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Made available in DSpace on 2022-04-29T08:35:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Background: Much of the complex anatomy of a holometabolous insect is built from disc-shaped epithelial structures found inside the larva, i.e., the imaginal discs, which undergo a rapid differentiation during metamorphosis. Imaginal discs-derived structures, like wings, are built through the action of genes under precise regulation. Results: We analyzed 30 honeybee transcriptomes in the search for the gene expression needed for wings and thoracic dorsum construction from the larval wing discs primordia. Analyses were carried out before, during, and after the metamorphic molt and using worker and queen castes. Our RNA-seq libraries revealed 13,202 genes, representing 86.2% of the honeybee annotated genes. Gene Ontology analysis revealed functional terms that were caste-specific or shared by workers and queens. Genes expressed in wing discs and descendant structures showed differential expression profiles dynamics in premetamorphic, metamorphic and postmetamorphic developmental phases, and also between castes. At the metamorphic molt, when ecdysteroids peak, the wing buds of workers showed maximal gene upregulation comparatively to queens, thus underscoring differences in gene expression between castes at the height of the larval-pupal transition. Analysis of small RNA libraries of wing buds allowed us to build miRNA-mRNA interaction networks to predict the regulation of genes expressed during wing discs development. Conclusion: Together, these data reveal gene expression dynamics leading to wings and thoracic dorsum formation from the wing discs, besides highlighting caste-specific differences during wing discs metamorphosis. Departamento de Genética Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo, Av. Bandeirantes 3900 Departamento de Tecnologia Faculdade de Ciências Agrárias e Veterinárias Universidade Estadual Paulista Júlio de Mesquita Filho Instituto Carlos Chagas FIOCRUZ Departamento de Biologia Faculdade de Filosofia Ciências e Letras de Ribeirão Preto Universidade de São Paulo, Av. Bandeirantes 3900 Departamento de Tecnologia Faculdade de Ciências Agrárias e Veterinárias Universidade Estadual Paulista Júlio de Mesquita Filho

Epitranscriptomics is an emerging field of investigation dedicated to the study of post-transcriptional RNA modifications. RNA methylations regulate RNA metabolism and processing, including changes in response to environmental cues. Although RNA modifications are conserved from bacteria to eukaryotes, there is little evidence of a epitranscriptomic pathway in insects. Here we identified genes related to RNA m6 A (N6-methyladenine) and m5 C (5-methylcytosine) methylation machinery in seven bee genomes (Apis mellifera, Melipona quadrifasciata, Frieseomelitta varia, Eufriesea mexicana, Bombus terrestris, Megachile rotundata and Dufourea novaeangliae). In A. mellifera, we validated the expression of methyltransferase genes and found that the global levels of m6 A and m5 C measured in the fat body and brain of adult workers differ significantly. Also, m6 A levels were differed significantly mainly between the fourth larval instar of queens and workers. Moreover, we found a conserved m5 C site in the honeybee 28S rRNA. Taken together, we confirm the existence of epitranscriptomic machinery acting in bees and open avenues for future investigations on RNA epigenetics in a wide spectrum of hymenopteran species. This article is protected by copyright. All rights reserved.

During the honeybee larval stage, queens develop larger brains than workers, with morphological differentiation appearing at the fourth larval phase (L4), just after a boost in nutritional difference both prospective females experience. The molecular promoters of this caste-specific brain development are already ongoing in previous larval phases. Transcriptomic analyses revealed a set of differentially expressed genes in the L3 brains of queens and workers, which represents the early molecular response to differential feeding females receive during larval development. Three genes of this set, hex70b, hex70c and hex110, are more highly transcribed in the brain of workers than in queens. The microRNAs miR-34, miR-210 and miR-317 are in higher levels in the queens' brain at the same phase of larval development. Here, we tested the hypothesis that the brain of workers expresses higher levels of hexamerins than that of queens during key phases of larval development and that this differential hexamerin genes expression is further enhanced by the repressing activity of miR-34, miR-210 and miR-317. Our transcriptional analyses showed that hex70b, hex70c and hex110 genes are differentially expressed in the brain of L3 and L4 larval phases of honeybee queens and workers. In silico reconstructed miRNA-mRNA interaction networks were validated using luciferase assays, which showed miR-34 and miR-210 negatively regulate hex70b and hex110 genes by directly and redundantly binding their 3'UTR (untranslated region) sequences. Taken together, our results suggest that miR-34 and miR-210 act together promoting differential brain development in honeybee castes by downregulating the expression of the putative antineurogenic hexamerin genes hex70b and hex110.

International audience; AbstractSex determination is one of the major developmental events in higher metazoans, where complex molecular mechanisms define two physiologically and behaviorally distinct organisms still genetically compatible. In honey bees, the sex determination cascade is initiated by the allelic composition of complementary sex determiner (csd) gene: males develop from hemi or homozygous embryos, whereas females develop from heterozygous embryos. In females, different alleles of csd lead to the formation of female-specific variants of feminizer (fem) and doublesex (dsx). In males, male-specific variants of fem and dsx are formed by default. In this paper, we investigated the genes of sex determination in the stingless bees Melipona quadrifasciata, Scaptotrigona postica, and Frieseomelitta varia. Our results revealed that the architecture of fem and dsx transcripts is highly conserved among the three stingless bee species, and also with honey bees.

Background Meiotic recombination is a fundamental genetic process that shuffles allele combinations and promotes accurate segregation of chromosomes. Analyses of the ubiquitous variation of recombination rates within and across species suggest that recombination is evolving adaptively. All studied insects with advanced eusociality have shown exceptionally high recombination rates, which may represent a prominent case of adaptive evolution of recombination. However, our understanding of the relationship between social evolution and recombination rates is incomplete, partly due to lacking empirical data. Here, we present a linkage map of the monandrous, advanced eusocial Brazilian stingless bee, Frieseomelitta varia, providing the first recombination analysis in the diverse Meliponini (Hymenoptera, Apidae). Results Our linkage map includes 1417 markers in 19 linkage groups. This map spans approximately 2580 centimorgans, and comparisons to the physical genome assembly indicate that it covers more than 75 % of the 275 Megabasepairs (Mbp) F. varia genome. Thus, our study results in a genome-wide recombination rate estimate of 9.3–12.5 centimorgan per Mbp. This value is higher than estimates from nonsocial insects and comparable to other highly social species, although it does not support our prediction that monandry and strong queen-worker caste divergence of F. varia lead to even higher recombination rates than other advanced eusocial species. Conclusions Our study expands the association between elevated recombination and sociality in the order Hymenoptera and strengthens the support for the hypothesis that advanced social evolution in hymenopteran insects invariably selects for high genomic recombination rates.

Young honey bee workers (0 to 2–3 weeks old) perform tasks inside the colony, including brood care (nursing), whereas older workers undergo foraging tasks during the next 3–4 weeks, when an intrinsic senescence program culminates in worker death. We hypothesized that foragers are less able to react to immune system stimulation than nurse bees and that this difference is due to an inefficient immune response in foragers. To test this hypothesis, we used an experimental design that allowed us to uncouple chronological age and behavior status (nursing/foraging). Worker bees from a normal age demography colony (where workers naturally transit from nursing to foraging tasks as they age) and of a single-cohort colony setup (composed of same-aged workers performing nursing or foraging tasks) were tested for survival and capability of activation of the immune system after bacterial injection. Expression of an antimicrobial peptide gene, defensin-1 (def-1), was used to assess immune system activation. We then checked whether the immune response includes changes in the expression of aging- and behavior-related genes, specifically vitellogenin (vg), juvenile hormone esterase (jhe), and insulin-like peptide-1 (ilp-1). We found a significant difference in survival rate between bees of different ages but carrying out the same tasks. Our results thus indicate that the bees' immune response is negatively affected by intrinsic senescence. Additionally, independent of age, foragers had a shorter lifespan than nurses after bacterial infection, although both were able to induce def-1 transcription. In the normal age demography colony, the immune system activation resulted in a reduction in the expression of vg, jhe and ilp-1 genes in foragers, but not in the nurse bees, demonstrating that age and behavior are both important influences on the bees' immune response. By disentangling the effects of age and behavior in the single-cohort colony, we found that vg, jhe and ilp-1 response to immune system stimulation was independent of behavior. Younger bees were able to mount a stronger immune response than older bees, thus highlighting age as an important factor for immunity. Taken together, our results provide new insights into how age and behavior affect the honey bee's immune response.

International audience; Vitellogenin (Vg) plays a vital role in the reproduction of oviparous species, as the precursor of the major egg storage protein. In bees, most of the studies of this protein rely on honey bees (Apis mellifera), and very little is known in solitary species. The solitary bees Centris tarsata and C. analis were chosen to characterize vg cDNA and better understand its expression. The Vg-deduced amino acid sequences from these bees have typical Vg features found in other hymenopterans. Besides its main site of synthesis, the fat body, transcripts of vg were also observed in ovaries. Males also showed expression of vg, although in lower levels compared with females. During development, vg expression was observed in late larvae, pupae, and female adults. The highest amounts of vg transcripts were observed in egg-laying females. Expression of vg increased rapidly in a few hours after female adult emergence, which indicates that its gene expression does not depend on mating to be triggered.

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.

Made available in DSpace on 2020-12-10T20:02:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-03 Brazilian National Science Fund (Conselho Nacional de Desenvolvimento Cientifico and Tecnologico) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) BackgroundMost 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.ResultsThe assembly of 243,974,526 high quality Illumina reads resulted in a predicted assembled genome size of 275Mb 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 15,144bp, 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.ConclusionsThe 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. Univ Sao Paulo, Fac Med Ribeirao Preto, Dept Genet, Ribeirao Preto, SP, Brazil Univ Fed Alfenas, Inst Ciencias Biomed, Dept Biol Celular & Desenvolvimento, Alfenas, MG, Brazil Univ Fed Vales Jequitinhonha & Mucuri, Fac Ciencias Biol & Saude, Dept Ciencias Biol, Diamantina, MG, Brazil Univ Fed Sao Carlos, Ctr Ciencias Biol & Saude, Dept Genet & Evolucao, Sao Carlos, SP, Brazil Univ Tecnol Fed Parana, Cornelio Procopio, PR, Brazil Univ Sao Paulo, Fac Med Ribeirao Preto, Dept Biol Celular & Mol & Bioagentes Patogen, Av Bandeirantes 3900, BR-14049900 Ribeirao Preto, SP, Brazil Univ, Estadual Paulista Julio de Mesquita Filho, Fac Ciencias Agr & Vet, Dept Tecnol, Jaboticabal, SP, Brazil Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Biol, Ribeirao Preto, SP, Brazil Univ, Estadual Paulista Julio de Mesquita Filho, Fac Ciencias Agr & Vet, Dept Tecnol, Jaboticabal, SP, Brazil Brazilian National Science Fund (Conselho Nacional de Desenvolvimento Cientifico and Tecnologico): 454103/2014-0 FAPESP: 2015/06657-0 CAPES: 001

Made available in DSpace on 2021-06-25T10:13:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-02-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Financiadora de Estudos e Projetos Apis mellifera adult workers feature more developed key brain regions than queens, which allows them to cope with the broad range of duties they need to perform in a colony. However, at the end of larval development, the brain of queens is largely more developed than that of workers. Major morphogenetic changes take place after metamorphosis that shift caste-specific brain development. Here, we tested the hypothesis that this phenomenon is hormonally governed and involves differential gene expression. Our molecular screening approach revealed a set of differentially expressed genes in Pp (first pharate-adult phase) brains between castes mainly coding for tissue remodelling and energy-converting proteins (e.g. hex 70a and ATPsynβ). An in-depth qPCR analysis of the transcriptional behaviour during pupal and pharate-adult developmental stage in both castes and in response to artificially augmented hormone titres of 18 genes/variants revealed that: i. subtle differences in hormone titres between castes might be responsible for the differential expression of the EcR and insulin/insulin-like signalling (IIS) pathway genes; ii. the morphogenetic activity of the IIS in brain development must be mediated by ILP-2, iii. which together with the tum, mnb and caspase system, can constitute the molecular effectors of the caste-specific opposing brain developmental trajectories. Departamento de Biologia Celular e do Desenvolvimento Instituto de Ciências Biomédicas Universidade Federal de Alfenas UNIFAL-MG Faculdade de Ciências Agrárias e Veterinárias UNESP - Universidade Estadual Paulista Departamento de Genética Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo Departamento de Biologia Faculdade de Filosofia Ciências e Letras de Ribeirão Preto Universidade de São Paulo Faculdade de Ciências Agrárias e Veterinárias UNESP - Universidade Estadual Paulista FAPESP: 2005/03926-5 FAPESP: 2011/03171-5 CNPq: 406734/2018-6 FAPEMIG: APQ-02134-14 CAPES: Finance Code 001 Financiadora de Estudos e Projetos: MCTI/FINEP/CT-INFRA-PROINFRA 01/2008 Financiadora de Estudos e Projetos: MCTI/FINEP/CT-INFRA-PROINFRA 01/2011

RNA interference (RNAi) is a powerful gene silencing technology, widely used in analyses of reverse genetics, development of therapeutic strategies and generation of biotechnological products. Here we present a free software tool for the rational design of RNAi effectors, named siRNA and shRNA designer (SSD). SSD incorporates our previously developed software Strand Analysis to construct template DNAs amenable for the large scale production of mono-, bi- and trivalent multimeric shRNAs, via in vitro rolling circle transcription. We tested SSD by creating a trivalent multimeric shRNA against the vitellogenin gene of Apis mellifera. RT-qPCR analysis revealed that our molecule promoted a decrease in more than 50% of the target mRNA, in a dose-dependent manner, when compared to the control group. Thus, SSD software allows the easy design of multimeric shRNAs, for single or multiple simultaneous knockdowns, which is especially interesting for studies involving large amounts of double-stranded molecules.

Ftz-f1 is an orphan member of the nuclear hormone receptor superfamily. A 20-hydroxyecdysone pulse allows ftz-f1 gene expression, which then regulates the activity of downstream genes involved in major developmental progression events. In honeybees, the expression of genes like vitellogenin (vg), prophenoloxidase and juvenile hormone-esterase during late pharate-adult development is known to be hormonally controlled in both queens and workers by increasing juvenile hormone (JH) titres in the presence of declining levels of ecdysteroids. Since Ftz-f1 is known for mediating intracellular JH signalling, we hypothesized that ftz-f1 could mediate JH action during the pharate-adult development of honeybees, thus controlling the expression of these genes. Here, we show that ftz-f1 has caste-specific transcription profiles during this developmental period, with a peak coinciding with the increase in JH titre, and that its expression is upregulated by JH and downregulated by ecdysteroids. RNAi-mediated knock down of ftz-f1 showed that the expression of genes essential for adult development (e.g. vg and cuticular genes) depends on ftz-f1 expression. Finally, a double-repressor hypothesis-inspired vg gene knock-down experiment suggests the existence of a positive molecular loop between JH, ftz-f1 and vg.

In social insects, juvenile hormone (JH) has acquired novel functions related to caste determination and division of labor among workers, and this is best evidenced in the honey bee. In contrast to honey bees, stingless bees are a much more diverse group of highly eusocial bees, and the genus Melipona has long called special attention due to a proposed genetic mechanism of caste determination. Here, we examined methyl farnesoate epoxidase (mfe) gene expression, encoding an enzyme relevant for the final step in JH biosynthesis, and measured the hemolymph JH titers for all life cycle stages of Melipona scutellaris queens and workers. We confirmed that mfe is exclusively expressed in the corpora allata. The JH titer is high in the second larval instar, drops in the third, and rises again as the larvae enter metamorphosis. During the pupal stage, mfe expression is initialy elevated, but then gradually drops to low levels before adult emergence. No variation was, however, seen in the JH titer. In adult virgin queens, mfe expression and the JH titer are significantly elevated, possibly associated with their reproductive potential. For workers we found that JH titers are lower in foragers than in nurse bees, while mfe expression did not differ. Stingless bees are, thus, distinct from honey bee workers, suggesting that they have maintained the ancestral gonadotropic function for JH. Hence, the physiological circuitries underlying a highly eusocial life style may be variable, even within a monophyletic clade such as the corbiculate bees.

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.

Made available in DSpace on 2019-10-04T12:36:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-03-14 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) 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. Univ Sao Paulo, Dept Genet, Fac Med Ribeirao Preto, Ribeirao Preto, Brazil Hosp Clin Porto Alegre, Nucleo Bioinformat, Porto Alegre, RS, Brazil Univ Estadual Paulista, Fac Ciencias Agr & Vet, Dept Tecnol, Jaboticabal, Brazil Univ Sao Paulo, Dept Biol, Fac Filosofia Ciencias & Letras Ribeirao Preto, Ribeirao Preto, Brazil Univ Sao Paulo, Dept Fis & Quim, Fac Ciencias Farmaceut Ribeirao Preto, Ribeirao Preto, Brazil Univ Estadual Paulista, Fac Ciencias Agr & Vet, Dept Tecnol, Jaboticabal, Brazil FAPESP: 2014/13136-0 FAPESP: 2011/03171-5 FAPESP: 2014/50265-3 CAPES: 88887.160608/2017-00 CAPES: 001 : FAPESP 2012/24284-5

Queen and worker honeybees differ profoundly in reproductive capacity. The queen of this complex society, with 200 highly active ovarioles in each ovary, is the fertile caste, whereas the workers have approximately 20 ovarioles as a result of receiving a different diet during larval development. In a regular queenright colony, the workers have inactive ovaries and do not reproduce. However, if the queen is sensed to be absent, some of the workers activate their ovaries, producing viable haploid eggs that develop into males. Here, a deep-sequenced ovary transcriptome library of reproductive workers was used as supporting data to assess the dynamic expression of the regulatory molecules and microRNAs (miRNAs) of reproductive and nonreproductive honeybee females. In this library, most of the differentially expressed miRNAs are related to ovary physiology or oogenesis. When we quantified the dynamic expression of 19 miRNAs in the active and inactive worker ovaries and compared their expression in the ovaries of virgin and mated queens, we noted that some miRNAs (miR-1, miR-31a, miR-13b, miR-125, let-7 RNA, miR-100, miR-276, miR-12, miR-263a, miR-306, miR-317, miR-92a and miR-9a) could be used to identify reproductive and nonreproductive statuses independent of caste. Furthermore, integrative gene networks suggested that some candidate miRNAs function in the process of ovary activation in worker bees.

Like all other insects, two key signalling pathways [Toll and immune deficiency (Imd)] regulate the induction of honey bee immune effectors that target microbial pathogens. Amongst these effectors are antimicrobial peptides (AMPs) that are presumed to be produced by the nuclear factors kappa B (NF-κB) Dorsal and Relish from the Toll and Imd pathways, respectively. Using in silico analysis, we previously proposed that the honey bee AMP defensin-1 was regulated by the Toll pathway, whereas hymenoptaecin was regulated by Imd and abaecin by both the Toll and Imd pathways. Here we use an RNA interference (RNAi) assay to determine the role of Dorsal in regulating abaecin and defensin-1. Honey bees have two dorsal genes (dorsal-1 and dorsal-2) and two splicing isoforms of dorsal-1 (dorsal-1A and dorsal-1B). Accordingly, we used both single and multiple (double or triple) isoform knockdown strategies to clarify the roles of dorsal proteins and their isoforms. Down-regulation of defensin-1 was observed for dorsal-1A and dorsal-2 knockdowns, but abaecin expression was not affected by dorsal RNAi. We conclude that defensin-1 is regulated by Dorsal (Toll pathway).

International audience; AbstractThe circadian clock is an advantageous adaptive system that enables organisms to predict and anticipate the daily environmental changes. The circadian rhythms are generated molecularly through the expression of clock genes, based on autoregulatory feedback loops. Honeybees are an excellent model to investigate how the circadian rhythms are modulated accordingly to the social context, behavioral plasticity, and task-related activities. Here, we show how the clock genes behave during the daily cycles in adult worker heads of Apis mellifera. Our results point to the clock genes period and cryptochrome as essential regulators of the circadian rhythms associated to the behavioral maturation in this social insect. We also identified putative miRNA-target and protein-protein interactions involving honeybee clock genes, indicating regulatory networks behind the adjustment of the molecular clock.

For bees, many roads lead to social harmony Eusociality, where workers sacrifice their reproductive rights to support the colony, has evolved repeatedly and represents the most evolved form of social evolution in insects. Kapheim et al. looked across the genomes of 10 bee species with varying degrees of sociality to determine the underlying genomic contributions. No one genomic path led to eusociality, but similarities across genomes were seen in features such as increases in gene regulation and methylation. It also seems that selection pressures relaxed after the emergence of complex sociality. Science , this issue p. 1139

Genetic exchange by hybridization or admixture can make an important contribution to evolution, and introgression of favourable alleles can facilitate adaptation to new environments. A small number of honeybees (Apis mellifera) with African ancestry were introduced to Brazil ~60 years ago, which dispersed and hybridized with existing managed populations of European origin, quickly spreading across much of the Americas in an example of a massive biological invasion. Here, we analyse whole-genome sequences of 32 Africanized honeybees sampled from throughout Brazil to study the effect of this process on genome diversity. By comparison with ancestral populations from Europe and Africa, we infer that these samples have 84% African ancestry, with the remainder from western European populations. However, this proportion varies across the genome and we identify signals of positive selection in regions with high European ancestry proportions. These observations are largely driven by one large gene-rich 1.4-Mbp segment on chromosome 11 where European haplotypes are present at a significantly elevated frequency and likely confer an adaptive advantage in the Africanized honeybee population. This region has previously been implicated in reproductive traits and foraging behaviour in worker bees. Finally, by analysing the distribution of ancestry tract lengths in the context of the known time of the admixture event, we are able to infer an average generation time of 2.0 years. Our analysis highlights the processes by which populations of mixed genetic ancestry form and adapt to new environments.

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.

The honeybee Apis mellifera has major ecological and economic importance. We analyze patterns of genetic variation at 8.3 million SNPs, identified by sequencing 140 honeybee genomes from a worldwide sample of 14 populations at a combined total depth of 634×. These data provide insight into the evolutionary history and genetic basis of local adaptation in this species. We find evidence that population sizes have fluctuated greatly, mirroring historical fluctuations in climate, although contemporary populations have high genetic diversity, indicating the absence of domestication bottlenecks. Levels of genetic variation are strongly shaped by natural selection and are highly correlated with patterns of gene expression and DNA methylation. We identify genomic signatures of local adaptation, which are enriched in genes expressed in workers and in immune system- and sperm motility-related genes that might underlie geographic variation in reproduction, dispersal and disease resistance. This study provides a framework for future investigations into responses to pathogens and climate change in honeybees.

Melipona subnitida, a tropical stingless bee, is an endemic species of the Brazilian northeast and exhibits great potential for honey and pollen production in addition to its role as one of the main pollinators of the Caatinga biome. To understand the genetic structure and better assist in the conservation of this species, we characterized the population variability of M. subnitida using geometric morphometrics of the forewing and cytochrome c oxidase I gene fragment sequencing. We collected workers from six localities in the northernmost distribution. Both methodologies indicated that the variability among the sampled populations is related both to the environment in which samples were collected and the geographical distance between the sampling sites, indicating that differentiation among the populations is due to the existence of at least evolutionary lineages. Molecular clock data suggest that this differentiation may have begun in the middle Pleistocene, approximately 396 kya. The conservation of all evolutionary lineages is important since they can present differential resistance to environmental changes, as resistance to drought and diseases.

Vitellogenin (Vg) is an egg yolk protein that is produced primarily in the fat body of most female insects. In the advanced social structure of eusocial honeybees, the presence of the queen inhibits egg maturation in the workers’ ovaries. However in the stingless bee Melipona quadrifasciata, the workers always develop ovaries and lay a certain amount of eggs while provisioning the brood cells with larval food during what is known as the worker nurse phase. The present work is a comparative study of the presence of Vg in homogenates of the fat bodies and ovaries of the nurse workers, and the virgin and physogastric queens of M. quadrifasciata. The presence of Vg was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting using Apis mellifera anti-egg antibody. Vg was not detected in the fat bodies or ovaries of the workers, but it was found in the ovaries of virgin and physogastric queens and in the fat body of physogastric queens. The results are discussed, taking into account the reproductive state of the individuals and the other possible roles of Vg, such as a storage protein for metoabolism of other organs.

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.

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 ORF of 2193 nucleotides. The deduced translation product is a 731 amino acid protein of 81.5 kDa and a pI 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.

Hexamerins and prophenoloxidases (PPOs) proteins are members of the arthropod-haemocyanin superfamily. In contrast to haemocyanin and PPO, hexamerins do not bind oxygen, but mainly play a role as storage proteins that supply amino acids for insect metamorphosis. We identified seven genes encoding hexamerins, three encoding PPOs, and one hexamerin pseudogene in the genome of the parasitoid wasp Nasonia vitripennis. A phylogenetic analysis of hexamerins and PPOs from this wasp and related proteins from other insect orders suggests an essentially order-specific radiation of hexamerins. Temporal and spatial transcriptional profiles of N. vitripennis hexamerins suggest that they have physiological functions other than metamorphosis, which are arguably coupled with its lifestyle.

In the Apis mellifera post-genomic era, RNAi protocols have been used in functional approaches. However, sample manipulation and invasive methods such as injection of double-stranded RNA (dsRNA) can compromise physiology and survival. To circumvent these problems, we developed a non-invasive method for honeybee gene knockdown, using a well-established vitellogenin RNAi system as a model. Second instar larvae received dsRNA for vitellogenin (dsVg-RNA) in their natural diet. For exogenous control, larvae received dsRNA for GFP (dsGFP-RNA). Untreated larvae formed another control group. Around 60% of the treated larvae naturally developed until adult emergence when 0.5 microg of dsVg-RNA or dsGFP-RNA was offered while no larvae that received 3.0 microg of dsRNA reached pupal stages. Diet dilution did not affect the removal rates. Viability depends not only on the delivered doses but also on the internal conditions of colonies. The weight of treated and untreated groups showed no statistical differences. This showed that RNAi ingestion did not elicit drastic collateral effects. Approximately 90% of vitellogenin transcripts from 7-day-old workers were silenced compared to controls. A large number of samples are handled in a relatively short time and smaller quantities of RNAi molecules are used compared to invasive methods. These advantages culminate in a versatile and a cost-effective approach.

Meiotic and mitotic chromosomes of Dichotomius nisus, D. semisquamosus and D. sericeus were analyzed after conventional staining, C-banding and silver nitrate staining. In addition, Dichotomius nisus and D. semisquamosus chromosomes were also analyzed after fluorescent in situ hybridization (FISH) with an rDNA probe. The species analyzed had an asymmetrical karyotype with 2n = 18 and meta-submetacentric chromosomes. The sex determination mechanism was of the Xy(p) type in D. nisus and D. semisquamosus and of the Xy (r) type in D. sericeus. C-banding revealed the presence of pericentromeric blocks of constitutive heterochromatin (CH) in all the chromosomes of the three species. After silver staining, the nucleolar organizer regions (NORs) were located in autosomes of D. semisquamosus and D. sericeus and in the sexual bivalent of D. nisus. FISH with an rDNA probe confirmed NORs location in D. semisquamosus and in D. nisus. Our results suggest that chromosome inversions and fusions occurred during the evolution of the group.

Though the replacement of European bees by Africanized honey bees in tropical America has attracted considerable attention, little is known about the temporal changes in morphological and genetic characteristics in these bee populations. We examined the changes in the morphometric and genetic profiles of an Africanized honey bee population collected near where the original African swarms escaped, after 34 years of Africanization. Workers from colonies sampled in 1968 and in 2002 were morphometrically analyzed using relative warps analysis and an Automatic Bee Identification System (ABIS). All the colonies had their mitochondrial DNA identified. The subspecies that mixed to form the Africanized honey bees were used as a comparison for the morphometric analysis. The two morphometric approaches showed great similarity of Africanized bees with the African subspecies, Apis mellifera scutellata, corroborating with other markers. We also found the population of 1968 to have the pattern of wing venation to be more similar to A. m. scutellata than the current population. The mitochondrial DNA of European origin, which was very common in the 1968 population, was not found in the current population, indicating selective pressure replacing the European with the African genome in this tropical region. Both morphometric methodologies were very effective in discriminating the A. mellifera groups; the non-linear analysis of ABIS was the most successful in identifying the bees, with more than 94% correct classifications.

The switch from within-hive activities to foraging behavior is a major transition in the life cycle of a honeybee (Apis mellifera) worker. A prominent regulatory role in this switch has long been attributed to juvenile hormone (JH), but recent evidence also points to the yolk precursor protein vitellogenin as a major player in behavioral development. In the present study, we injected vitellogenin double-stranded RNA (dsVg) into newly emerged worker bees of Africanized genetic origin and introduced them together with controls into observation hives to record flight behavior. RNA interference-mediated silencing of vitellogenin gene function shifted the onset of long-duration flights (>10 min) to earlier in life (by 3–4 days) when compared with sham and untreated control bees. In fact, dsVg bees were observed conducting such flights extremely precociously, when only 3 days old. Short-duration flights (

In preparing for metamorphosis, insect larvae store a huge amount of proteins in hemolymph, mainly hexamerins. Out of the four hexamerins present in the honeybee larvae, one, HEX 70a, exhibited a distinct developmental pattern, especially since it is also present in adults. Here, we report sequence data and experimental evidence suggesting alternative functions for HEX 70a, besides its well-known role as an amino acid resource during metamorphosis. The hex 70a gene consists of 6 exons and encodes a 684 amino acid chain containing the conserved hemocyanin N, M, and C domains. HEX 70a classifies as an arylphorin since it contains more than 15% of aromatic amino acids. In the fat body of adult workers, hex 70a expression turned out to be a nutrient-limited process. However, the fat body is not the only site for hex 70a expression. Both, transcript and protein subunits were also detected in developing gonads from workers, queens and drones, suggesting a role in ovary differentiation and testes maturation and functioning. In its putative reproductive role, HEX 70a however differs from the yolk protein, vitellogenin, since it was not detected in eggs or embryos.

For obtaining accurate and reliable gene expression results it is essential that quantitative realtime 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.

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.

Reproduction and development in insects and caste differentiation in bees are mainly governed by ecdysteroids and juvenile hormone. We characterized the ultraspiracle (nuclear hormone receptor) cDNA of the highly social bees Melipona scutellaris and Scaptotrigona depilis. The predicted Usp proteins (with 427aa) show a greater sequence similarity to its orthologs from the “ApLoTe” group than to the dipteran-lepidopteran group, suggesting that Usp proteins included in the bee group might share functional characteristics. The expression profiles in fat bodies (FB) and brains turned out not to be very similar between the species. In M. scutellaris FB and brain and in S. depilis brain usp expression anti-parallels pupal ecdysteroid titers, thus suggesting a repression of usp expression by this hormone.

In stingless bee species, workers regularly lay eggs even in queenright colonies, but a variety of patterns ranging from a high frequency to a complete absence of laying workers has been recognized. We investigated whether these differences could be linked to alternative expression patterns of vitellogenin (Vg), the major component of egg yolk. In Frieseomelitta varia workers, which never lay eggs, and in Melipona scutellaris and Scaptotrigona postica that frequently participates in egglaying activity, we found discrepant developmental patterns of Vg transcript and of the corresponding protein. Interestingly, only in the permanently sterile F. varia workers, Vg protein was found to be constitutively present throughout pupal and adult stages. In workers as well as in males of this species, the quality of the alimentary diet shaped the levels of Vg transcript. In conjunct, our data suggest differences in the regulatory mechanisms underlying vg gene expression in these stingless bee species, and point to alternative roles for Vg, in addition to its essential function in vitellogenesis.

In honeybees, the haplodiploid sex determination system promotes a unique embryogenesis process wherein females develop from fertilized eggs and males develop from unfertilized eggs. However, the developmental strategies of honeybees during early embryogenesis are virtually unknown. Similar to most animals, the honeybee oocytes are supplied with proteins and regulatory elements that support early embryogenesis. As the embryo develops, the zygotic genome is activated and zygotic products gradually replace the preloaded maternal material. The analysis of small RNA and mRNA libraries of mature oocytes and embryos originated from fertilized and unfertilized eggs has allowed us to explore the gene expression dynamics in the first steps of development and during the maternal-to-zygotic transition (MZT). We localized a short sequence motif identified as TAGteam motif and hypothesized to play a similar role in honeybees as in fruit flies, which includes the timing of early zygotic expression (MZT), a function sustained by the presence of the zelda ortholog, which is the main regulator of genome activation. Predicted microRNA (miRNA)-target interactions indicated that there were specific regulators of haploid and diploid embryonic development and an overlap of maternal and zygotic gene expression during the early steps of embryogenesis. Although a number of functions are highly conserved during the early steps of honeybee embryogenesis, the results showed that zygotic genome activation occurs earlier in honeybees than in Drosophila based on the presence of three primary miRNAs (pri-miRNAs) (ame-mir-375, ame-mir-34 and ame-mir-263b) during the cleavage stage in haploid and diploid embryonic development.

Made available in DSpace on 2015-10-21T20:31:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-04-24. Added 1 bitstream(s) on 2015-10-22T09:48:01Z : No. of bitstreams: 1 WOS000353676700001.pdf: 2518339 bytes, checksum: d1f8d18bd059b76504f3fcd9aa639a74 (MD5) National Institutes of Health (NIH) Agriculture and Food Research Initiative Competitive grant from the USDA National Institute of Food and Agriculture Research Council of Norway (NFR) PEW Charitable Trust University of East Anglia, UK Israel Science Foundation (ISF) Biotechnology and Biological Sciences Research Council, UK University of East Anglia University of Alabama College of Arts and Sciences Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Marie Curie International Outgoing Fellowship Swiss National Science Foundation New Faculty Initiative Grant (NFIG) from Illinois State University College of Arts and Sciences DFG Instituto Nacional de Bioinformatica (INB) from ISCIII in Spain Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats.Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits.Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation. Illinois State Univ, Sch Biol Sci, Normal, IL 61790 USA ETH, Inst Integrat Biol, Expt Ecol, CH-8092 Zurich, Switzerland E Carolina Univ, Dept Biol, Greenville, NC 27858 USA Hebrew Univ Jerusalem, Alexander Silberman Inst Life Sci, Dept Ecol Evolut &Behav, Jerusalem, Israel Univ Ghent, Fac Sci, Lab Zoophysiol, B-9000 Ghent, Belgium Univ Otago, Dept Biochem, Labo Evolut &Dev Genet, Dunedin 9054, New Zealand Univ Otago, Dept Biochem, Natl Res Ctr Growth &Dev, Dunedin 9054, New Zealand Univ Missouri, Div Plant Sci, Div Anim Sci, Columbia, MO 65211 USA Univ Missouri, MU Informat Inst, Columbia, MO 65211 USA Georgetown Univ, Dept Biol, Washington, DC 20057 USA Arizona State Univ, Sch Life Sci, Tempe, AZ 85287 USA Univ Copenhagen, Dept Biol, Ctr Funct &Comparat Insect Gen, Copenhagen, Denmark Univ Hohenheim, Inst Anim Sci, D-70599 Stuttgart, Germany Univ Alabama, Dept Biol Sci, Tuscaloosa, AL 35487 USA Univ Illinois, Dept Entomol, Urbana, IL 61801 USA Univ Ghent, Fac Biosci Engn, Dept Crop Protect, Lab Agrozool, B-9000 Ghent, Belgium Univ Halle Wittenberg, Inst Biol, Wittenberg, Germany Univ Geneva, Sch Med, Dept Genet Med &Dev, CH-1211 Geneva, Switzerland Swiss Inst Bioinformat, CH-1211 Geneva, Switzerland MIT, Comp Sci &Artificial Intelligence Lab, Cambridge, MA 02139 USA MIT &Harvard, Broad Inst, Cambridge Ctr 7, Cambridge, MA 02142 USA Univ Munster, Inst Evolut &Biodivers, D-48149 Munster, Germany Ctr Genom Regulat CRG, Barcelona 08003, Spain UPF, Barcelona, Spain Ernst Moritz Arndt Univ Greifswald, Inst Math &Comp Sci, D-17487 Greifswald, Germany Univ Calif Berkeley, Lawrence Berkeley Natl Lab, Genom Div, Berkeley, CA 94720 USA Natl Lib Med, Natl Ctr Biotechnol Informat, Bethesda, MD 20894 USA Norwegian Univ Food Sci, Dept Chem Biotechnol &Food Sci, N-1432 As, Norway Univ E Anglia, Sch Comp Sci, Norwich NR4 7TJ, Norfolk, England Univ Dusseldorf, Inst Evolut Genet, D-40225 Dusseldorf, Germany Univ Cologne, Inst Genet, Cologne, Germany Univ Sao Paulo, Dept Biol, Fac Filosofia Ciencias &Letras Ribeirao Preto, BR-14040901 Ribeirao Preto, Brazil Univ Edinburgh, Ashworth Labs, Inst Evolutionary Biol &Edinburgh Gen, Edinburgh EH9 3FL, Midlothian, Scotland Univ E Anglia, Sch Biol Sci, Norwich NR4 7TJ, Norfolk, England Royal Holloway Univ London, Sch Biol Sci, London, England Maynooth Univ, Dept Biol, Co, Kildare, Ireland Univ Bristol, Sch Biol Sci, Bristol BS8 1TQ, Avon, England CSIRO, Land &Water Flagship, Canberra, ACT, Australia Trin Coll Dublin, Sch Nat Sci, Dept Zool, Dublin, Ireland Zool Soc London, Inst Zool, London NW1 4RY, England ETH, Inst Integrat Biol, Theoret Biol, CH-8092 Zurich, Switzerland Swiss Inst Bioinformat, Lausanne, Switzerland Swiss Fed Inst Technol, Dept Biosyst Sci &Engn, Computat Evolut, Basel, Switzerland USDA ARS, Bee Res Lab, Washington, DC 20250 USA Univ Sao Paulo, Dept Genet, Fac Med Ribeirao Preto, BR-14040900 Ribeirao Preto, Brazil N Carolina State Univ, Ctr Res Sci Computat, Raleigh, NC 27695 USA Kyoto Univ, Grad Sch Agr, Lab Insect Ecol, Kyoto, Japan Univ Sao Paulo, Dept Biol Celular &Mol &Bioagentes Patogen, Fac Med Ribeirao Preto, BR-14040900 Ribeirao Preto, Brazil Inst Fed Educ Ciencia &Tecnol Sao Paulo, BR-15991502 Matao, Brazil Ohio State Univ, Dept Entomol, Wooster, OH 44791 USA Oxford Brookes Univ, Fac Hlth &Life Sci, Dept Biol &Med Sci, Oxford OX3 0BP, England Xian Jiaotong Liverpool Univ, Dept Biol Sci, Suzhou, Peoples R China Univ Hosp Halle Saale, Dept Lab Med, Halle, Germany German Ctr Integrat Biodivers Res iDiv, Leipzig, Germany Univ Southampton, Southampton, Hants, England Univ Leicester, Dept Biol, Leicester, Leics, England Univ Fed Sao Carlos, Ctr Ciencias Biol &Saude, Dept Genet &Evolucao, BR-13565905 Sao Carlos, SP, Brazil Univ Estadual Paulista, Fac Ciencias Agr &Vet, Dept Tecnol, BR-14884900 Jaboticabal, Brazil Univ N Carolina, Dept Biol, Greensboro, NC 27403 USA Natl Ctr Sci Res Demokritos, Inst Biosci &Applicat, Athens, Greece Univ Munich, Munich, Germany Baylor Coll Med, Dept Mol &Human Genet, Human Genome Sequencing Ctr, Houston, TX 77030 USA Univ Illinois, Roy J Carver Biotechnol, Urbana, IL 61801 USA Univ Illinois, Dept Crop Sci, Urbana, IL 61801 USA Univ Illinois, Inst Genom Biol, Urbana, IL 61801 USA MD Anderson Canc Ctr, Sch Hlth Profess, Mol Genet Technol Program, Unit 2, Houston, TX 77025 USA Univ Chicago, Dept Human Genet, Chicago, IL 60637 USA Univ Ghent, Dept Biochem &Microbiol, Lab Prot Biochem &Biomol Engn, B-9000 Ghent, Belgium Queen Mary Univ London, Sch Biol &Chem Sci, Dept Biol &Expt Psychol, London E1 4NS, England Guangzhou Univ, Sch Life Sci, Guangzhou, Guangdong, Peoples R China Johns Hopkins Univ, McKusick Nathans Inst Genet Med, Ctr Computat Biol, Baltimore, MD 21205 USA Univ Illinois, Neurosci Program, Dept Entomol, Carl R Woese Inst Genom Biol, Urbana, IL 61801 USA Departamento de Tecnologia, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista, Jaboticabal, 14884-900, Brazi National Institutes of Health (NIH): DP1 OD006416 National Institutes of Health (NIH): U54 HG003273 Agriculture and Food Research Initiative Competitive grant from the USDA National Institute of Food and Agriculture: 2010-65106-21301 FAPESP: 11/03171-5 Marie Curie International Outgoing Fellowship: PIOF-GA-2011-303312 Swiss National Science Foundation: 31003A-125350 Swiss National Science Foundation: 31003A-143936