DNA methylation and demethylation in honeybee long-term memory formation

Trabalho final de mestrado integrado em Medicina (Cardiologia), apresentado à Faculdade de Medicina da Universidade de Coimbra.A estenose aórtica paradoxal define-se por uma área valvular aórtica ≤ 1 cm2, um volume sistólico indexado < 35 mL/m2 e um gradiente médio transvalvular < 40 mmHg, apesar de uma fração de ejeção do ventrículo esquerdo preservada (> 50%). Caracteriza-se por uma marcada remodelagem concêntrica do ventrículo esquerdo, com predomínio de disfunção diastólica, e está associada a uma rigidez vascular sistémica aumentada. A prevalência desta doença varia entre os 3-35% dos doentes com estenose aórtica grave e afeta sobretudo doentes idosos, do género feminino e com multimorbilidade. A estenose aórtica paradoxal é um desafio diagnóstico, particularmente pelas inconsistências associadas à equação de continuidade. Têm surgido novos métodos de avaliação na literatura para auxiliar no diagnóstico do estado de baixo fluxo sistólico, bem como para estudar a remodelagem ventricular esquerda. Até há pouco tempo existia algum consenso na literatura sobre o facto da estenose aórtica paradoxal representar um estadio mais avançado da doença valvular aórtica. Como tal, na presença de sintomas, a substituição valvular aórtica parecia ser o tratamento mais indicado. No entanto, outros autores mostraram resultados diferentes e assim, aumentaram a discussão sobre a abordagem e gestão deste fenótipo da estenose aórtica degenerativa. A presente tese tem por objetivo elaborar uma revisão sistemática da literatura sobre a estenose aórtica paradoxal, e abordar aspetos relacionados com a sua demografia, semiologia, avaliação diagnóstica, implicações terapêuticas e prognóstico.Paradoxical aortic stenosis is defined by an aortic valve area ≤ 1cm2, an indexed systolic volume < 35 mL/m2 and a transvalvular gradient < 40 mmHg, despite a preserved left ventricular ejection fraction (> 50%). It’s characterized by an extensive concentric cardiac remodelling of the left ventricle, mainly impairing diastolic function, and has been associated with an increased systemic valvular stiffness. This disease prevalence varies from 3 up to 35% of the patients with severe aortic stenosis, specially affecting the elderly, women and those with multi-morbilities. Paradoxical aortic stenosis is a diagnostic challenge due to the inconsistencies associated with the continuity equation. In order to assess the low flow state and to study the left ventricle remodelling, new methods have emerged. Until now there has been some agreement between authors regarding paradoxical aortic stenosis being a more advanced form of aortic valve disease. Hence, in the presence of symptoms, aortic valve replacement presents as an appropriate therapeutic approach. On the contrary, other authors have shown different results, and so fired up the debate concerning the management of this degenerative aortic stenosis phenotype. The aim of the present paper is to systematically review the literature on paradoxical aortic stenosis and to cast light on its demography, semiology, diagnostic workup, therapeutic approach and prognosis

DNA methylation mediates neural processing after odor learning in the honeybee

DNA methyltransferases (Dnmts) - epigenetic writers catalyzing the transfer of methyl-groups to cytosine (DNA methylation) - regulate different aspects of memory formation in many animal species. In honeybees, Dnmt activity is required to adjust the specificity of olfactory reward memories and bees' relearning capability. The physiological relevance of Dnmt-mediated DNA methylation in neural networks, however, remains unknown. Here, we investigated how Dnmt activity impacts neuroplasticity in the bees' primary olfactory center, the antennal lobe (AL) an equivalent of the vertebrate olfactory bulb. The AL is crucial for odor discrimination, an indispensable process in forming specific odor memories. Using pharmacological inhibition, we demonstrate that Dnmt activity influences neural network properties during memory formation in vivo. We show that Dnmt activity promotes fast odor pattern separation in trained bees. Furthermore, Dnmt activity during memory formation increases both the number of responding glomeruli and the response magnitude to a novel odor. These data suggest that Dnmt activity is necessary for a form of homoeostatic network control which might involve inhibitory interneurons in the AL network

\u3ci\u3eJhe in Gryllus assimilis\u3c/i\u3e: Cloning, sequence-activity associations and phylogeny

The 458 amino acid sequence of a mature JHE protein from the cricket Gryllus assimilis was identified after isolating the partial cDNA sequence encoding this protein from a fat body and midgut cDNA library. This hemimetabolan JHE sequence shows over 40% amino acid similarity to the known JHE sequences of several holometabolous insects. It also includes previously determined peptide sequences for G. assimilis JHE as well as two other motifs associated with JHE enzymes in holometabolous insects. The predicted molecular weight of the protein agrees with that of the JHE previously purified from G. assimilis. Partial genomic sequence encoding the Jhe contains two large (1330 and 2918 bp) introns. No coding DNA sequence variation was observed over a 1293 bp region between selected lines differing six to eight-fold in hemolymph JHE activity. However, a 19 bp indel was found in one of the introns; the insertion was strongly associated with elevated hemolymph activity, both in the selected lines and in the F2 progeny of crosses between them. Phylogenetic analyses localised the G. assimilis JHE to a clade containing dipteran and coleopteran JHEs, with lepidopteran JHEs occurring in a separate clade

\u3ci\u3eJhe in Gryllus assimilis\u3c/i\u3e: Cloning, sequence-activity associations and phylogeny

The 458 amino acid sequence of a mature JHE protein from the cricket Gryllus assimilis was identified after isolating the partial cDNA sequence encoding this protein from a fat body and midgut cDNA library. This hemimetabolan JHE sequence shows over 40% amino acid similarity to the known JHE sequences of several holometabolous insects. It also includes previously determined peptide sequences for G. assimilis JHE as well as two other motifs associated with JHE enzymes in holometabolous insects. The predicted molecular weight of the protein agrees with that of the JHE previously purified from G. assimilis. Partial genomic sequence encoding the Jhe contains two large (1330 and 2918 bp) introns. No coding DNA sequence variation was observed over a 1293 bp region between selected lines differing six to eight-fold in hemolymph JHE activity. However, a 19 bp indel was found in one of the introns; the insertion was strongly associated with elevated hemolymph activity, both in the selected lines and in the F2 progeny of crosses between them. Phylogenetic analyses localised the G. assimilis JHE to a clade containing dipteran and coleopteran JHEs, with lepidopteran JHEs occurring in a separate clade

Genes of the antioxidant system of the honey bee: annotation and phylogeny

Antioxidant enzymes perform a variety of vital functions including the reduction of life-shortening oxidative damage. We used the honey bee genome sequence to identify the major components of the honey bee antioxidant system. A comparative analysis of honey bee with Drosophila melanogaster and Anopheles gambiae shows that although the basic components of the antioxidant system are conserved, there are important species differences in the number of paralogs. These include the duplication of thioredoxin reductase and the expansion of the thioredoxin family in fly; lack of expansion of the Theta, Delta and Omega GST classes in bee and no expansion of the Sigma class in dipteran species. The differential expansion of antioxidant gene families among honey bees and dipteran species might reflect the marked differences in life history and ecological niches between social and solitary species

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 Plasticity of the Drosophila Transcriptome

Phenotypic plasticity is the ability of a single genotype to produce different phenotypes in response to changing environments. We assessed variation in genome-wide gene expression and four fitness-related phenotypes of an outbred Drosophila melanogaster population under 20 different physiological, social, nutritional, chemical, and physical environments; and we compared the phenotypically plastic transcripts to genetically variable transcripts in a single environment. The environmentally sensitive transcriptome consists of two transcript categories, which comprise ∼15% of expressed transcripts. Class I transcripts are genetically variable and associated with detoxification, metabolism, proteolysis, heat shock proteins, and transcriptional regulation. Class II transcripts have low genetic variance and show sexually dimorphic expression enriched for reproductive functions. Clustering analysis of Class I transcripts reveals a fragmented modular organization and distinct environmentally responsive transcriptional signatures for the four fitness-related traits. Our analysis suggests that a restricted environmentally responsive segment of the transcriptome preserves the balance between phenotypic plasticity and environmental canalization

Deep Sequencing of Pyrethroid-Resistant Bed Bugs Reveals Multiple Mechanisms of Resistance within a Single Population

A frightening resurgence of bed bug infestations has occurred over the last 10 years in the U.S. and current chemical methods have been inadequate for controlling this pest due to widespread insecticide resistance. Little is known about the mechanisms of resistance present in U.S. bed bug populations, making it extremely difficult to develop intelligent strategies for their control. We have identified bed bugs collected in Richmond, VA which exhibit both kdr-type (L925I) and metabolic resistance to pyrethroid insecticides. Using LD50 bioassays, we determined that resistance ratios for Richmond strain bed bugs were ∼5200-fold to the insecticide deltamethrin. To identify metabolic genes potentially involved in the detoxification of pyrethroids, we performed deep-sequencing of the adult bed bug transcriptome, obtaining more than 2.5 million reads on the 454 titanium platform. Following assembly, analysis of newly identified gene transcripts in both Harlan (susceptible) and Richmond (resistant) bed bugs revealed several candidate cytochrome P450 and carboxylesterase genes which were significantly over-expressed in the resistant strain, consistent with the idea of increased metabolic resistance. These data will accelerate efforts to understand the biochemical basis for insecticide resistance in bed bugs, and provide molecular markers to assist in the surveillance of metabolic resistance