Transcriptional signature of host shift in the seed beetle Zabrotes subfasciatus.

In phytophagous insects, adaptation to a new host is a dynamic process, in which early and later steps may be underpinned by different features of the insect genome. Here, we tested the hypothesis that early steps of this process are underpinned by a shift in gene expression patterns. We set up a short-term artificial selection experiment (10 generations) for the use of an alternative host (Cicer arietinum) on populations of the bean beetle Zabrotes subfasciatus. Using Illumina sequencing on young adult females, we show the selected populations differ in the expression of genes associated to stimuli, signalling, and developmental processes. Particularly, the "C. arietinum" population shows upregulation of histone methylation genes, which may constitute a strategy for fine-tuning the insect global gene expression network. Using qPCR on body regions, we demonstrated that the "Phaseolus vulgaris" population upregulates the genes polygalacturonase and egalitarian and that the expression of an odorant receptor transcript variant changes over generations. Moreover, in this population we detected the existence of vitellogenin (Vg) variants in both males and females, possibly harbouring canonical reproductive function in females and extracellular unknown functions in males. This study provides the basis for future genomic investigations seeking to shed light on the nature of the proximate mechanisms involved in promoting differential gene expression associated to insect development and adaptation to new hosts.

Recombination mapping of the Brazilian stingless bee Frieseomelitta varia confirms high recombination rates in social hymenoptera.

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.

Transcriptome dynamics during metamorphosis of imaginal discs into wings and thoracic dorsum in Apis mellifera castes.

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.

SSD - a free software for designing multimeric mono-, bi- and trivalent shRNAs.

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.

Genomewide analysis of admixture and adaptation in the Africanized honeybee.

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.

Evidence of at least two evolutionary lineages in Melipona subnitida (Apidae, Meliponini) suggested by mtDNA variability and geometric morphometrics of forewings.

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.

Non-coding transcription characterization and annotation: a guide and web resource for non-coding RNA databases.

Large-scale transcriptome projects have shown that the number of RNA transcripts not coding for proteins (non-coding RNAs) is much larger than previously recognized. High-throughput technologies, coupled with bioinformatics approaches, have produced increasing amounts of data, highlighting the role of non-coding RNAs (ncRNAs) in biological processes. Data generated by these studies include diverse non-coding RNA classes from organisms of different kingdoms, which were obtained using different experimental and computational assays. This has led to a rapid increase of specialized RNA databases. The fast growth in the number of available databases makes integration of stored information a difficult task. We present here NRDR, a Non-coding RNA Databases Resource for information retrieval on ncRNA databases (www.ncrnadatabases.org). We performed a survey of 102 public databases on ncRNAs and we have introduced four categorizations to classify these databases and to help researchers quickly search and find the information they need: RNA family, information source, information content and available search mechanisms. NRDR is a useful databases searching tool that will facilitate research on ncRNAs.

Transcriptional expression of m6A and m5C RNA methyltransferase genes in the brain and fat body of honey bee adult workers.

Honey bee (Apis mellifera) adult workers change behaviors and nutrition according to age progression. Young workers, such as nurses, perform in-hive tasks and consume protein-rich pollen, while older workers (foragers) leave the colony to search for food, and consume carbohydrate-rich nectar. These environmentally stimulated events involve transcriptional and DNA epigenetic marks alterations in worker tissues. However, post-transcriptional RNA modifications (epitranscriptomics) are still poorly explored in bees. We investigated the transcriptional profiles of m6A and m5C RNA methyltransferases in the brain and fat body of adult workers of 1) different ages and performing different tasks [nurses of 8 days-old (N-8D) and foragers of 29 days-old (F-29D), sampled from wild-type colonies], and 2) same-aged young workers caged in an incubator and treated with a pollen-rich [PR] or a pollen-deprived [PD] diet for 8 days. In the brain, METTL3, DNMT2, NOP2, NSUN2, NSUN5, and NSUN7 genes increased expression during adulthood (from N-8D to F-29D), while the opposite pattern was observed in the fat body for METTL3, DNMT2, and NSUN2 genes. Regarding diet treatments, high expression levels were observed in the brains of the pollen-deprived group (DNMT2, NOP2, and NSUN2 genes) and the fat bodies of the pollen-rich group (NOP2, NSUN4, and NSUN5 genes) compared to the brains of the PR group and the fat bodies of the PD group, respectively. Our data indicate that RNA epigenetics may be an important regulatory layer in the development of adult workers, presenting tissue-specific signatures of RNA methyltransferases expression in response to age, behavior, and diet content.

Comparative cytogenetics of three species of Dichotomius (Coleoptera, Scarabaeidae).

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.

Immunosenescence in honey bees (Apis mellifera L.) is caused by intrinsic senescence and behavioral physiology.

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.

A honeybee storage protein gene, hex 70a, expressed in developing gonads and nutritionally regulated in adult fat body.

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.

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

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

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

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

Methyl farnesoate epoxidase (mfe) gene expression and juvenile hormone titers in the life cycle of a highly eusocial stingless bee, Melipona scutellaris.

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.

A comparative analysis of highly conserved sex-determining genes between Apis mellifera and Drosophila melanogaster.

A comparison of the most conserved sex-determining genes between the fruit fly, Drosophila melanogaster, and the honey bee, Apis mellifera, was performed with bioinformatics tools developed for computational molecular biology. An initial set of protein sequences already described in the fruit fly as participants of the sex-determining cascade was retrieved from the Gene Ontology database (http://www.geneontology.org/) and aligned against a database of protein sequences predicted from the honey bee genome. The doublesex (dsx) gene is considered one of the most conserved sex-determining genes among metazoans, and a male-specific partial cDNA of putative A. mellifera dsx gene (Amdsx) was identified experimentally. The theoretical predictions were developed in the context of sequence similarity. Experimental evidence indicates that dsx is present in embryos and larvae, and that it encodes a transcription factor widely conserved in metazoans, containing a DM DNA-binding domain implicated in the regulation of the expression of genes involved in sexual phenotype formation.

Transcriptome Analysis of Honeybee (Apis Mellifera) Haploid and Diploid Embryos Reveals Early Zygotic Transcription during Cleavage.

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.

Molecular characterization of a cDNA encoding prophenoloxidase and its expression in Apis mellifera.

Phenoloxidase (PO), a melanin-synthesizing enzyme known to play an important role in insect defense, is found as a zymogen (ProPO) in hemolymph and cuticle, where it is activated by proteolysis. We characterized the first proPO cDNA in an eusocial insect, the Apis mellifera honey bee. The AmproPO cDNA contains an ORF of 2079 bp encoding 693 amino acids, and is composed of 9 exons and 8 introns. Southern blot of digested genomic DNA suggested that only one copy of the proPO gene is present in A. mellifera. The molecular mass of the deduced ProPO and the active enzyme was predicted to be 80.1 and 74.4 kDa, respectively. The calculated pI was 6.28. BLASTp search of the deduced amino acid sequence, and neighbor-joining analysis, showed similarity with ProPOs from other insects, ranging from 47% to 63%. Protein signature analyses revealed four conserved regions, including the two copper binding sites characteristic of arthropod ProPOs. RT-PCR and Southern blot showed the highest amount of AmproPO transcripts in workers whole body, followed by queens and drones. Expression was also detected in hemocytes and integument. Real time RT-PCR showed higher amounts of AmproPO transcripts in adults and older pupae than in younger pupae and larvae, suggesting a function of AmproPO in adult exoskeleton melanization and differentiation.

Social evolution. Genomic signatures of evolutionary transitions from solitary to group living.

The evolution of eusociality is one of the major transitions in evolution, but the underlying genomic changes are unknown. We compared the genomes of 10 bee species that vary in social complexity, representing multiple independent transitions in social evolution, and report three major findings. First, many important genes show evidence of neutral evolution as a consequence of relaxed selection with increasing social complexity. Second, there is no single road map to eusociality; independent evolutionary transitions in sociality have independent genetic underpinnings. Third, though clearly independent in detail, these transitions do have similar general features, including an increase in constrained protein evolution accompanied by increases in the potential for gene regulation and decreases in diversity and abundance of transposable elements. Eusociality may arise through different mechanisms each time, but would likely always involve an increase in the complexity of gene networks.

Honey bee (Apis mellifera) transferrin-gene structure and the role of ecdysteroids in the developmental regulation of its expression.

Social life is prone to invasion by microorganisms, and binding of ferric ions by transferrin is an efficient strategy to restrict their access to iron. In this study, we isolated cDNA and genomic clones encoding an Apis mellifera transferrin (AmTRF) gene. It has an open reading frame (ORF) of 2136 bp spread over nine exons. The deduced protein sequence comprises 686 amino acid residues plus a 26 residues signal sequence, giving a predicted molecular mass of 76 kDa. Comparison of the deduced AmTRF amino acid sequence with known insect transferrins revealed significant similarity extending over the entire sequence. It clusters with monoferric transferrins, with which it shares putative iron-binding residues in the N-terminal lobe. In a functional analysis of AmTRF expression in honey bee development, we monitored its expression profile in the larval and pupal stages. The negative regulation of AmTRF by ecdysteroids deduced from the developmental expression profile was confirmed by experimental treatment of spinning-stage honey bee larvae with 20-hydroxyecdysone, and of fourth instar-larvae with juvenile hormone. A juvenile hormone application to spinning-stage larvae, in contrast, had only a minor effect on AmTRF transcript levels. This is the first study implicating ecdysteroids in the developmental regulation of transferrin expression in an insect species.

Effects of juvenile hormone and ecdysone on the timing of vitellogenin appearance in hemolymph of queen and worker pupae of Apis mellifera.

The caste-specific regulation of vitellogenin synthesis in the honeybee represents a problem with many yet unresolved details. We carried out experiments to determine when levels of vitellogenin are first detected in hemolymph of female castes of Apis mellifera, and whether juvenile hormone and ecdysteroids modulate this process. Vitellogenin levels were measured in hemolymph using immunological techniques. We show that in both castes the appearance of vitellogenin in the hemolymph occurs during the pupal period, but the timing was different in the queen and worker. Vitellogenin appears in queens during an early phase of cuticle pigmentation approximately 60h before eclosion, while in workers the appearance of vitellogenin is more delayed, initiating in the pharate adult stage, approximately 10h before eclosion. The timing of vitellogenin appearance in both castes coincides with a slight increase in endogenous levels of juvenile hormone that occurs at the end of pupal development. The correlation between these events was corroborated by topical application of juvenile hormone. Exogenous juvenile hormone advanced the timing of vitellogenin appearance in both castes, but caste-specific differences in timing were maintained. Injection of actinomycin D prevented the response to juvenile hormone. In contrast, queen and worker pupae that were treated with ecdysone showed a delay in the appearance of vitellogenin. These data suggest that queens and workers share a common control mechanism for the timing of vitellogenin synthesis, involving an increase in juvenile hormone titers in the presence of low levels of ecdysteroids.