The nuclear and mitochondrial genomes of Frieseomelitta varia - a highly eusocial stingless bee (Meliponini) with a permanently sterile worker caste.

BACKGROUND: Most of our understanding on the social behavior and genomics of bees and other social insects is centered on the Western honey bee, Apis mellifera. The genus Apis, however, is a highly derived branch comprising less than a dozen species, four of which genomically characterized. In contrast, for the equally highly eusocial, yet taxonomically and biologically more diverse Meliponini, a full genome sequence was so far available for a single Melipona species only. We present here the genome sequence of Frieseomelitta varia, a stingless bee that has, as a peculiarity, a completely sterile worker caste. RESULTS: The assembly of 243,974,526 high quality Illumina reads resulted in a predicted assembled genome size of 275 Mb composed of 2173 scaffolds. A BUSCO analysis for the 10,526 predicted genes showed that these represent 96.6% of the expected hymenopteran orthologs. We also predicted 169,371 repetitive genomic components, 2083 putative transposable elements, and 1946 genes for non-coding RNAs, largely long non-coding RNAs. The mitochondrial genome comprises 15,144 bp, encoding 13 proteins, 22 tRNAs and 2 rRNAs. We observed considerable rearrangement in the mitochondrial gene order compared to other bees. For an in-depth analysis of genes related to social biology, we manually checked the annotations for 533 automatically predicted gene models, including 127 genes related to reproductive processes, 104 to development, and 174 immunity-related genes. We also performed specific searches for genes containing transcription factor domains and genes related to neurogenesis and chemosensory communication. CONCLUSIONS: The total genome size for F. varia is similar to the sequenced genomes of other bees. Using specific prediction methods, we identified a large number of repetitive genome components and long non-coding RNAs, which could provide the molecular basis for gene regulatory plasticity, including worker reproduction. The remarkable reshuffling in gene order in the mitochondrial genome suggests that stingless bees may be a hotspot for mtDNA evolution. Hence, while being just the second stingless bee genome sequenced, we expect that subsequent targeting of a selected set of species from this diverse clade of highly eusocial bees will reveal relevant evolutionary signals and trends related to eusociality in these important pollinators.

The gene vitellogenin affects microRNA regulation in honey bee (Apis mellifera) fat body and brain.

In honey bees, vitellogenin (Vg) is hypothesized to be a major factor affecting hormone signaling, food-related behavior, immunity, stress resistance and lifespan. MicroRNAs, which play important roles in post-transcriptional gene regulation, likewise affect many biological processes. The actions of microRNAs and Vg are known to intersect in the context of reproduction; however, the role of these associations on social behavior is unknown. The phenotypic effects of Vg knockdown are best established and studied in the forager stage of workers. Thus, we exploited the well-established RNA interference (RNAi) protocol for Vg knockdown to investigate its downstream effects on microRNA population in honey bee foragers' brain and fat body tissue. To identify microRNAs that are differentially expressed between tissues in control and knockdown foragers, we used µParaflo microfluidic oligonucleotide microRNA microarrays. Our results showed that 76 and 74 microRNAs were expressed in the brain of control and knockdown foragers whereas 66 and 69 microRNAs were expressed in the fat body of control and knockdown foragers, respectively. Target prediction identified potential seed matches for a differentially expressed subset of microRNAs affected by Vg knockdown. These candidate genes are involved in a broad range of biological processes including insulin signaling, juvenile hormone (JH) and ecdysteroid signaling previously shown to affect foraging behavior. Thus, here we demonstrate a causal link between the Vg knockdown forager phenotype and variation in the abundance of microRNAs in different tissues, with possible consequences for the regulation of foraging behavior.

Worker bees (Apis mellifera) deprived of pollen in the first week of adulthood exhibit signs of premature aging.

Pollinator populations, including bees, are in rapid decline in many parts of the world, raising concerns over the future of ecosystems and food production. Among the factors involved in these declines, poor nutrition deserves attention. The diet consumed by adult worker honeybees (Apis mellifera) is crucial for their behavioral maturation, i.e., the progressive division of labor they perform, such as nurse bees initially and later in life as foragers. Poor pollen nutrition is known to reduce the workers' lifespan, but the underlying physiological and genetic mechanisms are not fully understood. Here we investigate how the lack of pollen in the diet of workers during their first week of adult life can affect age-related phenotypes. During the first seven days of adult life, newly emerged workers were fed either a pollen-deprived (PD) diet mimicking that of an older bee, or a control pollen-rich (PR) diet, as typically consumed by young bees. The PD-fed bees showed alterations in their fat body transcriptome, such as a switch from a protein-lipid based metabolism to a carbohydrate-based metabolism, and a reduced expression of genes involved with immune response. The absence of pollen in the diet also led to an accumulation of oxidative stress markers in fat body tissue and alterations in the cuticular hydrocarbon profiles, which became similar to those of chronologically older bees. Together, our data indicate that the absence of pollen during first week of adulthood triggers the premature onset of an aging-related worker phenotype.

Immunity and physiological changes in adult honey bees (Apis mellifera) infected with Nosema ceranae: The natural colony environment.

Nosema ceranae is a microsporidium that infects Apis mellifera, causing diverse physiological and behavioral alterations. Given the existence of individual and social mechanisms to reduce infection and fungal spread in the colony, bees may respond differently to infection depending on their rearing conditions. In this study, we investigated the effect of N. ceranae in honey bee foragers naturally infected with different fungal loads in a tropical region. In addition, we explored the effects of N. ceranae artificially infected young bees placed in a healthy colony under field conditions. Honey bees naturally infected with higher loads of N. ceranae showed downregulation of genes from Toll and IMD immune pathways and antimicrobial peptide (AMP) genes, but hemolymph total protein amount and Vitellogenin (Vg) titers were not affected. Artificially infected bees spread N. ceranae to the controls in the colony, but fungal loads were generally lower than those observed in cages, probably because of social immunity. Although no significant changes in mRNA levels of AMP-encoding were observed, N. ceranae artificially infected bees showed downregulation of miR-989 (an immune-related microRNA), lower vitellogenin gene expression, and decreased hemolymph Vg titers. Our results demonstrate for the first time that natural infection by N. ceranae suppresses the immune system of honey bee foragers in the field. This parasite is detrimental to the immune system of young and old bees, and disease spread, mitigation and containment will depend on the colony environment.

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.

Trade-off between immune stimulation and expression of storage protein genes.

Proteins stored in insect hemolymph may serve as a source of amino acids and energy for metabolism and development. The expression of the main storage proteins was assessed in bacterial-challenged honey bees using real-time (RT)-PCR and Western blot. After ensuring that the immune system had been activated by measuring the ensuing expression of the innate immune response genes, defensin-1 (def-1) and prophenoloxidase (proPO), we verified the expression of four genes encoding storage proteins. The levels of vitellogenin (vg) mRNA and of the respective protein were significantly lowered in bees injected with bacteria or water only (injury). An equivalent response was observed in orally-infected bees. The levels of apolipophorin II/I (apoLp-II/I) and hexamerin (hex 70a) mRNAs did not significantly change, but levels of Hex 70a protein subunit showed a substantial decay after bacterial challenge or injury. Infection also caused a strong reduction in the levels of apoLp-III transcripts. Our findings are consistent with a down-regulation of the expression and accumulation of storage proteins as a consequence of activation of the immune system, suggesting that this phenomenon represents a strategy to redirect resources to combat injury or infection.

Evaluation of reference genes for gene expression analysis by real-time quantitative PCR (qPCR) in three stingless bee species (Hymenoptera: Apidae: Meliponini).

Stingless bees are generalist pollinators distributed through the pantropical region. There is growing evidence that their wild populations are experiencing substantial decline in response to habitat degradation and pesticides. Policies for conservation of endangered species will benefit from studies focusing on genetic and molecular aspects of their development and behavior. The most common method for looking at gene expression is real-time quantitative polymerase chain reaction preceded by reverse transcription (RT-qPCR) of the mRNA of interest. This method requires the identification of reliable reference genes to correctly estimate fluctuations in transcript levels. To contribute to molecular studies on stingless bees, we used Frieseomelitta varia, Melipona quadrifasciata, and Scaptotrigona bipunctata species to test the expression stability of eight reference genes (act, ef1-α, gapdh, rpl32, rps5, rps18, tbp, and tbp-af) in RT-qPCR procedures in five physiological and experimental conditions (development, sex, tissues, bacteria injection, and pesticide exposure). In general, the rpl32, rps5 and rps18 ribosomal protein genes and tpb-af gene showed the highest stability, thus being identified as suitable reference genes for the three stingless bee species and defined conditions. Our results also emphasized the need to evaluate the stability of candidate genes for any designed experimental condition and stingless bee species.

Exploring integument transcriptomes, cuticle ultrastructure, and cuticular hydrocarbons profiles in eusocial and solitary bee species displaying heterochronic adult cuticle maturation.

Differences in the timing of exoskeleton melanization and sclerotization are evident when comparing eusocial and solitary bees. This cuticular maturation heterochrony may be associated with life style, considering that eusocial bees remain protected inside the nest for many days after emergence, while the solitary bees immediately start outside activities. To address this issue, we characterized gene expression using large-scale RNA sequencing (RNA-seq), and quantified cuticular hydrocarbon (CHC) through gas chromatography-mass spectrometry in comparative studies of the integument (cuticle plus its underlying epidermis) of two eusocial and a solitary bee species. In addition, we used transmission electron microscopy (TEM) for studying the developing cuticle of these and other three bee species also differing in life style. We found 13,200, 55,209 and 30,161 transcript types in the integument of the eusocial Apis mellifera and Frieseomelitta varia, and the solitary Centris analis, respectively. In general, structural cuticle proteins and chitin-related genes were upregulated in pharate-adults and newly-emerged bees whereas transcripts for odorant binding proteins, cytochrome P450 and antioxidant proteins were overrepresented in foragers. Consistent with our hypothesis, a distance correlation analysis based on the differentially expressed genes suggested delayed cuticle maturation in A. mellifera in comparison to the solitary bee. However, this was not confirmed in the comparison with F. varia. The expression profiles of 27 of 119 genes displaying functional attributes related to cuticle formation/differentiation were positively correlated between A. mellifera and F. varia, and negatively or non-correlated with C. analis, suggesting roles in cuticular maturation heterochrony. However, we also found transcript profiles positively correlated between each one of the eusocial species and C. analis. Gene co-expression networks greatly differed between the bee species, but we identified common gene interactions exclusively between the eusocial species. Except for F. varia, the TEM analysis is consistent with cuticle development timing adapted to the social or solitary life style. In support to our hypothesis, the absolute quantities of n-alkanes and unsaturated CHCs were significantly higher in foragers than in the earlier developmental phases of the eusocial bees, but did not discriminate newly-emerged from foragers in C. analis. By highlighting differences in integument gene expression, cuticle ultrastructure, and CHC profiles between eusocial and solitary bees, our data provided insights into the process of heterochronic cuticle maturation associated to the way of life.

Downregulation of ultraspiracle gene expression delays pupal development in honeybees.

Ecdysteroids regulate many aspects of insect physiology after binding to a heterodimer composed of the nuclear hormone receptor proteins ecdysone receptor (EcR) and ultraspiracle (Usp). Several lines of evidence have suggested that the latter also plays important roles in mediating the action of juvenile hormone (JH) and, thus, integrates signaling by the two morphogenetic hormones. By using an RNAi approach, we show here that Usp participates in the mechanism that regulates the progression of pupal development in Apis mellifera, as indicated by the observed pupal developmental delay in usp knocked-down bees. Knock-down experiments also suggest that the expression of regulatory genes such as ftz transcription factor 1 (ftz-f1) and juvenile hormone esterase (jhe) depend on Usp. Vitellogenin (vg), the gene coding the main yolk protein in honeybees, does not seem to be under Usp regulation, thus suggesting that the previously observed induction of vg expression by JH during the last stages of pupal development is mediated by yet unknown transcription factor complexes.

Molecular determinants of caste differentiation in the highly eusocial honeybee Apis mellifera.

BACKGROUND: In honeybees, differential feeding of female larvae promotes the occurrence of two different phenotypes, a queen and a worker, from identical genotypes, through incremental alterations, which affect general growth, and character state alterations that result in the presence or absence of specific structures. Although previous studies revealed a link between incremental alterations and differential expression of physiometabolic genes, the molecular changes accompanying character state alterations remain unknown. RESULTS: By using cDNA microarray analyses of >6,000 Apis mellifera ESTs, we found 240 differentially expressed genes (DEGs) between developing queens and workers. Many genes recorded as up-regulated in prospective workers appear to be unique to A. mellifera, suggesting that the workers' developmental pathway involves the participation of novel genes. Workers up-regulate more developmental genes than queens, whereas queens up-regulate a greater proportion of physiometabolic genes, including genes coding for metabolic enzymes and genes whose products are known to regulate the rate of mass-transforming processes and the general growth of the organism (e.g., tor). Many DEGs are likely to be involved in processes favoring the development of caste-biased structures, like brain, legs and ovaries, as well as genes that code for cytoskeleton constituents. Treatment of developing worker larvae with juvenile hormone (JH) revealed 52 JH responsive genes, specifically during the critical period of caste development. Using Gibbs sampling and Expectation Maximization algorithms, we discovered eight overrepresented cis-elements from four gene groups. Graph theory and complex networks concepts were adopted to attain powerful graphical representations of the interrelation between cis-elements and genes and objectively quantify the degree of relationship between these entities. CONCLUSION: We suggest that clusters of functionally related DEGs are co-regulated during caste development in honeybees. This network of interactions is activated by nutrition-driven stimuli in early larval stages. Our data are consistent with the hypothesis that JH is a key component of the developmental determination of queen-like characters. Finally, we propose a conceptual model of caste differentiation in A. mellifera based on gene-regulatory networks.

A cuticle protein gene in the honeybee: expression during development and in relation to the ecdysteroid titer.

A cDNA encoding a cuticle protein containing the R&R Consensus was characterized in the honeybee integument. AmelCPR14 developmental expression is distinguished by an on-off-on pattern, the transition from a low to a high level of transcripts occurring as the ecdysteroid titer is declining after the peak that triggers the onset of pharate (pupal and adult) development. The transcript is abundant during cuticle tanning and sclerotization, and persists even in the adult integument, suggesting that the corresponding protein is required for differentiation and maintenance of the adult cuticle. Such developmental pattern suggested that AmelCPR14 gene might be regulated by the titer of ecdysteroids. We confirmed this hypothesis using different experimental strategies. By tying a ligature in early pupae to prevent exposure of abdominal integument to a high ecdysteroid titer, we delayed the accumulation of AmelCPR14 transcripts in the abdominal integument. This is consistent with ecdysteroid priming being required in pupae for the increase in AmelCPR14 expression in pharate adults. By injecting 20-hydroxyecdysone (20E) in early pupae we demonstrated that hormone titer decay after the peak is critical for AmelCPR14 expression induction. Exposure of pupal integument in vitro to a 20E concentration mimicking the pupal ecdysteroid peak repressed AmelCPR14 expression, which was recovered by hormone removal. Taken together, these data are consistent with an ecdysteroid pulse (increase in hormone titer followed by its decline) being critical for a high AmelCPR14 gene expression in pharate adults.

MicroRNA-34 directly targets pair-rule genes and cytoskeleton component in the honey bee.

MicroRNAs (miRNAs) are key regulators of developmental processes, such as cell fate determination and differentiation. Previous studies showed Dicer knockdown in honeybee embryos disrupt the processing of functional mature miRNAs and impairs embryo patterning. Here we investigated the expression profiles of miRNAs in honeybee embryogenesis and the role of the highly conserved miR-34-5p in the regulation of genes involved in insect segmentation. A total of 221 miRNAs were expressed in honey bee embryogenesis among which 97 mature miRNA sequences have not been observed before. Interestingly, we observed a switch in dominance between the 5-prime and 3-prime arm of some miRNAs in different embryonic stages; however, most miRNAs present one dominant arm across all stages of embryogenesis. Our genome-wide analysis of putative miRNA-target networks and functional pathways indicates miR-34-5p is one of the most conserved and connected miRNAs associated with the regulation of genes involved in embryonic patterning and development. In addition, we experimentally validated that miR-34-5p directly interacts to regulatory elements in the 3'-untranslated regions of pair-rule (even-skipped, hairy, fushi-tarazu transcription factor 1) and cytoskeleton (actin5C) genes. Our study suggests that miR-34-5p may regulate the expression of pair-rule and cytoskeleton genes during early development and control insect segmentation.

Vitellogenin regulates hormonal dynamics in the worker caste of a eusocial insect.

Functionally sterile honey bee workers synthesize the yolk protein vitellogenin while performing nest tasks. The subsequent shift to foraging is linked to a reduced vitellogenin and an increased juvenile hormone (JH) titer. JH is a principal controller of vitellogenin expression and behavioral development. Yet, we show here that silencing of vitellogenin expression causes a significant increase in JH titer and its putative receptor. Mathematically, the increase corresponds to a dynamic dose-response. This role of vitellogenin in the tuning of the endocrine system is uncommon and may elucidate how an ancestral pathway of fertility regulation has been remodeled into a novel circuit controlling social behavior.

Molecular cloning and expression of a hexamerin cDNA from the honey bee, Apis mellifera.

A cDNA encoding a hexamerin subunit of the Africanized honey bee (Apis mellifera) was isolated and completely sequenced. In the deduced translation product we identified the N-terminal sequence typical of the honey bee HEX 70b hexamerin. The genomic sequence consists of seven exons flanked by GT/AT exon/intron splicing sites, which encode a 683 amino acid polypeptide with an estimated molecular mass of 79.5 kDa, and pI value of 6.72. Semi-quantitative RT-PCR revealed high levels of Hex 70b message in larval stages, followed by an abrupt decrease during prepupal-pupal transition. This coincides with decaying titers of juvenile hormone (JH) and ecdysteroids that is the signal for the metamorphic molt. To verify whether the high Hex 70b expression is dependent on high hormone levels, we treated 5th instar larvae with JH or 20-hydroxyecdysone (20E). In treated larvae, Hex 70b expression was maintained at high levels for a prolonged period of time than in the respective controls, thus indicating a positive hormone regulation at the transcriptional level. Experiments designed to verify the influence of the diet on Hex 70b expression showed similar transcript amounts in adult workers fed on a protein-enriched diet or fed exclusively on sugar. However, sugar-fed workers responded to the lack of dietary proteins by diminishing significantly the amount of HEX 70b subunits in hemolymph. Apparently, they use HEX 70b to compensate the lack of dietary proteins.

Ventral nerve cord remodeling in a stingless bee (Melipona quadrifasciata anthidioides, Hymenoptera, Apidae) depends on ecdysteroid fluctuation and programmed cell death.

The reorganization of the ventral nerve cord (VNC) during metamorphosis of M. quadrifasciata was observed to be characterized by shortening of connectives and subsequent fusion of the 2nd and 3rd thoracic and the 1st abdominal ganglia. Also, the 5th to 7th abdominal ganglia came into very close contact. These changes were accompanied by increasing levels of endogenous ecdysteroids, as determined by a radioimmunoassay. Incubation of VNC in the presence of 5 microg 20-hydroxyecdysone, caused significant shortening of connectives in the thoracic region, but not in the abdomen, evidencing a segment-specific response to this hormone. Cell death in the ventral ganglia was revealed by transmission electron microscopy and TUNEL-reaction. Detection of labeled cells in the region where contiguous ganglia come into close contact suggests that programmed cell death is involved in ganglionic fusion.

The use of Open Reading frame ESTs (ORESTES) for analysis of the honey bee transcriptome.

BACKGROUND: The ongoing efforts to sequence the honey bee genome require additional initiatives to define its transcriptome. Towards this end, we employed the Open Reading frame ESTs (ORESTES) strategy to generate profiles for the life cycle of Apis mellifera workers. RESULTS: Of the 5,021 ORESTES, 35.2% matched with previously deposited Apis ESTs. The analysis of the remaining sequences defined a set of putative orthologs whose majority had their best-match hits with Anopheles and Drosophila genes. CAP3 assembly of the Apis ORESTES with the already existing 15,500 Apis ESTs generated 3,408 contigs. BLASTX comparison of these contigs with protein sets of organisms representing distinct phylogenetic clades revealed a total of 1,629 contigs that Apis mellifera shares with different taxa. Most (41%) represent genes that are in common to all taxa, another 21% are shared between metazoans (Bilateria), and 16% are shared only within the Insecta clade. A set of 23 putative genes presented a best match with human genes, many of which encode factors related to cell signaling/signal transduction. 1,779 contigs (52%) did not match any known sequence. Applying a correction factor deduced from a parallel analysis performed with Drosophila melanogaster ORESTES, we estimate that approximately half of these no-match ESTs contigs (22%) should represent Apis-specific genes. CONCLUSIONS: The versatile and cost-efficient ORESTES approach produced minilibraries for honey bee life cycle stages. Such information on central gene regions contributes to genome annotation and also lends itself to cross-transcriptome comparisons to reveal evolutionary trends in insect genomes.

Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNA.

BACKGROUND: The ability to manipulate the genetic networks underlying the physiological and behavioural repertoires of the adult honeybee worker (Apis mellifera) is likely to deepen our understanding of issues such as learning and memory generation, ageing, and the regulatory anatomy of social systems in proximate as well as evolutionary terms. Here we assess two methods for probing gene function by RNA interference (RNAi) in adult honeybees. RESULTS: The vitellogenin gene was chosen as target because its expression is unlikely to have a phenotypic effect until the adult stage in bees. This allowed us to introduce dsRNA in preblastoderm eggs without affecting gene function during development. Of workers reared from eggs injected with dsRNA derived from a 504 bp stretch of the vitellogenin coding sequence, 15% had strongly reduced levels of vitellogenin mRNA. When dsRNA was introduced by intra-abdominal injection in newly emerged bees, almost all individuals (96%) showed the mutant phenotype. An RNA-fragment with an apparent size similar to the template dsRNA was still present in this group after 15 days. CONCLUSION: Injection of dsRNA in eggs at the preblastoderm stage seems to allow disruption of gene function in all developmental stages. To dissect gene function in the adult stage, the intra-abdominal injection technique seems superior to egg injection as it gives a much higher penetrance, it is much simpler, and it makes it possible to address genes that are also expressed in the embryonic, larval or pupal stages.

Hormonal control of the yolk precursor vitellogenin regulates immune function and longevity in honeybees.

A striking example of plasticity in life span is seen in social insects such as ants and bees, where different castes may display distinct ageing patterns. In particular, the honeybee offers an intriguing illustration of environmental control on ageing rate. Honeybee workers display a temporal division of labour where young bees (or 'hive bees') perform tasks within the brood nest, and older bees forage for nectar, pollen propolis and water. When bees switch from the hive bee to the forager stage, their cellular defence machinery is down-regulated by a dramatic reduction in the number of functioning haemocytes (immunocytes). This study documents that the yolk precursor vitellogenin is likely to be involved in a regulatory pathway that controls the observed decline in somatic maintenance function of honeybee foragers. An association between the glyco-lipoprotein vitellogenin and immune function has not previously been reported for any organism. Honeybee workers are functionally sterile, and via the expression of juvenile hormone, a key gonotrophic hormone in adult insects, their vitellogenin levels are influenced by social interactions with other bees. Our results therefore suggest that in terms of maintenance of the cellular immune system, senescence of the honeybee worker is under social control.

Phenoloxidase activity in Apis mellifera honey bee pupae, and ecdysteroid-dependent expression of the prophenoloxidase mRNA.

Phenoloxidase (monophenol, l-dopa: oxygen oxidoreductase, EC 1.14.18.1) is a multicopper oxidase, which plays an important role in melanin synthesis, necessary for defense against intruding microorganisms and parasites, wound healing and cuticle pigmentation. A phenoloxidase from the hemolymph of honey bee pupae exhibited an apparent molecular mass of 70 kDa, as estimated by gel filtration and SDS-PAGE. Optimal pH and temperature were 6.5 and 20 degrees C, respectively. Activity was fully stable for 30 min at 50 degrees C. Like phenoloxidases from the hemolymph of other insects, the honey bee enzyme was activated by trypsin and inhibited by protease inhibitors and phenylthiourea. Only high concentrations of sodium azide effectively inhibited the detected activity. A low concentration (5 microM) of Ca2+, Mg2+, and Mn2+ had a stimulatory effect on the activity. Single Michaelis-Menten curves were observed for l-dopa and dopamine oxidation, but the affinity of the enzyme for dopamine was greater than for L-dopa. Semiquantitative RT-PCR and Southern blot analysis using a 359 bp labeled probe, and quantification of the prophenoloxidase mRNA levels by real-time PCR showed increased amounts of transcripts in hemocytes and integument from young pupae injected with 20-hydroxyecdysone.

Neuroligin-associated microRNA-932 targets actin and regulates memory in the honeybee.

Increasing evidence suggests small non-coding RNAs (ncRNAs) such as microRNAs (miRNAs) control levels of mRNA expression during experience-related remodelling of the brain. Here we use an associative olfactory learning paradigm in the honeybee Apis mellifera to examine gene expression changes in the brain during memory formation. Brain transcriptome analysis reveals a general downregulation of protein-coding genes, including asparagine synthetase and actin, and upregulation of ncRNAs. miRNA-mRNA network predictions together with PCR validation suggest miRNAs including miR-210 and miR-932 target the downregulated protein-coding genes. Feeding cholesterol-conjugated antisense RNA to bees results in the inhibition of miR-210 and of miR-932. Loss of miR-932 impairs long-term memory formation, but not memory acquisition. Functional analyses show that miR-932 interacts with Act5C, providing evidence for direct regulation of actin expression by an miRNA. An activity-dependent increase in miR-932 expression may therefore control actin-related plasticity mechanisms and affect memory formation in the brain.