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.

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.

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.

Representational Difference Analysis (RDA) reveals differential expression of conserved as well as novel genes during caste-specific development of the honey bee (Apis mellifera L.) ovary

In highly eusocial insects, such as the honey bee, Apis mellifera, the reproductive bias has become embedded in morphological caste differences. These are most expressively denoted in ovary size, with adult queens having large ovaries consisting of 150-200 ovarioles each, while workers typically have only 1-20 ovarioles per ovary. This morphological differentiation is a result of hormonal signals triggered by the diet change in the third larval instar, which eventually generate caste-specific gene expression patterns. To reveal these we produced differential gene expression libraries by Representational Difference Analysis (RDA) for queen and worker ovaries in a developmental stage when cell death is a prominent feature in the ovarioles of workers, whereas all ovarioles are maintained and extend in length in queens. In the queen library, 48% of the gene set represented homologs of known Drosophila genes, whereas in the worker ovary, the largest set (59%) were ESTs evidencing novel genes, not even computationally predicted in the honey bee genome. Differential expression was confirmed by quantitative RT-PCR for a selected gene set, denoting major differences for two queen and two worker library genes. These included two unpredicted genes located in chromosome 11 (Group11.35 and Group11.31, respectively) possibly representing long non-coding RNAs. Being candidates as modulators of ovary development, their expression and functional analysis should be a focal point for future studies. (C) 2011 Elsevier Ltd. All rights reserved.

The insulin signaling pathway in honey bee (Apis mellifera) caste development - differential expression of insulin-like peptides and insulin receptors in queen and worker larvae

The insulin/insulin-like signaling (IIS) pathway is an evolutionarily conserved module in the control of body size and correlated organ growth in metazoans. In the highly eusocial bees, the caste phenotypes differ not only in size and several structural features but also in individual fitness and life history. We investigated the developmental expression profiles of genes encoding the two insulin-like peptides (AmILP-1 and AmILP-2) and the two insulin receptors (AmInR-1 and AmInR-2) predicted in the honey bee genome. Quantitative PCR analysis for queen and worker larvae in critical stages of caste development showed that AmILP-2 is the predominantly transcribed ILP in both castes, with higher expression in workers than in queens. Expression of both InR genes sharply declined in fourth instar queen larvae, but showed little modulation in workers. On first sight, these findings are non-intuitive, considering the higher growth rates of queens, but they can be interpreted as possibly antagonistic crosstalk between the IIS module and juvenile hormone. Analyzing AmInR-1 and AmInR-2 expression in ovaries of queen and worker larvae revealed low transcript levels in queens and a sharp drop in AmInR-2 expression in fifth instar worker larvae, indicating relative independence in tissue-specific versus overall IIS pathway activity. (C) 2008 Elsevier Ltd. All rights reserved.

Cytoplasmic and nuclear localization of cadherin in honey bee (Apis mellifera L.) gonads

Cadherins are crucial molecules mediating cell-cell interactions between somatic and germline cells in insect and mammalian male and female gonads. We analysed the presence and localization of cadherins in ovaries of honeybee queens and in testes of drones. Transcripts representing two classical cadherins, E-cadherin (shotgun) and N-cadherin, as well as three protocadherins (Starry night, Fat and Fat-like) were detected in gonads of both sexes. Pan-cadherin antibodies, which most probably detect a honeybee N-cadherin, were used in immunolocalization analyses. In the germarium of ovarioles, cadherin-IR (cadherin immunoreactivity) was evidenced as homogeneously distributed in the cytoplasm and as nuclear foci, in both germline and somatic cells. It was also detected in polyfusomes and ring canals. In testiolar tubules, cadherin-IR showed a cytoplasmic and nuclear distributon alike in ovaries. The unexpected nuclear localization and cytoplasmic distribution in ovaries and testes were corroborated by immunogold electron microscopy, which revealed cadherin aggregates associated with electron-dense nuclear structures. With respect to cadherin localization, the honeybee differs from Drosophila, the model for gametogenesis in insects, raising the question as to how differences among solitary and social species may be built into and generated from the general architecture of polytrophic meroistic ovaries. It also indicates the possibility of divergent roles for cadherin in the functional architecture of insect gonads, in general, especially in taxa with high reproductive output.

Differential expression of hypoxia pathway genes in honey bee (Apis mellifera L.) caste development

Diphenism in social bees is essentially contingent on nutrient-induced cellular and systemic physiological responses resulting in divergent gene expression patterns. Analyses of juvenile hormone (JH) titers and functional genomics assays of the insulin-insulin-like signaling (IIS) pathway and its associated branch, target-of-rapamycin (TOR), revealed systemic responses underlying honey bee (Apis mellifera) caste development. Nevertheless, little attention has been paid to cellular metabolic responses. Following up earlier investigations showing major caste differences in oxidative metabolism and mitochondrial physiology, we herein identified honey bee homologs of hypoxia signaling factors, HIF alpha/Sima, HIF beta/Tango and PHD/Fatiga and we investigated their transcript levels throughout critical stages of larval development. Amsima, Amtango and Amfatiga showed correlated transcriptional activity, with two peaks of occurring in both queens and workers, the first one shortly after the last larval molt and the second during the cocoon-spinning phase. Transcript levels for the three genes were consistently higher in workers. As there is no evidence for major microenvironmental differences in oxygen levels within the brood nest area, this appears to be an inherent caste character. Quantitative PCR analyses on worker brain, ovary, and leg imaginal discs showed that these tissues differ in transcript levels. Being a highly conserved pathway and linked to IIS/TOR, the hypoxia gene expression pattern seen in honey bee larvae denotes that the hypoxia pathway has undergone a transformation, at least during larval development, from a response to environmental oxygen concentrations to an endogenous regulatory factor in the diphenic development of honey bee larvae. (C) 2010 Elsevier Ltd. All rights reserved.

Mars is close to venus - Female reproductive proteins are expressed in the fat body and reproductive tract of honey bee (Apis mellifera L.) drones

Vitellogenin (Vg) and lipophorin (Lp) are lipoproteins which play important roles in female reproductive physiology of insects. Both are actively taken up by growing oocytes and especially Vg and its receptor are considered as female-specifically expressed. The finding that the fat body of in honey bee (Apis mellifera) drones synthesizes Vg and is present in hemolymph has long been viewed as a curiosity. The recent paradigm change concerning the role played by Vg in honey bee life history, especially social division of labor, has now led us to investigate whether a physiological constellation similar to that seen in female reproduction may also be represented in the male sex. By means of Western blot analysis we could show that both Vg and Lp are present in the reproductive tract of adult drones, including the accessory (mucus) glands, but apparently are not secreted. Furthermore, we analyzed the transcript levels of the genes encoding these proteins (vg and lp), as well as their putative receptors (Amvgr and Amlpr) in fat body and accessory glands. Whereas lp, vg and Amlpr transcript levels decreased with age in both tissues. Amvgr mRNA levels increased with age in fat body. To our knowledge this is the first report that vitellogenin and its receptor are co-expressed in the reproductive system of a male insect. We interpret these findings as a cross-sexual transfer of a social physiological trait, associated with the rewiring of the juvenile hormone/vitellogenin circuitry that occurred in the female sex of honey bees. (C) 2010 Elsevier Ltd. All rights reserved.

Africanized honey bee (Apis mellifera) venom profiling: Seasonal variation of melittin and phospholipase A(2) levels

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)