Evolutionary history of philaenus spumarius (hemiptera, aphrophoridae) and the adaptive significance and genetic basis of its dorsal colour polymorphism

Tese de doutoramento, Biologia (Biologia Evolutiva), Universidade de Lisboa, Faculdade de Ciências, 2017Understanding the genetic basis of adaptive traits and how natural populations adapt to their environment, are fundamental problems in evolutionary biology. Colour polymorphisms are good systems in which these issues can be addressed. This work exploits the adaptive significance of the dorsal colour polymorphism in Philaenus spumarius and describes the efforts to identify genomic region(s) linked to the dorsal colour variation in this species. It also involves the investigation of the evolutionary pattern of P. spumarius. The phylogeographic results showed that main demographic and evolutionary events for the European populations seem to have occurred during Pleistocene, probably as a consequence of the main climatic oscillations that characterised this period. Evidence of recent gene-flow among Mediterranean peninsulas, as well as a close relationship between Iberia and North Africa, a probable British origin for the populations of the Azores and New Zealand, and indication that both western and north-eastern Europe colonised North America, were also found. Captivity experiments, testing the adaptive function of P. spumarius' dorsal colour polymorphism, indicated a higher longevity, a higher number of oviposition events, and a higher number of eggs laid for trilineatus than for marginellus and typicus. A total of 1,837 genomic markers (SNPs) and 928 loci were obtained through RAD sequencing for 33 individuals of three colour phenotypes (trilineatus, marginellus and typicus), and a genome wide association study performed to identify regions related to dorsal colour variation. Single and multi-association analyses identified a total of 60 SNPs associated with dorsal colour phenotypes but none of these SNPs showed homology with colour genes described in other insect species. A stronger differentiation of the trilineatus colour phenotype was also found with these markers. New genomic and transcriptomic resources were developed, constituting important tools and a basis for future research in this species. The resources correspond to a genome draft (25% of the total estimated genome (5.3 Gb)) and to an 81.4 Mb transcriptome assembly. The analysis of the candidate gene yellow found no association with dorsal colour phenotypes. Although suggesting that yellow is not involved in colour variation in P. spumarius, a possible effect of this gene can not be totally excluded without, further analyses. Phylogenetic analyses found contrasting patterns between yellow and the neutral ITS2, indicating that, contrary to ITS2, yellow is conserved within the genus. A higher haplotype and nucleotide diversity was observed for P. spumarius. This could be related to a higher sample size or to the different ecology of the species. The present thesis showed that P. spumarius constitutes a potential good model system to study adaptation and the evolutionary mechanisms involved in the maintenance of polymorphisms.Compreender a base genética de características adaptativas e o modo como as populações naturais evoluem e se adaptam ao seu ambiente é fundamental e constitui um dos temas mais interessantes para a Biologia Evolutiva. Os polimorfismos de coloração são bastante comuns em animais e plantas e, quando combinados com dados genéticos e ecológicos, podem ser excelentes sistemas para estudar não só o processo de adaptação mas também as alterações moleculares subjacentes à variação fenotípica. O presente trabalho teve como principais objetivos a exploração da importância adaptativa de um polimorfismo de coloração e a identificação de genes ou regiões genómicas potencialmente envolvidos na variação de cor. O organismo escolhido para este estudo foi a espécie Philaenus spumarius (L.) (Hemiptera, Aphrophoridae), vulgarmente conhecida como cigarrinha-da-espuma. Este inseto, que se encontra amplamente distribuído pela região Holártica, apresenta vários padrões de coloração da zona dorsal do corpo, com 16 fenótipos melânicos e não melânicos descritos, e cuja base genética e significado adaptativo permanecem por investigar. Para melhor compreender o processo de adaptação e, neste caso, para melhor estudar a base molecular que está por trás do polimorfismo dorsal de coloração de P. spumarius, é importante perceber a história evolutiva da espécie. Ao comparar-se o padrão de genes neutrais com o padrão de genes adaptativos, os efeitos da história evolutiva e/ou demografia são passíveis de ser distinguidos dos efeitos da seleção natural. Embora existam vários estudos genéticos sobre a biogeografia da espécie, estes focam-se, maioritariamente, em populações Europeias e carecem de estimativas de tempo para os principais eventos evolutivos e demográficos. Assim, uma primeira fase deste trabalho envolveu a investigação do padrão biogeográfico da cigarrinha-da-espuma, usando marcadores mitocondriais (Citocromo Oxidase I, Citocromo Oxidase II e Citocromo b) e nucleares (“Elongation Factor 1α”). Pela primeira vez obtiveram-se estimativas de tempo para os principais eventos demográficos e evolutivos ocorridos para esta espécie nas penínsulas Mediterrânicas e, analisaram-se os seus padrões de colonização fora da Eurasia, nomeadamente, no norte de África e na América do norte. Os resultados indicam que a divergência entre as populações de P. spumarius é recente, tendo ocorrido no Pleistoceno Médio/Superior, e está muito provavelmente ligada às alterações climáticas do período Quaternário. Os marcadores mitocondriais mostram que terá ocorrido uma primeira separação da cigarrinha-da-espuma em duas grandes linhagens: a linhagem “ocidental”, na região Mediterrânica, e a linhagem “oriental”, na zona da Anatolia/Cáucaso. Posteriormente, ter-se-á dado a diferenciação da linhagem “ocidental” em duas sub-linhagens: a linhagem “Mediterrâneo-ocidental” na Península Ibérica e a linhagem “Mediterrâneo-oriental” localizada na região dos Balcãs. Este padrão, no entanto, difere do padrão nuclear, o que sugere a existência de cruzamentos e/ou separação incompleta de linhagens. Os eventos de divergência que tiveram lugar nas várias penínsulas Mediterrânicas (refúgios a sul) terão, provavelmente, ocorrido durante os períodos glaciares do Pleistoceno. Após esses períodos, as populações de P. spumarius ter-se-ão expandido em direcção ao norte, a partir da Península Ibérica para o centro e oeste da Europa, e da Anatolia/Cáucaso (ou zonas mais a este) para o leste e centro da Europa. Este estudo também detetou a existência de fluxo genético entre as penínsulas Mediterrânicas e uma relação próxima entre as populações Ibéricas e do norte de África. As populações dos Açores e Nova Zelândia tiveram muito provavelmente uma origem a partir das populações Britânicas e a colonisação da América do norte parece ter sido feita por indivíduos pertencentes não só à linhagem ocidental mas também pertencentes à linhagem presente no norte da Europa. Para tentar perceber porque é que o polimorfismo de cor se mantém nas populações naturais de P. spumarius e qual a sua importância adaptativa, foram realizadas experiências em cativeiro nas quais se testaram diferenças na sobrevivência, sucesso reprodutor e duração de maturação dos ovos em três fenótipos de cor (typicus, trilineatus e marginellus). Curiosamente, foi observada uma maior longevidade para a forma trilineatus, assim como um maior número de eventos de oviposição e maior número total de ovos. O estudo sugere que, na manutenção deste polimorfismo poderão estar envolvidos vários factores e que as diferenças observadas entre o trilineatus e os restantes fenótipos são, possivelmente, uma maneira destes indíviduos compensarem uma maior taxa de ataque por parasitóides e/ou uma maior refletância da radiação solar, ambos reportados em estudos anteriores. No presente trabalho foi investigada a base genética deste polimorfismo. O objetivo consistiu em tentar identificar uma ou várias regiões genómicas associadas à variação do padrão de coloração dorsal em P. spumarius. Duas abordagens foram usadas. A primeira abordagem implicou um estudo de associação no qual se usaram marcadores (“Single Nucleotide Polymorphisms (SNPs)) distribuídos ao longo do genoma, obtidos através da técnica “Restriction Associated DNA (RAD) sequencing”. Com este método de sequenciação, um conjunto de 1837 marcadores foi obtido para 33 indivíduos e, associações com três padrões de cor (typicus, trilineatus e marginellus), foram testadas. As análises identificaram um total de 60 SNPs associados com o padrão de coloração dorsal e revelaram uma maior diferenciação dos indivíduos trilineatus. Os indivíduos deste morfotipo também se revelaram os mais diferenciados em várias características fisiológicas e de história de vida testadas nas experiências de cativeiro. Não foi encontrada homologia entre as regiões associadas e genes da cor já descritos para outras espécies. Os dados sugerem que loci de grande efeito, correspondendo a várias regiões do genoma, podem estar envolvidos na variação encontrada entre os três fenótipos investigados. Para além disso, indicam que uma arquitetura genética complexa pode estar a controlar quer a variação dos padrões de cor quer características da história de vida e que, a selecção natural pode não estar a atuar diretamente na cor. Para ajudar na caracterização das regiões genómicas associadas à variação dos padrões de coloração, o genoma parcial, correspondendo a 25% dos 5.3 Gb estimados, e o transcriptoma de P. spumarius (81.4 Mb) foram sequenciados e “assemblados”. Dos SNPs associados com a cor, 35% alinharam com o genoma e 10% com o transcriptoma indicando que, caso o transcriptoma esteja bem representado, a maioria dos SNPs associados está em regiões não codificantes. Uma abordagem de genes candidatos foi igualmente usada para investigar genes que estão envolvidos na coloração em outros insectos, e que podem potencialmente contribuir para o padrão de coloração em P. spumarius. Os padrões de cor em P. spumarius variam desde totalmente melânicos a totalmente pálidos. Em Drosophila, o gene yellow é um gene envolvido na síntese de melanina e, como tal, constitui um potencial candidato para a variação dos padrões de cor em P. spumarius e nas restantes espécies do género, também elas polimórficas para o padrão de coloração. Uma possível associação entre o yellow e os morfotipos typicus, trilineatus e marginellus foi testada mas nenhuma relação foi encontrada, sugerindo que este gene pode não estar diretamente envolvido na variação de cor nesta espécie. No entanto, como só uma parte do gene foi investigada, o seu envolvimento não pode ser totalmente excluído. A análise filogenética envolvendo o P. spumarius e algumas espécies próximas detetou a existência de padrões contrastantes entre este gene e o gene nuclear Internal Transcribed Spacer 2 (ITS2). Isto indica que, contrariamente ao ITS2, que separa as espécies em dois grandes grupos, o gene yellow é conservado dentro do género. Para o yellow foi encontrado o mesmo haplotipo em quase todas as espécies de Philaenus excepto para o P. maghresignus e P. arslani. Uma maior diversidade genética foi observada para o P. spumarius, podendo ser resultado da diferente ecologia da espécie. Este estudo demonstrou que, apesar de a aplicação de uma abordagem de genes candidatos, numa espécie como o P. spumarius, ser difícil, é importante investigar outros genes que possam estar envolvidos na determinação deste polimorfismo. A presente tese mostrou que a cigarrinha-da-espuma, P. spumarius, é potencialmente um bom modelo para estudar o processo de adaptação e os mecanismos evolutivos envolvidos na manutenção dos polimorfismos nas populações naturais

Extreme Value Laws for dynamical systems with countable extremal sets

We consider stationary stochastic processes arising from dynamical systems by evaluating a given observable along the orbits of the system. We focus on the extremal behaviour of the process, which is related to the entrance in certain regions of the phase space, which correspond to neighbourhoods of the maximal set $\mathcal M$, i.e. the set of points where the observable is maximised. The main novelty here is the fact that we consider that the set $\mathcal M$ may have a countable number of points, which are associated by belonging to the orbit of a certain point, and may have accumulation points. In order to prove the existence of distributional limits and study the intensity of clustering, given by the Extremal Index, we generalise the conditions previously introduced in \cite{FFT12,FFT15}.Comment: arXiv admin note: text overlap with arXiv:1505.0155

Ferulic acid exerts antidepressant-like effect in the tail suspension test in mice: Evidence for the involvement of the serotonergic system

AbstractFerulic acid (4-hydroxy-3-methoxycinnamic acid) is a phenolic compound present in several plants with claimed beneficial effects in prevention and treatment of disorders linked to oxidative stress and inflammation. In this study, we aimed to verify the possible antidepressant-like effect of acute oral administration of ferulic acid in the forced swimming test (FST) and tail suspension test (TST) in mice. Additionally, the mechanisms involved in the antidepressant-like action and the effects of the association of ferulic acid with the antidepressants fluoxetine, paroxetine, and sertraline in the TST were investigated. Ferulic acid produced an antidepressant-like effect in the FST and TST (0.01–10mg/kg, p.o.), without accompanying changes in ambulation. The pretreatment of mice with WAY100635 (0.1mg/kg, s.c., a selective 5-HT1A receptor antagonist) or ketanserin (5mg/kg, i.p., a 5-HT2A receptor antagonist) was able to reverse the anti-immobility effect of ferulic acid (0.01mg/kg, p.o.) in the TST. The combination of fluoxetine (5mg/kg, p.o.), paroxetine (0.1mg/kg, p.o.) or sertraline (1mg/kg, p.o.) with a sub-effective dose of ferulic acid (0.001mg/kg, p.o.) produced a synergistic antidepressant-like effect in the TST, without causing hyperlocomotion in the open-field test. Taken together, these results demonstrate that ferulic acid exerts antidepressant-like effect in the FST and TST in mice through modulation of the serotonergic system

Low Serum Levels of DKK2 Predict Incident Low-Impact Fracture in Older Women

There are currently no robust noninvasive markers of fragility fractures. Secreted frizzled related protein-1 (sFRP-1), dickkopf-related protein 1 (DKK1) and DKK2, and sclerostin (SOST) inhibit Wnt signaling and interfere with osteoblast-mediated bone formation. We evaluated associations of serum levels of sFRP-1, DKK1, DKK2, and SOST with incident low-impact fracture and BMD in 828 women aged ≥65 years from EpiDoC, a longitudinal population-based cohort. A structured questionnaire during a baseline clinical appointment assessed prevalent fragility fractures and clinical risk factors (CRFs) for fracture. Blood was collected to measure serum levels of bone turnover markers and Wnt regulators. Lumbar spine and hip BMD were determined by DXA scanning. Follow-up assessment was performed through a phone interview; incident fragility fracture was defined by any new self-reported low-impact fracture. Multivariate Cox proportional hazard models were used to analyze fracture risk adjusted for CRFs and BMD. During a mean follow-up of 2.3 ± 1.0 years, 62 low-impact fractures were sustained in 58 women. A low serum DKK2 level (per 1 SD decrease) was associated with a 1.5-fold increase in fracture risk independently of BMD and CRFs. Women in the two lowest DKK2 quartiles had a fracture incidence rate of 32 per 1000 person-years, whereas women in the two highest quartiles had 14 fragility fractures per 1000 person-years. A high serum sFRP1 level was associated with a 1.6-fold increase in fracture risk adjusted for CRFs, but not independently of BMD. Serum levels of SOST (r = 0.191; p = 0.0025) and DKK1(r = -0.1725; p = 0.011) were correlated with hip BMD, but not with incident fragility fracture. These results indicate that serum DKK2 and sFRP1 may predict low-impact fracture. The low number of incident fractures recorded is a limitation and serum levels of Wnt regulators should be further studied in other populations as potential noninvasive markers of fragility fractures. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.publishersversionpublishe

Rare events for Cantor target sets

We study the existence of limiting laws of rare events corresponding to the entrance of the orbits on certain target sets in the phase space. The limiting laws are obtained when the target sets shrink to a Cantor set of zero Lebesgue measure. We consider both the presence and absence of clustering, which is detected by the Extremal Index, which turns out to be very useful to identify the compatibility between the dynamics and the fractal structure of the limiting Cantor set. The computation of the Extremal Index is connected to the box dimension of the intersection between the Cantor set and its iterates

Characterization of a new Staphylococcus aureus Kayvirus harboring a lysin active against biofilms

Staphylococcus aureus is one of the most relevant opportunistic pathogens involved in many biofilm-associated diseases, and is a major cause of nosocomial infections, mainly due to the increasing prevalence of multidrug-resistant strains. Consequently, alternative methods to eradicate the pathogen are urgent. It has been previously shown that polyvalent staphylococcal kayviruses and their derived endolysins are excellent candidates for therapy. Here we present the characterization of a new bacteriophage: vB_SauM-LM12 (LM12). LM12 has a broad host range (>90%; 56 strains tested), and is active against several MRSA strains. The genome of LM12 is composed of a dsDNA molecule with 143,625 bp, with average GC content of 30.25% and codes for 227 Coding Sequences (CDSs). Bioinformatics analysis did not identify any gene encoding virulence factors, toxins, or antibiotic resistance determinants. Antibiofilm assays have shown that this phage significantly reduced the number of viable cells (less than one order of magnitude). Moreover, the encoded endolysin also showed activity against biofilms, with a consistent biomass reduction during prolonged periods of treatment (of about one order of magnitude). Interestingly, the endolysin was shown to be much more active against stationary-phase cells and suspended biofilm cells than against intact and scraped biofilms, suggesting that cellular aggregates protected by the biofilm matrix reduced protein activity. Both phage LM12 and its endolysin seem to have a strong antimicrobial effect and broad host range against S. aureus, suggesting their potential to treat S. aureus biofilm infections.This study was supported by Lisando GmbH and by the Portuguese Foundation for Science and Technology (FCT), under the scope of the scope of the project the Project PTDC/BBB-BSS/6471/2014 (POCI-01-0145-FEDER-016678), the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684), and BioTecNorte operation (NORTE-01-0145-FEDER-000004), funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte. Ana Brandão and Ergun Akturk acknowledge FCT for grants SFRH/BD/133193/2017 and PD/BD/13524/2017, respectively. The authors declare that they have no competing financial interests.info:eu-repo/semantics/publishedVersio

Reference values for lysosomal enzymes activities using dried blood spots samples - a Brazilian experience

Background: Lysosomal storage diseases (LSD) are inherited disorders caused by deficiency of lysosomal enzymes in which early diagnosis is essential to provide timely treatment. This study reports interval values for the activity of lysosomal enzymes that are deficient in Mucopolysaccharidosis type I, Fabry, Gaucher and Pompe disease, using dried blood spots on filter paper (DBS) samples in a Brazilian population.Results: Reference activity values were obtained from healthy volunteers samples for alpha-galactosidase A (4.57 +/- 1.37 umol/L/h), beta-glucosidase (3.06 +/- 0.99 umol/L/h), alpha-glucosidase (ratio: 13.19 +/- 4.26; % inhibition: 70.66 +/- 7.60), alpha-iduronidase (3.45 +/- 1.21 umol/L/h) and beta-galactosidase (14.09 +/- 4.36 umol/L/h).Conclusion: Reference values of five lysosomal enzymes were determined for a Brazilian population sample. However, as our results differ from other laboratories, it highlights the importance of establishing specific reference values for each center

results from a nationwide population-based study

Funding Information: This study is funded by national funds through FCT - Fundação para a Ciência e Tecnologia, I. P. under the PhD grant SFRH/BD/148420/2019 awarded to the first author. This protocol was included in the PhD previously approved project. EpiReumpaPt was supported by unrestricted grants from Direcção-Geral da Saúde, Fundação Calouste Gulbenkian, Fundação Champalimaud, Fundação AstraZeneca, AbbVie, Merck, Sharp & Dohme, Pfizer, Roche, Servier, Bial, D3A Medical Systems, Happybrands, Center de Medicina Laboratorial Germano de Sousa, Clínica Médica da Praia da Vitória, CAL-Clínica, Galp Energia, Açoreana Seguros, and individual rheumatologists.Background: Worldwide, the current management of knee osteoarthritis appears heterogeneous, high-cost and often not based on current best evidence. The absence of epidemiological data regarding the utilisation of healthcare services may conceal the need for improvements in the management of osteoarthritis. The aim of this study is to explore the profiles of healthcare services utilisation by people with knee osteoarthritis, and to analyse their determinants, according to Andersen’s behavioural model. Methods: We analysed a sample of 978 participants diagnosed with knee osteoarthritis from the population-based study EpiReumaPt, in Portugal. Data was collected with a structured interview, and the diagnosis of knee osteoarthritis was validated by a rheumatologist team. With the Two-step Cluster procedure, we primarily identified different profiles of healthcare utilisation according to the services most used by patients with knee osteoarthritis. Secondly, we analysed the determinants of each profile, using multinomial logistic regression, according to the predisposing characteristics, enabling factors and need variables. Results: In our sample, a high proportion of participants are overweight or obese (82,6%, n = 748) and physically inactive (20,6%, n = 201) and a small proportion had physiotherapy management (14,4%, n = 141). We identified three profiles of healthcare utilisation: “HighUsers”; “GPUsers”; “LowUsers”. “HighUsers” represents more than 35% of the sample, and are also the participants with higher utilisation of medical appointments. “GPUsers” represent the participants with higher utilisation of general practitioner appointments. Within these profiles, age and geographic location – indicated as predisposing characteristics; employment status and healthcare insurance - as enabling factors; number of comorbidities, physical function, health-related quality of life, anxiety and physical exercise - as need variables, showed associations (p < 0,05) with the higher utilisation of healthcare services profiles. Conclusions: Healthcare utilisation by people with knee osteoarthritis is not driven only by clinical needs. The predisposing characteristics and enabling factors associated with healthcare utilisation reveal inequities in the access to healthcare and variability in the management of people with knee osteoarthritis. Research and implementation of whole-system strategies to improve equity in the access and quality of care are paramount in order to diminish the impact of osteoarthritis at individual-, societal- and economic-level.publishersversionpublishe