Characterization of an antennal carboxylesterase from the pest moth Spodoptera littoralis degrading a host plant odorant.

Background: Carboxyl/cholinesterases (CCEs) are highly diversified in insects. These enzymes have a broad range of proposed functions, in neuro/developmental processes, dietary detoxification, insecticide resistance or hormone/pheromone degradation. As few functional data are available on purified or recombinant CCEs, the physiological role of most of these enzymes is unknown. Concerning their role in olfaction, only two CCEs able to metabolize sex pheromones have been functionally characterized in insects. These enzymes are only expressed in the male antennae, and secreted into the lumen of the pheromone-sensitive sensilla. CCEs able to hydrolyze other odorants than sex pheromones, such as plant volatiles, have not been identified. Methodology: In Spodoptera littoralis, a major crop pest, a diversity of antennal CCEs has been previously identified. We have employed here a combination of molecular biology, biochemistry and electrophysiology approaches to functionally characterize an intracellular CCE, SlCXE10, whose predominant expression in the olfactory sensilla suggested a role in olfaction. A recombinant protein was produced using the baculovirus system and we tested its catabolic properties towards a plant volatile and the sex pheromone components. Conclusion: We showed that SlCXE10 could efficiently hydrolyze a green leaf volatile and to a lesser extent the sex pheromone components. The transcript level in male antennae was also strongly induced by exposure to this plant odorant. In antennae, SlCXE10 expression was associated with sensilla responding to the sex pheromones and to plant odours. These results suggest that a CCE-based intracellular metabolism of odorants could occur in insect antennae, in addition to the extracellular metabolism occurring within the sensillar lumen. This is the first functional characterization of an Odorant- Degrading Enzyme active towards a host plant volatile.

Mathematical modeling and optimal control of malaria

Malaria continues to infect millions and kill hundreds of thousands of people worldwide each year, despite over a century of research and attempts to control and eliminate this infectious disease. Challenges such as the development and spread of drug resistant malaria parasites, insecticide resistance to mosquitoes, climate change, the presence of individuals with subpatent malaria infections which normally are asymptomatic and behavioral plasticity in the mosquito hinder the prospects of malaria control and elimination. In this thesis, mathematical models of malaria transmission and control that address the role of drug resistance, immunity, iron supplementation and anemia, immigration and visitation, and the presence of asymptomatic carriers in malaria transmission are developed. A within-host mathematical model of severe Plasmodium falciparum malaria is also developed. First, a deterministic mathematical model for transmission of antimalarial drug resistance parasites with superinfection is developed and analyzed. The possibility of increase in the risk of superinfection due to iron supplementation and fortification in malaria endemic areas is discussed. The model results calls upon stakeholders to weigh the pros and cons of iron supplementation to individuals living in malaria endemic regions. Second, a deterministic model of transmission of drug resistant malaria parasites, including the inflow of infective immigrants, is presented and analyzed. The optimal control theory is applied to this model to study the impact of various malaria and vector control strategies, such as screening of immigrants, treatment of drug-sensitive infections, treatment of drug-resistant infections, and the use of insecticide-treated bed nets and indoor spraying of mosquitoes. The results of the model emphasize the importance of using a combination of all four controls tools for effective malaria intervention. Next, a two-age-class mathematical model for malaria transmission with asymptomatic carriers is developed and analyzed. In development of this model, four possible control measures are analyzed: the use of long-lasting treated mosquito nets, indoor residual spraying, screening and treatment of symptomatic, and screening and treatment of asymptomatic individuals. The numerical results show that a disease-free equilibrium can be attained if all four control measures are used. A common pitfall for most epidemiological models is the absence of real data; model-based conclusions have to be drawn based on uncertain parameter values. In this thesis, an approach to study the robustness of optimal control solutions under such parameter uncertainty is presented. Numerical analysis of the optimal control problem in the presence of parameter uncertainty demonstrate the robustness of the optimal control approach that: when a comprehensive control strategy is used the main conclusions of the optimal control remain unchanged, even if inevitable variability remains in the control profiles. The results provide a promising framework for the design of cost-effective strategies for disease control with multiple interventions, even under considerable uncertainty of model parameters. Finally, a separate work modeling the within-host Plasmodium falciparum infection in humans is presented. The developed model allows re-infection of already-infected red blood cells. The model hypothesizes that in severe malaria due to parasite quest for survival and rapid multiplication, the Plasmodium falciparum can be absorbed in the already-infected red blood cells which accelerates the rupture rate and consequently cause anemia. Analysis of the model and parameter identifiability using Markov chain Monte Carlo methods is presented.

Determination of the susceptibility of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) to ivermectin and fipronil by Larval Immersion Test (LIT) in Uruguay

Rhipicephalus (Boophilus) microplus is an important cattle pest in Uruguay, and the law regulates its control. It is resistant to organophosphates, synthetic pyrethroids and, as recently discovered, to fipronil. Resistance to macrocyclic lactones (MLs) and amitraz have not been documented; however, veterinarians and farmers have reported treatment failures. The objective of the present work was to study the susceptibility of cattle tick strains from different Uruguayan counties to ivermectin (IVM) and fipronil by using the Larval Immersion Test (LIT). The Mozo strain was used as the susceptible reference strain. From 2007 to 2009, twenty-eight tick populations were collected from different cattle farms with and without history of IVM or fipronil use. A probit analysis estimated dose-mortality regressions, lethal concentrations (LC), and confidence intervals. The resistance ratio (RR) was determined at the LC(50) and LC(90) estimates. To classify a tick population in relation to resistance, three categories based on a statistical analysis of LC and RR between field populations and Mozo strains were defined: susceptible (no differences), incipient resistance (differences and RR(50) < 2) and resistant (differences and RR(50) >= 2). Eighteen field populations were tested with IVM and five of them presented a RR(50) range between 1.35 and 1.98 and the LC(50/90), which is statistically different from the Mozo strain (incipient resistance). However, the RR(90) increases >= 2 in four of the populations, confirming that tick resistance to IVM is emergent. The low RR values obtained could be a result of a low frequency of treatments with IVM. Twenty-seven tick populations were tested with fipronil and six were diagnosed as resistant according to the LIT. Cross-resistance was not observed between fipronil and IVM on these tick populations. The current study presents different R. (B.) microplus populations with an incipient resistance to IVM, and indicates that the fipronil tick resistance is restricted to certain areas in Uruguay. (c) 2011 Elsevier B.V. All rights reserved.

Mortality of Bemisia tabaci biotype B (sternorrhyncha: aleyrodidae) adults by aliphatic and aromatic synthetic sucrose esters

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genetic variation along time in a brazilian population of Aedes aegypti (Diptera : Culicidae), detected by changes in the esterase patterns

Aedes aegypti from the Brazilian cities of Sao Jose do Rio Preto (SJ) and Goiania (GO) were analyzed as to their esterase patterns and the results were compared with data obtained about 5 years before for SJ population. Esterase bands not detected in the previous study were now observed in mosquitoes from both SJ and GO populations, being the last considered a population resistant to insecticides. Other similarities between SJ and GO populations in this study, and some differences in comparison with the previous data on SJ were observed, involving, in addition to changes in band type, changes in frequency of mosquitoes expressing them and differential gene activation during development. As it is generally true for genetic features, changes in the esterase patterns are expected to be the result of factors such as selection by environmental conditions and genetic drift. In the present case, continuous use of insecticides aiming mosquito population size control in SJ by sanitary authorities could be involved in the observed changes. Changed esterases were classified as carboxylesterases and cholinesterases, which are enzymes already shown to take part in the development of resistance in several organisms. In addition, data obtained in the elapsed time by authorities responsible for the mosquito control has shown increasing insecticide resistance of SJ population mosquitoes parallel to increase in the total amount of esterases, reinforcing the mentioned possibility.

Variation at the Est3 locus and adaptability to organophosphorous compounds in Zaprionus indianus populations

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The Genome of Anopheles darlingi, the main neotropical malaria vector

Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vectorhuman and vectorparasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles- darlingi. © 2013 The Author(s).

Componentes do valor adaptativo e investigação do polimorfismo genético do gene Cyp6g1 (exons 1 e 2) em linhagens resistentes e suscetíveis de Drosophila melanogaster e de Drosophila simulans

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Análise da ocorrência de transposição em regiões reguladoras dos genes da família Cyp em espécies de Drosophila

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo da variabilidade genética de populações de Aedes aegypti (Diptera,Culicidae), resistente e suscetíveis a inseticidas

Pós-graduação em Genética - IBILCE

Desenvolvimento de diferentes populações de Cydia pomonella (Lepidoptera: Tortricidae) em temperaturas variáveis e consequências na modelagem fenológica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Padrões de esterases em populações resistentes e suscetíveis de Aedes aegypti (Diptera, Culicidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Aedes aegypti on Madeira Island (Portugal): genetic variation of a recently introduced dengue vector

The increasing population of Aedes aegypti mosquitoes on Madeira Island (Portugal) resulted in the first autochthonous dengue outbreak, which occurred in October 2012. Our study establishes the first genetic evaluation based on the mitochondrial DNA (mtDNA) genes [cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 4 (ND4)] and knockdown resistance ( kdr ) mutations exploring the colonisation history and the genetic diversity of this insular vector population. We included mosquito populations from Brazil and Venezuela in the analysis as putative geographic sources. The Ae. aegyptipopulation from Madeira showed extremely low mtDNA genetic variability, with a single haplotype for COI and ND4. We also detected the presence of two important kdr mutations and the quasi-fixation of one of these mutations (F1534C). These results are consistent with a unique recent founder event that occurred on the island of Ae. aegyptimosquitoes that carry kdr mutations associated with insecticide resistance. Finally, we also report the presence of the F1534C kdr mutation in the Brazil and Venezuela populations. To our knowledge, this is the first time this mutation has been found in South American Ae. aegypti mosquitoes. Given the present risk of Ae. aegypti re-invading continental Europe from Madeira and the recent dengue outbreaks on the island, this information is important to plan surveillance and control measures.

LOW GENETIC DIVERSITY IN Wolbachia-INFECTED Culex quinquefasciatus (DIPTERA: CULICIDAE) FROM BRAZIL AND ARGENTINA

Culex quinquefasciatus is a vector of human pathogens, including filarial nematodes and several viruses. Although its epidemiological relevance is known to vary across geographical regions, an understanding of its population genetic structure is still incipient. In light of this, we evaluated the genetic diversity of Cx. quinquefasciatus and Cx. pipiens x Cx. quinquefasciatus hybrids collected from nine localities in Brazil and one site in Argentina. We used mitochondrial genes cox1 and nd4, along with the coxA and wsp genes of the maternally-inherited Wolbachia endosymbiont. The nd4 fragment was invariant between samples, whilst cox1 exhibited four haplotypes that separated two types of Cx. quinquefasciatus, one clustered in southern Brazil. Low sequence diversity was generally observed, being discussed. Both Brazilian and Argentinian mosquitoes were infected with a single Wolbachia strain. As reported in previous studies with these populations, cox1 and nd4 diversity is not congruent with the population structure revealed by nuclear markers or alar morphology. Future Cx. quinquefasciatus research should, if possible, evaluate mtDNA diversity in light of other markers.

Genetic Control of Mosquitoes: population suppression strategies

Over the last two decades, morbidity and mortality from malaria and dengue fever among other pathogens are an increasing Public Health problem. The increase in the geographic distribution of vectors is accompanied by the emergence of viruses and diseases in new areas. There are insufficient specific therapeutic drugs available and there are no reliable vaccines for malaria or dengue, although some progress has been achieved, there is still a long way between its development and actual field use. Most mosquito control measures have failed to achieve their goals, mostly because of the mosquito's great reproductive capacity and genomic flexibility. Chemical control is increasingly restricted due to potential human toxicity, mortality in no target organisms, insecticide resistance, and other environmental impacts. Other strategies for mosquito control are desperately needed. The Sterile Insect Technique (SIT) is a species-specific and environmentally benign method for insect population suppression, it is based on mass rearing, radiation mediated sterilization, and release of a large number of male insects. Releasing of Insects carrying a dominant lethal gene (RIDL) offers a solution to many of the drawbacks of traditional SIT that have limited its application in mosquitoes while maintaining its environmentally friendly and species-specific utility. The self-limiting nature of sterile mosquitoes tends to make the issues related to field use of these somewhat less challenging than for self-spreading systems characteristic of population replacement strategies. They also are closer to field use, so might be appropriate to consider first. The prospect of genetic control methods against mosquito vectored human diseases is rapidly becoming a reality, many decisions will need to be made on a national, regional and international level regarding the biosafety, social, cultural and ethical aspects of the use and deployment of these vector control methods.