This procedure impaired the natural increase in transcript levels in the abdominal integument. Both tweedle genes were expressed at higher levels in the empty gut than in the thoracic integument and trachea of pharate adults. In contrast, Ampxd transcripts were found in higher levels in the thoracic integument and trachea than in the gut. Together, the data strongly suggest that these three genes play roles in ecdysteroid-dependent exoskeleton construction and differentiation and also point to a possible role for the two tweedle genes in the formation of the cuticle (peritrophic membrane) that internally lines the gut.

Rearing Africanized honey bee (Apis mellifera L.) brood under laboratory conditions

We developed a method for rearing larvae of Africanized bees under laboratory conditions to determine the amount of diet needed during larval development to obtain a worker bee. We started with larvae 18-24 h old, which were transferred to polyethylene cell cups and fed for five days. We found that the amount of diet needed for successful larval development was: 4, 15, 25, 50, and 70 mu L during the first to fifth days, respectively. The survival rate to the adult stage was 88.6% when the larvae received the daily amount of diet divided into two feedings, and 80% when they received only one feeding per day. The adult weight obtained in the laboratory, when the larvae received the daily amount of diet in a single dose, did not differ from those that were developed under field conditions (our control). All adults that we obtained in laboratory appeared to be normal. This technique has the potential to facilitate studies on brood pathogens, resistance mechanisms to diseases and also might be useful to test the impacts of transgenic products on honey bee brood.

Differential gene expression profiling in mucus glands of honey bee (Apis mellifera) drones during sexual maturation

The mating sign that each drone leaves when mating with a queen essentially consists of mucus gland proteins. We employed a Representational Difference Analysis (RDA) methodology to identify genes that are differentially expressed in mucus glands during sexual maturation of drones. The RDA library for mucus glands of newly emerged drones was more complex than that of 8 day-old drones, with matches to 20 predicted genes. Another 26 reads matched to the Apis genome but not to any predicted gene. Since these ESTs were located within ORFs they may represent novel honey bee genes, possibly fast evolving mucus gland proteins. In the RDA library for mucus glands of 8 day-old drones, most reads corresponded to a capsid protein of deformed wing virus, indicating high viral loads in these glands. The expression of two genes encoding venom allergens, acid phosphatase-1 and hyaluronidase, in drone mucus glands argues for their homology with the female venom glands, both associated with the reproductive system.

Effects of stingless bee and honey bee propolis on four species of bacteria

We examined the antibacterial activities of several types of propolis, including Africanized honey bee green propolis and propolis produced by meliponini bees. The antibacterial activity of green propolis against Micrococcus luteus and Staphylococcus aureus was superior to that of Melipona quadrifasciata and Scaptotrigona sp propolis. Only two samples of propolis (green propolis and Scaptotrigona sp propolis) were efficient against Escherichia coli. Melipona quadrifasciata propolis was better than green propolis and Scaptotrigona sp propolis against Pseudomonas aeruginosa. We concluded that these resins have potential for human and veterinary medicine.

An oligonucleotide primer set for PCR amplification of the complete honey bee mitochondrial genome

Mitochondrial DNA markers have been widely used to address population and evolutionary questions in the honey bee Apis mellifera. Most of the polymorphic markers are restricted to few mitochondrial regions. Here we describe a set of 24 oligonucleotides that allow PCR amplification of the entire mitochondrial genome of the honey bee A. mellifera in 12 amplicons. These fragments have important applications for the study of mitochondrial genes in different subspecies of A. mellifera and as heterospecific probes to characterize mitochondrial genomes in other bee species.

Expression analysis of putative vitellogenin and lipophorin receptors in honey bee (Apis mellifera L.) queens and workers

Two members of the low density lipoprotein receptor (LDLR) family were identified as putative orthologs for a vitellogenin receptor (Amvgr) and a lipophorin receptor (Amlpr) in the Apis mellifera genome. Both receptor sequences have the structural motifs characteristic of LDLR family members and show a high degree of similarity with sequences of other insects. RT-PCR analysis of Amvgr and Amlpr expression detected the presence of both transcripts in different tissues of adult female (ovary, fat body, midgut, head and specifically hypopharyngeal gland), as well as in embryos. In the head RNA samples we found two variant forms of AmLpR: a full length one and a shorter one lacking 29 amino acids in the O-linked sugar domain. In ovaries the expression levels of the two honey bee LDLR members showed opposing trends: whereas Amvgr expression was upregulated as the ovaries became activated, Amlpr transcript levels gradually declined. In situ hybridization analysis performed on ovaries detected Amvgr mRNA exclusively in germ line cells and corroborated the qPCR results showing an increase in Amvgr gene expression concomitant with follicle growth. (C) 2008 Elsevier Ltd. All rights reserved.

Identification of a juvenile hormone esterase-like gene in the honey bee, Apis mellifera L. - Expression analysis and functional assays

Tight control over circulating juvenile hormone (JH) levels is of prime importance in an insect`s life cycle. Consequently, enzymes involved in JH metabolism, especially juvenile hormone esterases (JHEs), play major roles during metamorphosis and reproduction. In the highly eusocial Hymenoptera, JH has been co-opted into additional functions, primarily in the development of the queen and worker castes and in age-related behavioral development of workers. Within a set of 21 carboxylesterases predicted in the honey bee genome we identified one gene (Amjhe-like) that contained the main functional motifs of insect JHEs. Its transcript levels during larval development showed a maximum at the switch from feeding to spinning behavior, coinciding with a JH titer minimum. In adult workers, the highest levels were observed in nurse bees, where a low JH titer is required to prevent the switch to foraging. Functional assays showed that Amjhe-like expression is induced by JH-III and suppressed by 20-hydroxyecdysone. RNAi-mediated silencing of Amjhe-like gene function resulted in a six-fold increase in the JH titer in adult worker bees. The temporal profile of Amjhe-like expression in larval and adult workers, the pattern of hormonal regulation and the knockdown phenotype are consistent with the function of this gene as an authentic JHE. (C) 2008 Elsevier Inc. All rights reserved.

The juvenile hormone (JH) epoxide hydrolase gene in the honey bee (Apis mellifera) genome encodes a protein which has negligible participation in JH degradation

Epoxide hydrolases are multifunctional enzymes that are best known in insects for their role in juvenile hormone (JH) degradation. Enzymes involved in JH catabolism can play major roles during metamorphosis and reproduction, such as the JH epoxide hydrolase (JHEH), which degrades JH through hydration of the epoxide moiety to form JH diol, and JH esterase (JHE), which hydrolyzes the methyl ester to produce JH acid. In the honey bee, JH has been co-opted for additional functions, mainly in caste differentiation and in age-related behavioral development of workers, where the activity of both enzymes could be important for JH titer regulation. Similarity searches for jheh candidate genes in the honey bee genome revealed a single Amjheh gene. Sequence analysis, quantification of Amjheh transcript levels and Western blot assays using an AmJHEH-specific antibody generated during this study revealed that the AmJHEH found in the fat body shares features with the microsomal JHEHs from several insect species. Using a partition assay we demonstrated that AmJHEH has a negligible role in JH degradation, which, in the honey bee, is thus performed primarily by JHE. High AmJHEH levels in larvae and adults were related to the ingestion of high loads of lipids, suggesting that AmJHEH has a role in dietary lipid catabolism. (C) 2010 Elsevier Ltd. All rights reserved.

Developmental characterization, function and regulation of a Laccase2 encoding gene in the honey bee, Apis mellifera (Hymenoptera, Apinae)

In insects, exoskeleton (cuticle) formation at each molt cycle includes complex biochemical pathways wherein the laccase enzymes (EC 1.10.3.2) may have a key role. We identified an Amlac2 gene that encodes a laccase2 in the honey bee, Apis mellifera, and investigated its function in exoskeleton differentiation. The Amlac2 gene consists of nine exons resulting in an ORE of 2193 nucleotides. The deduced translation product is a 731 amino acid protein of 81.5 kDa and a pl of 6.05. Amlac2 is highly expressed in the integument of pharate adults, and the expression precedes the onset of cuticle pigmentation and the intensification of sclerotization. In accordance with the temporal sequence of exoskeleton differentiation from anterior to posterior direction, the levels of Amlac2 transcript increase earlier in the thoracic than in the abdominal integument. The gene expression lasts even after the bees emerge from brood cells and begin activities in the nest, but declines after the transition to foraging stage, suggesting that maturation of the exoskeleton is completed at this stage. Post-transcriptional knockdown of Amlac2 gene expression resulted in structural abnormalities in the exoskeleton and drastically affected adult eclosion. By setting a ligature between the thorax and abdomen of early pupae we could delay the increase in hemolymph ecdysteroid levels in the abdomen. This severely impaired the increase in Amlac2 transcript levels and also the differentiation of the abdominal exoskeleton. Taken together, these results indicate that Amlac2 expression is controlled by ecdysteroids and has a critical role in the differentiation of the adult exoskeleton of honey bees. (C) 2010 Elsevier Ltd. All rights reserved.

Pollen substitutes increase honey bee haemolymph protein levels as much as or more than does pollen

Adequate substitutes for pollen are necessary for maintaining healthy bee colonies during periods of pollen dearth, but testing them objectively is both time consuming and expensive. We compared two commercial diets with bee collected pollen and acacia pod flour (used by beekeepers in some parts of Brazil) by measuring their effect on haemolymph protein contents of young bees exclusively fed on these diets, which is a fast and inexpensive assay. The commercial diets included a new, non-soy-based, pollen substitute diet (named Feed-Bee (R)) and a soy-based diet, named Bee-Pro (R). The diets were each given in patty form to groups of 100 Africanized honey bees in hoarding cages, maintained and fed from emergence until six days of age. Sucrose, in the form of sugar syrup, was used as a protein free control. Feed-Bee (R), Bee-Pro (R), pollen and acacia pod flour diets increased protein titers in the haemolymph by factors of 2.65, 2.51, 1.76 and 1.69, respectively, over protein titers in bees fed only sucrose solution. The bees fed Feed-Bee (R) and Bee-Pro (R) had their haemolymph significantly enriched in protein compared to the controls and those fed acacia pod flour and to titers slightly higher than those fed pollen. All four proteinaceous diets were significantly superior to sucrose alone.