Дисертації з теми "Invasive mosquitoes"

Thesis (Ph. D.)--Illinois State University, 2007.
Title from title page screen, viewed on February 11, 2008. Dissertation Committee: Steven A. Juliano (chair), Diane L. Byers, L. Philip Lounibos, Charles F. Thompson, William L. Perry. Includes bibliographical references (leaves 152-163) and abstract. Also available in print.

Las enfermedades transmitidas por mosquitos (ETM) son una creciente preocupación para la salud pública mundial. El aumento de las actividades humanas a escala internacional y el calentamiento global han permitido la rápida difusión y establecimiento de las especies invasoras de mosquitos en nuevas áreas geográficas. Además, la globalización facilita la introducción de arbovirus exóticos en áreas naïve, donde el mosquito está presente, incrementando la probabilidad de los brotes de ETM. Los brotes de ETM, especialmente aquellos causados por arbovirus como el virus del Dengue, el virus del Zika, el virus de la fiebre del valle del Rift (VFVR) o el virus de la fiebre del Nilo Occidental (VFNO), tienen graves consecuencias para la salud pública y/o veterinaria. Las pérdidas económicas debidas a las ETM zoonóticas pueden comprometer severamente la economía del país afectado. Este aspecto es particularmente relevante en los países en desarrollo, donde enfermedades como la fiebre del valle del Rift pueden provocar la muerte de un número extremadamente elevado de animales, tanto domésticos como salvajes. Por estos motivos, es necesario adquirir un profundo conocimiento de las especies de mosquito presentes en un país y cuáles de ellas pueden ser vectores de los distintos agentes patógenos, con el fin de realizar eficientes planes de vigilancia y controles entomológicos. La presente tesis está dividida en tres partes y cada una de ellas está subdividida en capítulos. La primera parte se compone de dos apartados: Chapter 1 General Introduction y Chapter 2 Objectives. El objetivo de esta primera parte es ofrecer una visión general sobre la importancia de las ETM, el rol de los vectores y el concepto de competencia vectorial. Además se proporciona una descripción del VFNO y del VFVR. A continuación, se exponen los objetivos de la tesis. Hasta ahora se desconoce la dinámica poblacional y la estructura genética de la especie invasora Aedes albopictus en España. Además, no se ha realizado ningún estudio de competencia vectorial de poblaciones españolas de Cx. pipiens y Ae. albopictus para arbovirus zoonóticos (VFNO y VFVR). El objetivo de esta tesis fue investigar estos dos puntos. La segunda parte está formada por tres capítulos. Cada uno es un estudio original, cuyo objetivo es responder a las preguntas propuestas en el segundo capítulo. En el primer estudio (Chapter 3) se analizó la estructura genética de Ae. albopictus en España usando distintas técnicas de análisis genético. En base a los resultados obtenidos se formularon dos hipótesis para explicar la introducción y la difusión de esta especie en el país. Además, se realizó un estudio sobre la dispersión global de Ae. albopictus. Los resultados confirman la importancia de las actividades humanas en la dispersión local y global de Ae. albopictus. El segundo y el tercer estudio (respectivamente, Chapter 4 y Chapter 5) se demostró que las especies Cx. pipiens y Ae. albopictus de España son vectores competentes para VFNO y VFVR. En ambos estudios se demostró también la importancia de usar temperaturas variables durante el periodo extrínseco de incubación. Los resultados indican que la utilización de temperaturas variables permite estimar con más precisión la competencia vectorial. Además, se utilizaron distintos métodos para la detección de partículas virales en la saliva de los mosquitos infectados. La tercera parte incluye Chapter 6- General discussion y Chapter 7- Conclusions. En esta parte, se discutieron todos los resultados, se propusieron futuros estudios y se enumeraron las conclusiones. Al final de cada capítulo, se han enumerados todas las referencias usadas.
Mosquito-borne diseases (MBDs) are an increasing global public health concern. The increase of international human activities and the global warming, have allowed the rapid spread and establishment of invasive mosquito species in new geographical areas. Moreover, the globalization also cause the introduction of exotic arboviruses in naïve areas where the mosquito vector is present, enhancing MBDs outbreaks. The outbreaks of MBDs, especially those caused by arbovirus like Dengue virus, Zika virus (ZIKV), Rift Valley fever virus (RVFV) or West Nile virus (WNV), have severe consequences for public and/or veterinary health. The economic losses linked to the zoonotic MBDs can severely compromise the economy of the affected country. This is particularly relevant in the developing countries, where diseases such as Rift Valley fever can provoke the death of a huge number of domestic and wild animals. For these reasons a deep knowledge of which mosquito species are present in a country and which of them are competent vectors of different pathogen agents is needed in order to set up appropriate surveillance programs and entomological control strategies. The present thesis is divided in three parts and each one is further divided in chapters. The first part consists of two sections: Chapter 1- General Introduction and Chapter 2- Objectives. The aim of this first part is to give a general overview on the importance of MBDs, the role of the vectors and the concept of vector competence. The descriptions of WNV and RVFV are also provided. Afterwards, the objectives of the thesis are exposed. So far, the population dynamics and the genetic structure of invasive Aedes albopictus, the Asian tiger mosquito, were unknown in Spain. Moreover, no studies on the vector competence of Spanish populations of Culex pipiens and Ae. albopictus for zoonotic arboviruses (WNV and RVFV) have been performed until now. The aim of the present thesis is to clarify all these points. The second part is formed by three chapters. Each of them is an original study aimed to answer the objectives. In the first study (Chapter 3), the genetic structure of Ae. albopictus in Spain has been analyzed with the use of different genetic tools. Based on the results obtained, two different hypotheses have been postulated to explain the introduction and the spread of this species across the country. Moreover, a global analysis about the worldwide spread of Ae. albopictus has been performed. The results of both analyses confirmed the importance of human activities for the local and global dispersion of Ae. albopictus. The second and third studies (Chapter 4 and Chapter 5, respectively), demonstrated how Spanish Cx. pipiens and Ae. albopictus are competent vectors for WNV and RVFV. In both studies the importance of using variable cycling temperature during the extrinsic period of incubation have been verified. The results suggested that variable cycling temperature allows to estimate a more realistic vector competence. Moreover, different approaches have been used for the detection of viral particles in the saliva of infected mosquitoes. The third includes Chapter 6- General discussion and Chapter 7- Conclusions. In this part all the findings are discussed, further studies are proposed and the major conclusions are detailed. At the end of each chapter all the references used are listed.

Molecular dating (or molecular clock) is a powerful technique that uses the mutation rate of biomolecules to estimate divergence times among organisms. In the last two decades, the theory behind the molecular clock has been intensively developed, and it is now possible to employ sophisticated evolutionary models on genome-scaled datasets in a Bayesian framework. The molecular clock has been successfully applied to virtually all types of organisms and molecules to estimate timing of speciation, timing of gene duplications, and generation times: this knowledge allows contextualizing past and present events in the light of (paleo)ecological scenarios. Molecular clock studies are routinely used in evolutionary and ecological studies, but their use in applied fields such as agricultural and medical entomology is still scarce in particular because of a paucity of genome data. Genome-scaled clocks have been successfully applied, for example, to various model organisms such as Anopheles and Drosophila, as well as to invasive mosquitoes Aedes aegypti and Aedes albopictus. Many other invasive pests are emerging worldwide aided by global trade, increased connectivity among countries, lack of prevention, and flawed invasive species management. Among them, there is Aedes koreicus and Aedes japonicus, two invasive mosquito species which are monitored for public health concerns because of their harboured human pathogenic viruses. For these, as well as for other insects of societal relevance, such as the parasitoid Trissolcus japonicus, there is a paucity of gene markers and no genome data for large scale molecular clock studies. Invasive pests are typically studied using microevolutionary approaches that tackle events at an intraspecific level: these approaches provide important information for the pest management, for example, by revealing invasion routes and insecticide resistances. Approaches that tackle the deep-time evolution of the pest, such as the molecular clock, are instead less used in pest science. Many important traits associated with invasiveness have evolved by speciation over a long time frame: the molecular clock can reveal the paleo-ecological conditions that favoured these traits helping a better understanding of pest biology. Molecular clock, when coupled with phylogenomics, can further identify genes and patterns that characterize the pest: this knowledge can be used to enhance management practices. Although this is a data-driven thesis, its major aim is to provide new results to demonstrate the utility of the molecular clock in pest science. This has been done by systematically apply the molecular clock to various neglected organisms of medical and agricultural relevance. To this aim, I generated new genome data and/or assembled the largest genome-scaled data to date. I studied the molecular clock in mosquitoes, focusing on the Aedini radiation (Chapter 2) and identified a strong incongruence between the mitochondrial and nuclear phylogeny for what concerns their molecular clock. This result highlighted the importance of employing genome scaled data for these species to exclude stochastic effects due to poor/inaccurate sampling in clock studies. To tackle the absence of data, I further assembled the whole mitogenome of emerging invasive species Aedes koreicus and Aedes japonicus with the aim of producing useful data for molecular typing and of inferring divergence estimates using whole mitogenomes (Chapter 3). Dated phylogenies point toward more recent diversification of Aedini and Culicini compared to estimates from previous works, addressing the issue of taxon sampling sensitivity in dated phylogeny. Although it is possible to perform molecular clock studies on single/few gene markers, the current trend is to couple this methodology with genome-scaled datasets to reduce the stochastic effect of using few genes. For this reason, I sequenced the draft genome of A. koreicus and A. japonicus (Chapter 4). The assemblies were extremely fragmented, highlighting the problem of sequencing large genomes using short reads. The assemblies provided, however enough information for genome skimming allowing extraction of BUSCO genes for downstream analyses, whole mitogenome assemblies (used in Chapter 3), and characterisation of the associated metagenome. These data need to be integrated by long reads; it provides, however a first framework to investigate the genome evolution of these species. I further sequenced and assembled the genome of Trissolcus japonicus, the parasitoid wasp of the invasive pest Halyomorpha halys. To elucidate its divergence, estimate and define an intraspecific typing system to differentiate strains for biocontrol strategies, I reconstructed the mitochondrial genomes of two populations: the mitogenomes were surprisingly identical, suggesting that they belong to the same de facto population. I further provide a detailed clock investigation of Zika, a virus harboured and transmitted by some Aedes species (Chapter 5). Using the largest set of genomes to date, I could set the origin of ZIKV in the middle age and its first diversification in the mid-19th century. From a methodological point of view, the clocking of this virus highlighted the importance of checking for recombination and for cell-passages to obtain correct divergence estimates. I finally show my contributions to molecular clock studies of three other invasive species (Chapter 6): I helped disentangle the divergence times of Bactrocera, a genus of invasive fruit files pest of agriculture; I contributed in performing a phylogenomics study of opsin genes in Diptera; I used chloroplast and nuclear genome data to reconstruct the divergences of the invasive reed Arundo. In the various Chapters of my thesis, I highlighted the limits and the problems of current molecular clock methodologies and identified the best practices for different types of organisms in order to develop a cross-discipline understanding of the molecular clock techniques. The various results presented in this thesis further demonstrate the utility of the molecular clock approach in pest studies.

Populations of Crassostrea virginica within Mosquito Lagoon, Florida have recently undergone significant die-offs, which are a subject of major concern. Restoration efforts within Mosquito Lagoon are focusing on reconstructing the three-dimensional reef habitats. Before effective protocols can be established, however, important questions about the sources of juvenile and adult oyster mortality must be answered. Potential causes of Crassostrea virginica mortality in the Indian River Lagoon system include sediment loads, competition, predation, and disease. My research focused on the interactions between oysters and the competitors that may affect the settlement, growth, and survival of Crassostrea virginica. The four objectives of my thesis research were to: 1) identify potential oyster competitors in Mosquito Lagoon, 2) determine if the sessile species recruiting to oyster shells have changed over time, 3) determine how the dominant competitors, barnacles, affect oyster settlement, growth and survival, and 4) determine if oyster or barnacle larvae are better able to settle in increased sediment and flow conditions that are associated with high levels of recreational boating. Lift nets were deployed within Mosquito Lagoon to determine available competing species. I collected species inventory data at six sites to determine the sessile invertebrate species (competitors) present on oyster reefs. Nets were deployed intertidally, just above mean low water, on living oyster reefs. One and a half liters of live and dead oysters were placed within the nets upon deployment. The nets were picked up monthly and surveyed for all fauna. Upon retrieval, all oysters within each net were brought back to the lab where all sessile organisms were immediately identified and returned to the lagoon. This survey began June 2004 and continued for one year. Shells from historic shell middens (up to 15,000 years old) were examined to determine if the sessile species settling on oyster reefs have changed over time. Similar species were found on both shells of historic and extant reefs. One notable exception was the appearance of Balanus amphitrite, an invasive barnacle, on the extant reefs. Balanus amphitrite is thought to have invaded Mosquito Lagoon approximately 100 years ago. This has resulted in a five fold increase in barnacle abundance per oyster shell. Balanus spp. were identified as important potential competitors and thus my research focused on spatial competition between C. virginica and native versus invasive barnacles of the area. Over 300 barnacles, including a native species, Balanus eburneus, and an invasive, Balanus amphitrite, have been counted on a single oyster shell. To determine how Balanus spp. affected settlement, growth, and survivorship of C. virginica, laboratory and field experiments were conducted in which densities of Balanus amphitrite and Balanus eburneus were manipulated. Density treatments included: no barnacles (control), low, medium, and high coverage of barnacles. Laboratory settlement trials with cultured oyster larvae were run in still water and flow (recirculating flume) using all barnacle density treatments. Additionally, all treatments with 7-day oyster spat were deployed in the field to follow oyster spat growth and survivorship. Settlement was counted by microscopy, and growth and survivorship were measured every 3 days for 4 weeks. Settlement of oysters was affected by barnacle presence only in flowing water. Still water trials showed no oyster preference related to any barnacle density or species. The presence of barnacles affected the growth and survivorship of oyster spat. However, there were no species specific differences. Studies suggest that recreational boating activities, especially boat wakes that cause sediment resuspension, may decrease recruitment and this may then provide an advantage to sessile competitors less affected by flow and sediment loads. To address these issues, replicated laboratory trials were run in a laboratory flume to quantify the effects of water motion (0, 5, 10 cm/s) and sediment loads (0, 8, 16 g/ml) on oyster recruitment and the recruitment of an important, relatively new competitor in the system, the barnacle Balanus amphitrite. If B. amphitrite settles in a wider variety of flow rates and sediment conditions, it may have a competitive advantage over the native oyster in this space-limited habitat. I found that high flow and sediment loads reduced larval settlement of C. virginica. Alternatively, settlement of cyprids of B. amphitrite did not differ among treatments. Thus, continuous boat traffic during settlement times should favor recruitment of the invasive barnacle Balanus amphitrite over the native oyster Crassostrea virginica. Determination of the competitive interactions of Crassostrea virginica in Mosquito Lagoon gives us important insights into the ecological conditions necessary for reestablishment of these oyster populations. Crassostrea virginica in Mosquito Lagoon was significantly impacted by barnacles; settlement, growth, and survivorship were all reduced by Balanus spp. This information will help resource managers in planning restoration techniques to minimize oyster and barnacle competitive interactions and increase Crassostrea virgininca success.
M.S.
Department of Biology
Arts and Sciences
Biology

A new strategy to fight mosquito-borne disease is based on infections of the maternally-transmitted, intracellular bacterium Wolbachia pipientis. Estimates predict that Wolbachia infects nearly half of all insect species, as well as other arthropods and some nematodes. Wolbachia manipulates the reproduction of its host to promote infection, most commonly causing a form of conditional sterility known as cytoplasmic incompatibility. Generally, Wolbachia infections are benign and do not inflict significant costs upon its host. However, studies demonstrate that some infections are associated with substantial costs to its host. These same infections can also induce pathogen interference and decrease vector competency of important disease vectors. Theory predicts that organisms that incur costs relative to conspecifics are less competitive and their competitive exclusion is expected. In the case of Wolbachia, the bacterium can influence reproduction such that phenotypes with lower fitness may still reach fixation in natural populations. In this dissertation, I describe theoretical and empirical experiments that aim to understand the invasion and stability of Wolbachia infections that impose costs on their host. Particular attention is paid to immature insect lifestages, which have been previously marginalized. These results are discussed in relation to ongoing vector control strategies that would use Wolbachia to manipulate vector populations. Specifically, I discuss the cost of novel Wolbachia infections in Aedespolynesiensis, which decreases larval survival and overall fitness relative to wild-type mosquitoes. Then, a theoretical framework was developed to determine the significance of reductions in larval viability in relation to the population replacement disease control strategy. Further theoretical studies determined that Wolbachia infections, once established, resist re-invasion by uninfected individuals despite relatively high costs associated with infection so long as the infection produces reproductive manipulations. Additional studies determined that larvae hatched from old eggs experience reduced survival in mosquito strains with novel Wolbachia infections when compared to the wild-type. To validate the theoretical studies, model predictions were tested empirically to determine the importance of the larval viability. Finally, a COPAS PLUS machine was evaluated and its role in understanding early larval development in mosquitoes is discussed. The importance of integrated research in disease control is highlighted.

Despite the recent establishment and spread of Aedes koreicus mosquitoes in Europe, its natural history and its potential public health impact remain poorly described. This thesis provides the first detailed insights into the biology of Aedes koreicus and its capacity to transmit arboviral diseases. Field work in Italy evaluated a variety of surveillance techniques for this species and its propensity to bite humans. A laboratory colony established in Australia was used to characterise its reproductive biology and its ability to transmit chikungunya virus. The findings help us understand the invasion risks and the public health threat posed by Aedes koreicus

Le moustique tigre, Aedes albopictus, originaire d’Asie du Sud-Est, a colonisé l’ensemble des continents excepté l’Antarctique depuis les dernières décennies. En revanche, les raisons du succès invasif des populations à l’échelle mondiale sont encore peu connues. Nous nous sommes concentrés sur l’invasion de l’Europe, et avons combiné différentes méthodes d’analyse et des données multi-sources afin de distinguer le rôle des processus historiques et contemporains, neutres et adaptatifs, dans la structuration de la variabilité génétique des populations invasives. L’analyse de la variabilité génétique de 1000 individus appartenant à 150 populations invasives et natives a révélé trois introductions indépendantes en Europe (en Albanie, au Nord de l’Italie, et au Centre de l’Italie), à partir des Etats-Unis (aire envahie) et de la Chine (aire native). Les populations initialement introduites ont constitué des centres de dispersion en Europe et les voies de migration corrèlent avec la géographie des transports humains. Différents évènements d’admixture au moment de l’introduction ou durant l’expansion subséquente, ainsi qu’une forte connectivité des populations, ont favorisé le maintien d’une forte diversité génétique. Des adaptations au froid préexistantes dans l’aire native de l’espèce et un fort conservatisme de niche entre les populations introduites et leurs sources suggèrent que les populations introduites étaient déjà pré-adaptées pour coloniser les environnements tempérés de l’Europe. Néanmoins, des changements de fréquences alléliques le long des gradients environnementaux en Europe suggèrent également une réponse adaptative après l’introduction. Le potentiel adaptatif des populations, ainsi que la dispersion longue distance assistée par l’homme, ont favorisé l’expansion rapide en Europe. Bien que souvent négligée dans le contexte des invasions biologiques, la dispersion naturelle semble également jouer un rôle dans l’expansion des populations à l’échelle du paysage. L’étude des caractéristiques démo-génétiques des populations invasives et des caractéristiques environnementales de l’aire envahie en Europe a permis d’identifier les processus favorisant deux étapes clés du processus d’invasion: l’établissement et l’expansion
The Asian tiger mosquito, Aedes albopictus, is native to Southeast Asia and has colonized all continents but Antarctica in the last decades. However, the factors determining the invasive success of populations at the global scale remain to be elucidated. Focusing on the European invasion, we used a comprehensive framework and multi-source data for distinguishing the role of historical and contemporary processes, both neutral and adaptive, in structuring the genetic variability of invasive populations. Examining the genetic variability of 1,000 individuals from 150 invasive and native populations revealed three independent introduction events in Europe (in Albania, North Italy, and Central Italy), from the United States (previously invaded area) and from China (native range). Primary introduced populations constituted dispersal centers for the colonization of Europe, and migration routes correlate with the geography of human transportation networks. Several admixture events either during introduction or subsequent expansion, as well as high connectivity between invasive populations, promoted the maintenance of high levels of genetic diversity. Pre-existing cold adaptation within the native range of the species and niche conservatism between introduced populations and their sources suggest that these populations were already prepared for establishing under temperate European climate. Nonetheless, shifts in allele frequencies along environmental gradients within Europe suggest post-introduction adaptive changes. The adaptive potential of populations and long-distance human-aided dispersal facilitated the rapid expansion of populations. Although often neglected in the context of biological invasions, natural dispersal at the landscape scale further contributed to range filling in range edge populations. The study of the demo-genetic and environmental characteristics of the European invasion allows a better understanding of processes at play during two key stages of the invasion process: establishment and expansion

Understanding the factors that make non-native species successful invaders is an important step towards mitigating spread. At the same time, species invasions can serve as natural experiments to test range-limit theory. Range-limit theory postulates declines in local abundance (abundant center model) and genetic diversity (central-peripheral hypothesis) towards range edges because of underlying environmental gradients. Such declines constrain adaptation to marginal habitats via gene swamping. However, broader evolutionary theory predicts intermediate rates of immigration into range-edge populations can relieve genetic drift and improve adaptive potential. I tested hypotheses generated from theory while illuminating aspects affecting of the invasion of the Asian tiger mosquito (Aedes albopictus Skuse) into the US. Using reciprocal distribution modeling, I found US populations occupied significantly different climate and habitat than in their native range (SE Asia). Most inconsistencies were found in the northern US range, where Ae. albopictus has recently crept northward, providing an opportunity to test range-limit theory as the range reaches its limit. Because of its limited natural dispersal ability, rapid spread after the 1985 US introduction pointed to human-aided dispersal. I tested the current role of human-aided versus natural dispersal using a landscape genetics framework, and found that natural dispersal dominated current patterns. Some distant localities were highly genetically similar, indicating potential human-aided transport in limited cases. Asymmetric gene flow from core to edge localities supported the abundant center model, but uniformly high genetic diversity contrasted with the central-marginal hypothesis. I detected a significant signature of local adaptation by overwintering diapause-induced eggs in multiple field sites using reciprocal transplants. Surprisingly, most genotypes from throughout the range produced large offspring when overwintered at the range edge. Relative offspring mass between home and away winters peaked at an intermediate immigration rate. These results show that rapid adaptation has occurred in US populations of Ae. albopictus and highlight the potential for further spread. Genetic admixture from multiple introductions may explain high genetic diversity throughout the US range and contribute to high offspring size for all genotypes overwintered at the range edge. Finally, my work highlights the need for a better understanding of contemporary ecological and evolutionary processes leading to range-limits (or expansion) to more accurately reflect processes occurring in a human-dominated world.
Ph.D.
Doctorate
Biology
Sciences
Conservation Biology; Ecology and Organismal Biology

Actualment una de les principals amenaces a la biodiversitat és la introducció d'espècies. Revisant 26 variables de les 69 espècies de peixos continental de la Península Ibèrica concloem que la filogènia, variabilitat i els usos de l'home són necessaris per entendre millor les diferències entres les espècies natives i invasores.
Entre les especies més afectades per la introducció de peixos es troben els ciprinodontiformes endèmics del Mediterrani. Aportem les primers dades sobre l'ús d'hàbitats ocasionalment inundats i la selecció de preses del fartet (Aphanius iberus), observant un canvi ontogenètic, clarament relacionat amb el microhàbitat.
També demostrem que la salinitat influeix en l'èxit invasor de la gamúsia, afectant la seva densitat i biologia reproductiva. Per altra banda, demostrem experimentalment que amb l'increment de salinitat la gambúsia disminueix la seva agressivitat i captura menys preses, reduint la seva eficàcia competitiva respecte dels ciprinodonts natius.
One of the main current threats to biodiversity is the introduction of invasive species. By reviewing 26 life-history and ecological variables of the 69 inland fish species of the Iberian Peninsula we conclude that phylogeny, variability and human use are needed to a better understanding of the differences between native and invasive species.
Among the most threatened species by invasive fish are the Mediterranean endemic cyprinodontiform fish. We report the first data on the use of occasionally-inundated habitats ad prey electivity by the Spanish toothcarp (Aphanius iberus). We observed an ontogenetic diet shift clearly linked to a microhabitat change.
We also demonstrate that salinity limits the invasive success of mosquitofish (Gambusia holbrooki), affecting density and life history traits. ON the ohter hand, we experimentally demonstrate that mosquitofish decreases its aggresive behavior and capture less prey, reducing its competitive efficiency with salinity increases regarding to native ciprinodonts.

Biological invasions are a side effect of globalisation and human modification of natural systems. Invasive mosquitoes and the pathogens that they carry are leading causes of human suffering. Mosquito vectors of pathogens are often ecologically plastic and able to exploit human movements to disperse. As such they spread across wide geographical areas and are among the most successful species to have become invasive. Mosquito-borne pathogens have complex life cycles which involve a rich set of reservoir and vector hosts. They are evolutionarily very dynamic, ready to take advantage of new opportunities offered by anthropogenic environmental changes. Many pathogens associated with mosquitoes are indeed changing their ecology or modifying their geographical and biological distribution, being considered as invasive species. Despite this impressive plasticity, invasive mosquitoes and mosquito-borne pathogens are still part of a natural system, and as such are limited by environmental conditions. The main target of my research was to apply ecological theory and environmental modelling to gain knowledge on these limiting conditions, by establishing mechanistic and correlative associations between the environment, invasive vectors and pathogen demographics, as well as physiological and spatial dynamics. Understanding these mechanisms is key to design effective health policies which aim to mitigate the emergence of new pathogens, mainly by hindering the population that vectors them. The core of this thesis is formed by a series of stand-alone scientific papers, published, submitted or close to submission to peer-reviewed journals; the core chapters are backed by the General Introduction, and the General Summary and Outlook.

Background: Mosquito-borne diseases are a growing concern for temperate regions including the northeastern US. There the two primary mosquito vectors, Cx. pipiens and Ae. albopictus are widespread, endemic circulation of West Nile virus causes sporadic outbreaks, and imported arboviruses such as dengue, chikungunya, and Zika are on the rise. With temperate mosquito-borne disease outbreaks likely to increase in frequency, it is critical to reduce mosquito populations in the northeastern US. Community-based source reduction is heralded as the most sustainable component of integrated mosquito management. Yet mosquitoes develop rapidly, requiring weekly maintenance of mosquito habitat. This is onerous and community commitment flags. The development of predictive models to inform focused vector-control efforts is therefore of great utility. Objectives and Methods: The overarching objective of this research is to make robust predictive modeling frameworks based on empirically derived relationships of the ecology and epidemiology of mosquito-borne disease systems in the northeastern US. We aim to quantify the relationships between local environmental and meteorological conditions and mosquito vectors. In Chapters 2 and 4 we use lengthy surveillance records to develop models and use model ensembles to generate predictions based on out-of-sample data. For chapter 3 we use more spatially refined data to investigate the influence of intra-urban heterogeneities and how climatic conditions influence mosquito populations across these defined differences. Results: In Chapter 2, we model and forecast WNV infection rates among mosquito vectors using meteorological and hydrological conditions. We show that real-time climate information can predict WNV Culex infection rates prior to when human risk is greatest. In Chapter 3, we link infrastructure degradation and vegetation patterns with Ae. albopictus infestation levels as well as the interactive effect of precipitation across these environmental conditions. In Chapter 4, we identify key land use characteristics and meteorological conditions associated with annual Ae. albopictus abundance. Further we use imported chikungunya cases to delineate areas of high arboviral importation and, in combination with areas of high Ae. albopictus abundance, areas at heightened risk for arboviral transmission. Conclusions: While temperate outbreaks are often self-limiting they may be increasing in frequency and severity. Due to the multitude of invasive vectors and arboviruses, vector control techniques that work for multiple mosquito species are likely more effective and sustainable. Here we build build empirical models that accurately predict mosquito dynamics before populations peak which is critical for vector control. We recommend integrating predictive modeling into mosquito management guidelines as this could focus valuable resources to when and where mosquito-borne transmission risk is greatest. Further we find social and ecological determinants of mosquito dynamics, supporting further study that combine socio-ecological processes into model frameworks.

碩士
國立中興大學
昆蟲學系
90
The pathogenicity of entomopathogenic nematodes, Steinernema abbasi and Steinernema carpocapsae, to Aedes albopictus larvae and pupae showed that both S. abbasi and S. carpocapsae were pathogenic to 3rd and 4th instar larvae, there was no any pathogenic to 1st, 2nd instar larvae and pupae. The pathogenicity of S. carpocapsae was higher than that of pathogenicity in the experiment which there was only a larva in the container. However, the pathogenicity of S. abbasi was higher than that of S. carpocapsae in the experiment which there were thirty larvae in the container. The different combinations of nematodes and mosquitoes in inoculation caused the different mosquito mortalities; the more larvae inoculated in one container, the lower mortality was caused. The invasion route of S. abbasi into the hemocoel of 4th instar larvae of Ae. albopictus was found via the mouth and gastric caecum mainly. In pupa of Ae. albopictus, S. abbasi was able to penetrate into it through the spiracle or intersegmental membrane, although this nematode lacks mouth or penetrative hook. Encapsulation was induced by both S. abbasi and S. carpocapsae in the hemocoel of Ae. albopictus larva. The encapsulation capacity of Ae. albopictus larva was maximal 31 menalized-capsules per larva in S. abbasi and 15 in S. carpocapsae. Both S. abbasi and S. carpocapsae were able to kill the mosquito larvae by only one nematode invaded, in this context, the percentage of dead mosquito larvae was 81% as invaded by S. abbasi and 94% by S. carpocapsae. Ultrastructural examination of encapsulation of S. abbasi by Ae. albopictus larva was observed using transmission electron microscopy (TEM). The humoral encapsulation was primarily formed by electron dense homogenous structure without cellular material, which formed an inner layer of capsule enclosing S. abbasi 10 min after inoculation. About 1 hr after inoculation, the humoral encapsulaton was formed completely when plasmatocyte started to attach onto the surface of humoral capsule, which formed the outer layer of cellular capsule. Encapsulation process was terminated at 24-48 hr after IJs inoculation by formation of the basement membrane-like structure around the whole capsule.

Introdução: Compreender a interação entre Plasmodium e o mosquito Anopheles é crucial para o desenvolvimento de estratégias de controlo e eliminação da malária em áreas endêmicas. A invasão das glândulas salivares do mosquito vetor é uma das etapas mais importantes para a transmissão da malária. Compreender como Plasmodium interage com esse órgão é fundamental para o controle e eliminação da malária. Muitos glicanos estão implicados na interação célula-célula e podem desempenhar um papel central na interação do parasita com o hospedeiro. O glicano Galα1-3Galβ1-4GlcNAc-R ou simplesmente α-Gal é um antígeno importante para o sistema imunológico humano e está presente no Plasmodium. Objetivos: Neste trabalho pretendemos compreender o papel da α-Gal durante a invasão das glândulas salivares de Anopheles spp. e estabelecer a origem da α-Gal presente no Plasmodium. Métodos: Para deteção da α-Gal usou-se a técnica de microscopia de imunofluorescência em esporozoítos de Plasmodium bergei ANKA-GFP (SPZs), oocineto e oócitos, fixados com paraformaldeído a 4% e incubados com o mAbs anti-α-Gal_IgG2b e o secundários IgG anti-rato conjugado com Alexa fluor- 647, com mAbs anti-CSP conjugado com Alexa fluor-647 ou com a Isolectina BSI-IB4 conjugado com o fluoróforo Alexa fluor-647. O antígeno α-Gal foi detectado no intestino médio (MGs) e nas glândulas salivares (SGs) dos mosquitos por microscopia de imunofluorescência e Western Blots. Esporozoítos (SPZs) expressando α-Gal, coletados do MGs (8º aos 23º dias pós-infeção), hemolinfa (14º ao 23º dpi) e SGs (14º ao 23º dpi) de An. Stephensi, foram detetados e quantificados por citometria de fluxo. A expressão do gene UDP-GalT em SPZs obtidos a partir de MGs e SGs, de diferentes dias pós-infeção, foi quantificada por RT-qPCR e comparado usando o método 2-ΔΔct. O papel de α-Gal na interação Plasmodium e as glândulas salivares foi investigado. Os genes do UDP-GalT e das lecitinas do tipo C (CTL) do mosquito foram silenciados usando o método RNA de interferência. RNA(s) de fita dupla específico foi injetado em mosquitos com 8º dia após-infeção. O silenciamento dos genes foi confirmado por RT-qPCR e no 18º dpi as SGs e as MGs foram dissecados e os SPZs quantificados usando o hemocitómetro. O número de SPZs foi comparado com os grupos controle injetados com dsRNA não relacionado. A presença de α-Gal no ensaio de gliding dos SPZs foi investigado usando mAbs anti-α-Gal conjugados com Alexa fluor-647. Além disso, o efeito de mAbs anti-α-Gal e Leciona BSI-IB4 no movimento de glinding dos SPZs foi testado usando microscópica. Resultados: A presença de α-Gal foi detetada por microscopia de imunofluorescência no intestino médio e nas glândulas salivares do mosquito e em esporozoítos e oocinetes de Plasmodium. A percentagem de SPZs que expressaram α-Gal em citometria de fluxo em diferentes dias pós-infeção, não apresentaram diferença significativa entre esses, mas quando SPZs de diferentes origens (SGs, HL e MGs) foram comparados, os SPZs das SGs apresentaram expressão significativamente mais elevada de α-Gal. O número de SPZs de SGs foi reduzido quando o gene UDP-GalT dos mosquitos foi silenciado, enquanto nenhuma diferença foi observada no número de SPZs das MGs, mas uma percentagem reduzida de SPZs expressando α-Gal e menor intensidade de α-Gal foi observada nas provenientes das MGs, mas não nas SG. Dos três genes alvo da lectina do tipo C (CTL)-galactose silenciados com dsRNA, apenas um foi silenciado com sucesso. O silenciamento do gene de ligação à lectina do tipo C (CTL) -galactose resultou em uma redução significativa da invasão de SGs pelos Plasmódios quando comparado ao controlo. No entanto, um incremento na expressão de α-Gal foi observado nesses SPZs. α-Gal não pôde ser detetado no ensaio de glinding dos SPZs e nenhum efeito no movimento dos SPZs foi observado quando encubados com mAbs anti-α-Gal ou lectina BSI-IB4. Conclusão: O antígeno α-Gal está presente em todo o ciclo esporogónio do Plasmodium, apresentando uma origem não exclusiva dos mosquitos. Os dados sugerem que α-Gal está envolvida na invasão da glândula salivar dos mosquitos, possível por meio da interação do recetor, mas não por processo de glinding.
Introduction: Understanding Plasmodium and mosquito vector interactions is crucial to develop strategies for control and malaria elimination in endemic areas. Mosquito’s salivary glands invasion is one of the most important steps for malaria transmission. Understanding how the Plasmodium parasite interacts with this organ is fundamental to achieve malaria elimination. Many glycans are implicated in cell-to-cell interactions and could play a central role in parasite-host interactions. The Galα1-3Galβ1-4GlcNAc-R glycan or simply α-Gal epitope is a major antigen for the human immunologic system and its present on Plasmodium, the malaria parasite. Aims: In this work, we aimed to understand the role of α-Gal during Anopheles spp. salivary glands invasion by Plasmodium and to establish the origin of Plasmodium α-Gal. Methods: To detect α-Gal immunofluorescence was used, Plasmodium bergei ANKA-GFP sporozoites (SPZs), ookinetes, and oocytes were fixed with 4% paraformaldehyde and stained with the anti-α-Gal_IgG2b with secondary anti-mouse IgG conjugated with Alexa fluor-647, anti-CSP mAbs conjugated with Alexa fluor-647, or with Isolectin BSI-IB4 conjugated with Alexa fluor-647. The α-Gal antigen was detected on mosquitoes’ midgut (MG) and salivary glands (SGs) by immunofluorescence microscopy and western blots. SPZs expressing α-Gal from infected An. stephensi collected from MG (8th to the 23rd days post-infection), hemolymph (14th to the 23rd dpi), and SGs (14th to the 23rddpi) were detected and quantified using flow cytometry. The Plasmodium UDP-Gal transporter gene expression in SPZs from the MGs and SGs was accessed by RT-qPCR and the 2-ΔΔct at different days post-infection. The role of α-Gal on the Plasmodium mosquito interactions was investigated. Mosquitoes` UDP-Gal transporter and C-Type lectins (CTL)-galactose-binding genes were targets using RNA interference. The specific double-stranded RNA was injected on the 8th day post-infection. Gene silence was confirmed using RT-qPCR and at the 18th dpi, SGs and MGs were dissected and SPZs quantified by haemocytometer. The number of SPZs were compared with the control groups injected with unrelated dsRNA. The presence of α-Gal on SPZ gliding trial was investigated using anti-α-Gal mAbs conjugated with Alexa fluor-647. Additionally, the effect of anti-α-Gal mAbs and Isolectin BSI-IB4 on SPZs gliding movement was tested using live microscopic images. Results: The presence of α-Gal was detected by immunofluorescence microscopy in the mosquito’s midguts and salivary glands and on Plasmodium sporozoites and ookinetes. The percentage of SPZs expressing α-Gal, by flow cytometry at different days post-infection showed a non-significant difference between them, but when SPZ from different organs (SG, HL, and MG) were compared, the SPZs from SGs presented significantly higher expression of α-Gal. The number of SPZs from SGs was reduced when mosquitoes UDP-Gal transporter gene was silenced while no differences were observed on the number SPZs from MG, but a reduced percentage of SPZs expressing α-Gal and lower α-Gal intensity was observed in MG but not SG. From the three C-Types lectins (CTL)-galactose-binding gene target with dsRNA, only one was successfully silenced. The silencing of the C-Types lectin (CTL)-galactose-binding gene resulted in a significant reduction of SGs invasion when compared with the control. However, an increment in α-Gal expression was observed on those SPZs. α-Gal could not be detected on SPZs gliding trial and non-effect on SPZs movement were observed when SPZs were incubated with anti-α-Gal mAbs or Isolectin BSI-IB4. Conclusion: The α-Gal antigen is present throughout the Plasmodium sporogonic cycle, presenting a non-mosquito exclusive origin. The data suggest that α-Gal is involved in mosquito salivary gland invasion possible via receptor interaction but not via gliding.