Differential Proteomics in Dequeened Honeybee Colonies Reveals Lower Viral Load in Hemolymph of Fertile Worker Bees

The eusocial societies of honeybees, where the queen is the only fertile female among tens of thousands sterile worker bees, have intrigued scientists for centuries. The proximate factors, which cause the inhibition of worker bee ovaries, remain largely unknown; as are the factors which cause the activation of worker ovaries upon the loss of queen and brood in the colony. In an attempt to reveal key players in the regulatory network, we made a proteomic comparison of hemolymph profiles of workers with completely activated ovaries vs. rudimentary ovaries. An unexpected finding of this study is the correlation between age matched worker sterility and the enrichment of Picorna-like virus proteins. Fertile workers, on the other hand, show the upregulation of potential components of the immune system. It remains to be investigated whether viral infections contribute to worker sterility directly or are the result of a weaker immune system of sterile workers

The genomes of two key bumblebee species with primitive eusocial organization

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

Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa

Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch.One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10(-5) on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels.By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system

Application of post-genomic approaches for unravelling physiological aspects of locust phase polyphenism.

We are not far from celebrating the centennial of the introduction of the concept of locust density-dependent phase polymorphism (Uvarov, 1921) which causes the same locust species to occur in either a solitarious or gregarious phase. All current findings concerning this topic have been summarized recently (Pener and Simpson, 2009). Although both phases and some aspects of gregarisation are well described, the underlying molecular events of this phenotypic plasticity remain to be unravelled.During the past twenty years, the synthesis of several macromolecules (mRNA transcripts, peptides, proteins) have been shown to be differentially regulated or expressed in both phases. Although these molecules are clearly linked to the polyphenic phenotypes none of them acquired the status of being the primary inducer that triggers the phase-transition cascade. Neither can they explain how these two phases (phenotypes) are generated from one genotype, nor how the gregarious trait is passed on from generation to generation, nor how solitarious locusts can acquire gregarious characteristics during life. The center for regulation of behavioural gregarisation is likely to reside in the central nervous system (CNS). The first indications for grounding the latter were recently provided and indicated that the neurotransmitter serotonin was both necessary and sufficient to induce aggregating behaviour by topical application of serotonin solution on the metathoracic ganglion (Anstey et al., 2009). Nevertheless, more research is needed in order to elucidate both upstream and downstream players of the phase-transition cascade in order to target specific compounds towards the development of an unambiguous plague control strategy.Upon realizing that for drug delivery in the body, lipidization is a technique used in the pharmaceutical industry, we took into consideration that corazonin melanizes the cuticle of albino Locusta migratoria only when injected in an emulsion in oil and not when applied in an aqueous solution. In Boerjan et al., (2011a) we have shown that oral administration of corazonin in oil was not only active, it also induced red cuticular colouration in some animals, and it was also unexpectedly lethal for nymphs, but not for adults. These results necessitate the revision of the potential of (some) peptides for insect control. Also, they suggest practical recommendations for the application of other peptides in physiological assays where oil could be used as a simple slow release formula.The 21st century is in the biological field also called the post-genomic era. With the completion of full genomes of different organisms new analytical and bioinformatical approaches have emerged. One of the aims of this work was to explore whether the recently established locust EST-databases (Kang et al., 2004; Badisco et al., 2011a) might be used in combination with post-genomic approaches.We used the desert locust EST-database in order to analyse 90 selected protein spots of the prothoracic gland proteome. Of the latter fifty could be identified with an EST-based identification strategy. This implicates that the lack of genomic sequence information for the desert locust does not necessarily have to be a bottleneck for further proteomic experiments. Making use of the reverse genetics approach in the form of RNA interference, we are among the first to show that the function for the fruitless transcription factor has been conserved in hemimetabolous insects (Boerjan et al., 2011b). Copulation and copulation success are clearly affected by knocking down fruitless mRNA in early nymphal males. Analysis of post-mating effects in females suggests that sperm or seminal fluids might be affected by the fru RNAi treatment of males. Finally, we propose the possible involvement of an epigenetic model, involving DNA methylation, for locust phenotypic plasticity. The rationale and parallels with both vertebrate and invertebrate phenotypic plasticity are depicted. We show that the epigenetic machinery and functional DNA methylation are present in the desert locust. Our DNA methyltransferase expression profiles provide the first step in supporting this novel conceptual framework. Furthermore, our data suggests that longevity gets imprinted during the early larval stages, but further research will tell whether locust phenotypic plasticity is a true case of imprinting mediated by DNA methylation.nrpages: 212status: publishe

Mass spectrometric profiling of (neuro)-peptides in the worker honeybee, Apis mellifera

The honeybee is the economically most important beneficial insect and a model for studying immunity, development and social behavior. Hence, this species was selected for genome sequencing and annotation. An intensive interplay between bioinformatics and mass spectrometry (MS) resulted in the annotation of 36 neuropeptide genes (Hummon et al., 2006). Exactly 100 peptides were demonstrated by a variety of MS techniques. In this follow-up study we dissected and analysed separately all ganglia of the central nervous system (CNS) of adult worker bees in three repeats. The combined MALDI-TOF spectra enabled the accurate mapping of 67 peptides, encoded by 20 precursors. We also demonstrated the expression of an additional but already predicted peptide. In addition to putative bioactive peptides we also list and discuss spacer peptides, propeptides and truncated peptides. The majority of such peptides have a more restricted distribution pattern. Their presence provides some information on the precursor turnover and/or the location of neural cell bodies in which they are produced. Of a given precursor, the (neuro)-peptides with the widest distribution pattern are likely to be the best candidates to interact with receptors. The separate analysis of a neuroendocrine complex and the mushroom body yields suggestions as to which (neuro)-peptides might act as hormones and which neuropeptides might be involved in the complex spectrum of non-hormone driven honeybee behaviour, at these sites. Our data complement immunohistochemical studies of (neuro)-peptides in the honeybee, and form a reference for comparative studies in other insect or arthropod models, in particular in the light of recent or upcoming genome projects. Finally, they also form a firm basis for physiological, functional and/or differential peptidomics studies in the honeybee.status: publishe

Gel based proteomics without a genome: an EST based approach in Schistocerca gregaria

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The mode of action of juvenile hormone and ecdysone: Towards an epi-endocrinological paradigm

In some insect species, two sites of juvenile hormone (JH) synthesis have been reported: the very well documented corpora allata that secrete JH for "general use", and the reproductive system, in particular the male accessory glands, in which the function of the sometimes huge amounts of JH (e.g. in Hyalophora cecropia) remains to be clarified. A recent finding in Schistocerca gregaria, namely that suppression of the ecdysteroid peak preceding a molt by RNAi of the Halloween genes spook, phantom and shade does not impede normal molting, challenges the (never experimentally proven) classical concept that such a peak is causally linked to a molt. Recent developments in epigenetic control of gene expression in both the honey bee and in locusts suggest that, in addition to the classical scheme of hormone-receptor (membrane- and/or nuclear) mode of action, there may be a third way. Upon combining these and other orphan data that do not fit in the commonly accepted textbook schemes, we here advance the working hypothesis that both JH and ecdysone might be important but overlooked players in epigenetic control of gene expression, in particular at extreme concentrations (peak values or total absence). In this review, we put forward how epi-endocrinology can complement classical arthropod endocrinology.status: publishe

Dispersion of peptides in vegetable oil as a simple slow release formula for both injection and oral uptake in insects: A case study with [His7]-corazonin in an albino Locusta migratoria deficient in corazonin

Upon realizing that for drug delivery in the body, lipidization is a technique used in the pharmaceutical industry, we took in consideration that corazonin melanizes the cuticle of albino Locusta migratoria only when injected in an emulsion in oil, not when applied in a watery solution. In this study, we investigate the possibility for oral uptake of corazonin dispersed in oil, and validated the activity by a melanization assay. Not only was it active, it also induced red cuticular coloration in some animals, and it was also unexpectedly lethal for nymphs, but not for adults. These results necessitate the revision of the potential of (some) peptides for insect control. Also, they suggest practical recommendations for the application of other peptides in physiological assays where oil could be used as a simple slow release formula.status: publishe

Male reproduction is affected by RNA interference of period and timeless in the desert locust Schistocerca gregaria

In all living organisms, behavior, metabolism and physiology are under the regulation of a circadian clock. The molecular machinery of this clock has been conserved throughout the animal kingdom. Besides regulating the circadian timing of a variety of processes through a central oscillating mechanism in the brain, these circadian clock genes were found to have a function in peripheral tissues in different insects. Here, we provide evidence that the circadian clock genes period (per) and timeless (tim) have a role in the male locust reproduction. A knockdown of either of the two genes has no effect on male sexual maturation or behavior, but progeny output in their untreated female copulation partners is affected. Indeed, the fertilization rates of the eggs are lower for females with a per or tim RNAi copulation partner as compared to the eggs deposited by females that mated with a control male. As the sperm content of the seminal vesicles is higher in per or tim knockdown males, we suggest that this phenotype could be caused by a disturbance of the circadian regulated sperm transfer in the male reproductive organs, or an insufficient maturation of the sperm after release from the testes.status: publishe