Phytophthora infestans enzymes and their role in the interaction with its hosts

Pathogens use a huge arsenal of proteins and metabolites in order to infect plants and establish disease. They function in facilitating pathogen entry or suppressing plant defence triggered upon pathogen attack. The proteins that pathogens employ to infect the host include enzymes such as cell wall degrading enzymes, phospholipid modifying enzymes and proteases, but also effectors that target and disable the host cell machinery. To facilitate disease these proteins function at several levels. First of all inside the pathogen, for example in processing or maturation of pathogenicity or virulence factors. Secondly in the apoplast, for example in cell wall degradation or receptor modification. And lastly, inside the host cell where they can have a role in a variety of processes such as disrupting vesicle transport, inhibiting transcription factors or deregulating the hormone balance. The oomycete Phytophthora infestans is the causal agent of late blight, the most important disease in potato crops worldwide. Since its appearance in Europe in the mid-nineteenth century, when it destroyed the potato crop all over Western Europe and gave rise to the Irish potato famine, this pathogen has been studied extensively. Since the early 1990’s, when the first molecular tools became available to study oomycete plant pathogens, attempts have been made to identify genes in P. infestans that play a role in pathogenicity or virulence and to unravel in depth the molecular and cellular mechanisms underlying pathogenicity. Despite rapid progress and the information provided by Phytophthora genome sequencing projects, these mechanisms are still poorly understood. The research described in this thesis deals with three different groups of enzymes in P. infestans that are anticipated to have a role in host-pathogen interactions and have not been studied previously. In Chapter 1 we first introduce potato and potato late blight and give a glimpse of the history of the disease and its societal impact. We then describe the life cycle of P. infestans and the molecular toolbox that is currently available to study this pathogen and the interaction with its host. Subsequently, we address the current practices of late blight control with emphasis on resistance genes and the ability of the pathogen to rapidly escape recognition. This is followed by an overview of enzymes and enzyme inhibitors that have been shown to play a role in virulence in a variety of pathogens and finally we present the scope of this thesis. In Chapter 2 we present an inventory of metalloproteases (MPs) in P. infestans. MPs are a very diverse group of proteases that function by virtue of a divalent metal cation positioned at their catalytic site. Several MPs have been shown to be involved in the pathogenicity of mammalian and plant pathogens. Thorough genome mining and gene model corrections based on RNA-Seq data, revealed 99 MPs, divided over 20 families. Searches for homologs in other oomycetes and Stramenopiles species showed that some MPs are expanded in Phytophthora species. Analyses of the domain compositions of MPs revealed a few MPs with a novel domain architecture exclusively found in Phytophthora species. Gene expression analyses showed that several MPs are highly expressed in infectious propagules. To our knowledge, this is the first systematic inventory of MPs in an oomycete. Based on these results MP genes can be selected as candidates for future functional studies in P. infestans and other oomycetes. Chapters 3 and 4 deal with aspartic proteases (APs). In other pathogens APs were found to be involved in effector modification and virulence. The Plasmodium falciparum AP PlasmepsinV (PMV) has been shown to modify PEXEL effectors of Plasmodium prior to their translocation into the host cell. Of the twelve P. infestans APs three are highly homologous to PMV Due to this homology as well as the homology between the host translocation motifs in Plasmodium PEXEL effectors and Phytophthora RXLR effectors, it has been suggested that these three APs, PiAP10, PiAP11 and PiAP12, play a similar role in effector modification as PMV. In order to test their involvement in virulence we generated P. infestans transformants in which the PiAPs genes are silenced or overexpressed (Chapter 3). PiAP11 transformants showed no obvious changes in virulence but transformants silenced for PiAP10 and PiAP12, showed reduced infection efficiency and smaller lesions upon inoculation on potato leaves. Overexpression of these two genes also resulted in transformants with compromised virulence, while in several cases, they triggered cell death upon inoculation. A small reduction in colony growth and sporulation of the transformants was also observed. In-gel zymography assays with gelatin showed that all PiAPs have enzymatic activity that was inhibited by pepstatin. Moreover, PiAP10 and PiAP12 showed proteolytic activity against the RXLR effector AVR4 with its authentic RXLR motif, but not against AVR4 with a mutated RXLR motif showing that the modification only occurs when the RXLR motif is intact. PiAP11 had no proteolytic activity against AVR4 effector, suggesting a different function for the particular PiAP. These results show that PiAP10 and PiAP12 are involved in virulence and suggest their involvement in effector modification. One of the P. infestans APs named PiAP5, is an AP with a unique domain architecture. It has a G-protein coupled receptor (GPCR) domain adjacent to the AP domain and its topology suggests that PiAP5, with its seven transmembrane spanning regions, is integrated in the membrane with the N-terminal AP domain as extracellular domain. Such an AP-GPCR is exclusively found in oomycetes. In Chapter 4 we describe that alteration of PiAP5 expression in P. infestans strongly reduced growth and sporulation and resulted in malformed germinating sporangia, especially in P. infestans transformants overexpressing the gene. Protease activity assays with general substrates and AVR4 did not show any activity of PiAP5 as a protease. These results show that PiAP5 is involved in fitness and sporulation of the pathogen, and consequently affects its virulence. Whether or not PiAP5 is active as a protease is unknown. Chapter 5 focuses on the function of a subclass of phospholipase D’s (PLDs). PLDs are enzymes that are involved in the hydrolysis of phospholipids, the main structural components of cell membranes, and in the production of the second messenger phosphatidic acid (PA). Three small PLD genes were selected for functional analysis by means of transient expression in Nicotiana benthamiana leaves The presence of PLD-like-1, sPLD-like-1 or sPLD-like-12 in the leaves gave rise to calcium-dependent cell death and, when the leaves were inoculated with P. infestans it enhanced lesion growth. Mutations in the catalytic HKD motifs of the PLDs or removal of the signal peptide strongly reduced the cell death responses and abolished the virulence promotion demonstrating that the enzymatic activity of the PLDs is the major determinant and that the PLDs likely function outside the cell. These results show that that PLD-likes play a role in virulence, either by modifying the host membranes or through PA signalling. To gain more insight in the detailed mechanisms underlying Phytophthora-plant interactions, it is desirable to have experimental systems that enable high quality data generation. The establishment of model systems that fit that purpose, yet resembling the natural infection process are necessary. In Chapter 6 we describe the development of a new infection system using the tomato cell line MsK8 as host for Phytophthora pathogens. The infection system was optimized by studying the interaction of MsK8 cells with several Phytophthora species over time. The experiments included infection assays, microscopy, gene expression profiling and ROS production measurements. The results show that the MsK8 infection system offers a versatile platform that can be used in studies ranging from analysing a single gene, testing chemical compounds, to large -omics studies. Chapter 7 addresses the main findings of this thesis, and puts them into a broader perspective. The function and potential involvement of MPs, APs and PLDs in fitness or virulence of P. infestans are discussed, as well as the possible effects of the enzymatic activity in relation to virulence. Overall, this thesis highlights the importance of enzymes in growth and virulence of P. infestans and gives insights in three different types of enzymes. The potential roles of these enzymes in Phytophthora-host interactions could serve as food for thought for further studies.</p

Cryptic reservoirs of micro-eukaryotic parasites in ecologically relevant intertidal invertebrates from temperate coastal ecosystems unveiled by a combined histopathological, ultrastructural, and molecular approach

271 p.La mayoría de los eucariotas son organismos unicelulares (protistas), muchos de ellos pertenecientes a linajes que divergieron temprano en la historia evolutiva de este Dominio de organismos nucleados. Microscópicos, enormemente diversos y fenotípicamente convergentes, su clasificación cladística ha sido históricamente compleja, dejando atrás un extenso registro de taxones y de términos parafiléticos y polifiléticos. Teniendo que investigar atributos estructurales, celulares, biológicos y ecológicos en un mundo de rápidas interacciones y difícilmente accesible a simple vista, la protistología es particularmente dependiente de la sistemática. Ésta permite inferir rasgos de especies crípticas a partir de especies evolutivamente relacionadas.Las moléculas de ADN (y ARN), representan un "registro" preciso de estos eventos de diversificación, que preceden incluso a los más antiguos registros fósiles. En los últimos años, la maduración de los métodos de filogenia molecular, catalizados por una mayor accesibilidad a la secuenciación de próxima generación (NGS), está permitiendo resolver preguntas e hipótesis sobre la evolución y la especiación de estos organismos micro-eucariotas que no se habían podido responder mediante otros métodos. Por una parte, arboles filogenéticos construidos mediante concatenaciones de cientos, incluso miles de genes, están permitiendo rastrear la historia evolutiva de los linajes protistas hasta el último ancestro común de todos los eucariotas (LECA). Concomitantemente, análisis moleculares basados en genes e incluso fragmentos cortos (especialmente 18S rRNA), recuperados principalmente de matrices ambientales u orgánicas (eDNA o ADN ambiental), están revelando una ¿caja de Pandora¿ de diversidad micro-eucariota. Ésta diversidad ¿oculta¿ está transformando nuestra percepción de los protistas en la cadena trófica y la estructura ecológica. En el medio marino, sus papeles como autótrofos, heterótrofos (predadores, saprófitos, parásitos) o mixótrofos crece en importancia día a día. El aumento simultáneo de diversidad e importancia ha sido particularmente pronunciado entre los linajes de parásitos protistas, que adaptados a la vida dentro de un huésped son más inaccesibles y morfológicamente indistinguibles que sus homólogos de vida libre. Muy competitivo como estilo de vida, el parasitismo ha evolucionado de forma independiente varias veces en prácticamente todos los grupos eucariotas, en algunos incluso cientos de veces. De hecho, es posible que el efecto parapátrico que implica una existencia endosimbiótica, haya exacerbado la especiación entre los parásitos, que representan la que posiblemente sea la más común estrategia de consumo entre los organismos vivos. Es más, el número de especies crípticas que están, a día de hoy, siendo descubiertas en la mayoría de los linajes de parásitos protistas sigue aumentando abruptamente o apenas comienza a mostrar una desaceleración. Cabe destacar, que el descubrimiento de esta diversidad oculta, incluidas las especies crípticas, va más allá de la escalada en el número de especies; afecta los estudios sobre biología celular, ciclos biológicos, y ecología. Inexorablemente, esta fuerza mostrada por los métodos de análisis y secuenciación del ADN está abriendo una brecha entre la diversidad genética existente y nuestra comprensión de la morfología, patología, transmisión y posibles hospedadores de los parásitos protistas que la constituyen. Este desequilibrio es particularmente evidente entre los parásitos que infectan linajes de invertebrados, los cuales, salvo algunos taxones con interés comercial, permanecen en gran parte sin analizar, a pesar de constituir un grupo mucho más diverso que los vertebrados. Por una parte, es lógico que los parásitos protistas causantes de infecciones en especies marinas de interés comercial (peces, bivalvos, crustáceos¿) hayan sido priorizadas, pero hay que tener en cuenta que muchos de estos micro-eucariotas tienen ciclos de vida complejos, en los que pequeños invertebrados actúan muchas veces como vectores o reservorios. Descubrir y contextualizar estas asociaciones puede ser determinante a la hora de comprender cuándo y dónde puede variar la presión y capacidad infectiva de algunas de estas infecciones en la comunidad o huéspedes específicos. Al mismo tiempo que su diversidad e importancia aumenta, la inclusión progresiva de parásitos en modelos ecológicos está experimentando variaciones de gran alcance en la dinámica poblacional de las especies animales, vegetales o fúngicas en los ecosistemas. En consecuencia, las asociaciones entre parásitos y hospedadores se investigan cada vez más como una parte importante de la estructura de la comunidad, y no exclusivamente como una "molestia" para el ser humano y sus intereses. Por desgracia la inclusión de parásitos en modelos ecológicos está siendo lastrada por un profundo desconocimiento de estas interacciones. A diferencia de los organismos multicelulares, que han podido ser observados por científicos y aficionados durante siglos, la distribución espaciotemporal de la mayoría de los organismos unicelulares sigue siendo un profundo misterio. No obstante, dadas sus importantes funciones como vectores, huéspedes intermediarios y reservorios, una comprensión mucho más profunda del patobioma (patógenos asociados a un hospedador) y su variabilidad espacio-temporal es de suma importancia para un mayor poder de predicción de los factores de presión causantes de epidemias o zoonosis en el huésped, la población y el medioambiente.En este contexto, la hipótesis de este estudio plantea que especies de invertebrados comunes en la zona inter-mareal de ecosistemas costeros en climas templados son reservorios crípticos de un número significativo de parásitos micro-eucariotas (protistas) de interés para el medio y los recursos marinos. Eldescubrimiento progresivo de estas asociaciones ocultas de parásitos-huéspedes (mediante exámenes combinando técnicas histopatológicas, ultraestructurales y moleculares) permite una mejor comprensión de la morfología, patología, biología celular y ciclo de vida de dichos patógenos, lo que a su vez consiente un seguimiento más estrecho de los factores y presiones que promueven epidemias y zoonosis en una escala espacio-temporal.PIE:Plentziako Itsas Estazio

Quantitative proteomic responses of macrophages to Leishmania mexicana infection

The dynamics of protein turnover is central to the regulation of protein expression. The steady-state level of a protein is the net outcome of the change in its rate of synthesis and degradation. Different biological states or perturbations cause changes in the expression of specific proteins, which can be assessed by proteomic analysis to reveal links between genotype and phenotype. Unlike other conventional proteomic methods, which measure the amount of proteins in the system at a specific point in time, pulse-chase stable isotope labelling by amino acids in cell culture (pcSILAC) can reveal changes in the rates of protein synthesis and degradation over time. The causative agent of Leishmaniasis, Leishmania, has a digenetic lifestyle involving an extracellular flagellated promastigote living in the mid-gut of the sand fly vector and an aflagellated intracellular amastigote residing in the macrophage of the mammalian host. As they live in a different niche their protein expression could give insight into their adaptation and survival. The intricate interaction between the human host and the Leishmania parasite is key to pathology and may present new targets for chemotherapeutic development. Employing high-resolution mass spectrometry coupled with pulse-chase SILAC technique, we delved into the investigation of proteome changes in L. mexicana-infected macrophages. The first part of the thesis discusses the quantitative proteomic analysis of L. mexicana promastigote and amastigote stage. In this work, stable isotope dimethyl labelling was employed to differentially labelled promastigotes and axenic amastigotes. Our results revealed transformation from promastigote to amastigote were accompanied by: i) reduced glycolytic and gluconeogenesis pathway, ii) increased fatty acid oxidation, iii) increased mitochondrial respiration, iv) reduced expression of proteins that may have flagellar role (e.g. flagellar connector protein, flagellum targeting protein KHARON1), v) reduced stress response proteins, vi) increased protein synthesis, and vii) increased proteolytic proteins. The findings reported here substantially advance our knowledge on the differences of protein expression in different life cycle stage of L. mexicana and could be useful in finding drug targets. Another part of the thesis discusses the establishment and application of pulse-chase SILAC. In this work, a human macrophage-like cell line (THP-1) was grown in media containing L-Arg-13C6 and L-Lys-13C6 until isotope incorporation of >98% was achieved. Media was then replaced with light arginine and lysine so that light amino acids were pulsed into cells for 24 and 48 hours. In other words, protein synthesis is ‘chased’ with unlabelled amino acids. Synchronous with the switch from pulse to chase, the macrophages were infected with L. mexicana. This approach provides the ability to monitor the rates of heavy-label loss, hence determining protein degradation rates and half-lives. At 24-hour post-infection, when compared to mock-infected cells, 2016 proteins were identified, 761 were quantified, and 51 were significantly modulated at p-value < 0.05. Interestingly, proteins involved in glycolysis were markedly downregulated in synthesis after infection while oxidative phosphorylation and fatty acid β-oxidation had increased synthesis, suggesting a subversion of host cell metabolism by Leishmania which proposed to play a key role in microbial growth and persistence. Additionally, pro-apoptotic proteins such as apoptosis regulator BAX and caspase 3 had increased translation in cells infected for 24 hour. This was accompanied by the overexpression of STAT1 which could result in modulation of apoptotic pathways. These characteristics advocate that THP-1 cells most likely exhibit an M2 macrophage phenotype following 24-hour infection. Temporal proteomic data revealed some striking changes in metabolisms of the host at 24 and 48-hour post-infection. After 48 hours of infection, 2104 proteins were identified, and 84 were significantly modulated post-infection at p-value < 0.05. After 48 hours of infection, relative to levels at 24 hours of infection, host cells increased the synthesis of glycolytic enzymes and reduced oxidative phosphorylation synthesis. Further, a total of 400 newly synthesized proteins were selected based on stringent criteria to measure synthesis rates, degradation rate constant (kdeg) and half-lives. These include several ribosomal proteins, pyruvate kinase, L-lactate dehydrogenase, moesin, several glycolytic enzymes such as glucose-6-phosphate isomerase and alpha enolase, gelsolin, galectin-9, catalase and lamin-B. We found that globally kdeg values in THP-1 were low ranging from 0.01 to 0.04 h-1. Our degradation data indicated that proteins involved in mitochondrial related functions (TCA, oxidative phosphorylation) as well as other energy production pathways were more stable and have longer half-lives. For the 400 proteins, the mean half-life for uninfected 24 h, 24 hpi, uninfected 48h and 48 hpi were 21.74 h, 20.51 h, 47.39 h and 47.33 h, respectively. Intriguingly, newly synthesized proteins involved in immune responses, including HLA complexes, were rapidly degraded in infected cells, despite having decreased synthesis rates after 48 hours of infection. Collectively, most proteins in the present study had decreased kdeg and longer half-lives following longer exposure of THP-1 to L. mexicana. Our data show the potential of pulse-chase SILAC to dissect the response of macrophages to Leishmania infection. To our knowledge, no studies have reported the proteome turnover of macrophage in response to Leishmania infection

Analyse des Proteoms von Trypanosoma brucei unter besonderer Berücksichtigung des Stoffwechsels von Prostaglandinen

African trypanosomiasis is caused by extra cellular parasitic protozoa that can be transmitted by the bite of a tsetse fly. The disease is caused by sub-species of Trypanosoma brucei, an extra-cellular eukaryotic flagellated parasite. There is no prophylactic chemotherapy or prospect of a vaccine and current treatment is inadequate. Drugs for late-stage disease are highly toxic. Livestock trypanosomiasis is caused by closely related Trypanosoma species. It has the greatest impact in sub-Saharan Africa, where the tsetse fly vector is common (WHO, 2002). The complication of genomics results and analysis caused a big shift of interest from genomics to proteomics. The main analytical tool used for protein separation in proteomics is two-dimensional gel electrophoresis (2DGE) (O'Farrell, 1975 and Fey and Larsen, 2001). This technique separates proteins according to their masses and charges. These independent attributes enables separation of thousands of proteins in a single run. Since numerous proteins of a whole cell (its 'proteome') connect the genotype with the phenotype, we set out to study the proteome of the bloodstream form in T brucei which are infectious and known to cause diseases in human and animals. Deviating protein patterns between the different stages could direct the attention to disease-specific proteins and genes, which might be involved in the expression of infection and cause of disease. After separation, most proteins can be identified by mass spectroscopy. In studies performed to identify specific proteins related to a given metabolic process or disease it is, however, much more efficient to detect and identify only differentiated protein groups of samples. In order to understand the molecular basis of the parasites differentiation, we were interested in characterization of specific proteins expressed in bloodstream forms. However, the vast evolutionary distance between trypanosomes and the higher eukaryotes presents significant problems with functional assignment based on sequence similarities, and frequently homologues cannot be identified with sufficient confidence to be informative. Direct identification of proteins in isolated organelles has the potential of providing robust functional insight and is a powerful approach for initial assignment. The cytoplasm protein fraction and membrane protein fraction of T. brucei were used in this work to analyse the protozoan proteomics. Proteomics is a rapidly developing technique, which allows the efficient isolation of multiple protein families and it is a valuable tool for global patterns of gene expression. It allows the studies of membrane as well as cytosolic proteins. The rapidly growing development of bioinformatics, for example the use of software's like The JVirGel will also transform the handling of the multitude of data accumulating in proteomic experiments. The JVirGel software creates and visualizes virtual two-dimensional (2D) protein gels based on the migration behaviour of proteins in dependence of their theoretical molecular weights in combination with their calculated isoelectric points. Although, 9068 protein-coding genes and many pseudo genes have been identified, (science 2005) a number of potential new proteins are yet to be characterized. Analysis of T. brucei proteins using two-dimension gel electrophoresis, may offer prognostic analysis and information on disease mechanism. These results suggest the first step towards the generation of proteome profiles for use in future studies on protein expression, especially those accompanying the differentiation of the parasite. Comparison of the different stage-specific protein profiles will allow us to identify candidates as targets for drug action. Protein data using JVirGel software together with the provision of the complete genome sequence for T. brucei, the application of the genomic and post-genomic technologies should provide advances in the understanding of the biology of this parasite and the identification of key factors for virulence, drug resistance and infectivity. In this study the slender bloodstream form of T. brucei 221 were used because they are easy to grow in rats. These parasites can equally be cultivated at 37°C to grow to a cell density of about 1x106/ml. This approach allows the cultivation of high cell numbers without excessive expenditure of work and cost. I first had to establish a protocol for separating trypanosomal proteins by iso-electric focusing to produce a reference map for the first time. In this project, it was possible to identify many cytoplasmic and membrane proteins on the proteome-map of T. bruce. In proteomics the combination of the high-resolution two-dimensional electrophoresis and matrix assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOFMS) is currently the method of choice for protein identification. More than 300 protein spots were detected on a silver-stained two-dimensional gel. Analysis of 50 spots among them those highly expressed when T. brucei when grown in the presence of arachidonic acid was carried out. The protein spots from gel were digested with trypsin in-gel digestion followed by subsequent MALDI-TOF mass spectrometry in a process termed 'peptide mass fingerprinting'. Following a database search, 27 protein spots were identifield (belonging to different functional groups of proteins). 20 of the identified proteins are components of the main biological and cell regulation pathways located in T. brucei With this study, I established a partial data base and reference proteome map of the bloodstream form T. bruce. These data will facilitate further addition of information to T. brucei proteome, which may aid drug design in the treatment of patients with and control of trypanosomiasis and the control of infection. Future research based on this dissertation should mainly focus in the identification of different proteins between the bloodstream forms, the procyclic forms and the short stumpy forms of T. brucei.Trypanosoma brucei ist die Ursache für die afrikanische Schlafkrankheit (Trypanosomiasis), welche hauptsächlich die ärmsten Teile der Bevölkerung in den Entwicklungsländern in Zentralafrika und der Subsahara-Region bedroht. Trypanosoma brucei hat einen Mechanismus entwickelt, um der Immunabwehr des Wirts zu entkommen, der auf der Antigenvariation der über Glycosylphosphatidylinositol-Anker verbundenen Oberflächenproteine beruht. Die Genexpression ist hauptsächlich auf post-transkriptioneller Ebene reguliert. Die vollständige Aufklärung dieses Organismus wurde immer wieder erschwert durch Herausforderungen technischer Art. Dennoch ermöglicht der Fortschritt verschiedener Disziplinen wie Genomik, Proteomik und der Erstellung und Analyse von 2D-Gelen verbunden mit Methoden wie der matrix-assisted laser desorption/ionisation-time-of-flight (MALDI-TOF) mass spectrometry (MS) einen neuen Ansatz um schnell funktionelle Daten über Proteine zu bekommen. Im Gegensatz zu konventionellen biochemischen Ansätzen, die auf die Beobachtung einiger weniger Proteine abzielen, lässt sich durch proteomische Methoden die globale Proteinexpression im großen Maßstab untersuchen. Aus der direkten Messung der verschiedenen Proteinexpressionslevel lassen sich Rückschlüsse auf die Aktivität relevanter Proteine unter verschiedenen Bedingungen ziehen. Die Methoden der Proteomik bieten ein wichtiges Werkzeug um differentielle Expression von Proteinen während der verschiedenen Lebenscyclus-Phasen und verschiedenen Kulturbedingungen des Parasiten zu beobachten

Understanding the Mechanisms of Insecticide Resistance in Phlebotomus papatasi and Lutzoymia longipalpis Sand Flies (Diptera: Psychodidae: Phlebotominae)

The prevalence of insecticide resistance in vector species around the world is a continuous threat for any success at mitigating the spread of vector-borne diseases. With a limited arsenal of new insecticides, it is crucial for public health programs to understand the geographic range and the genetic mechanisms of resistance to best approach controlling insect vectors. Insecticide resistance is being increasingly observed in phlebotomine sand fly (Diptera: Psychodidae) populations in both the Old World and New World. Sand flies transmit the protozoans that cause leishmaniasis, a disfiguring disease that kills tens of thousands of people each year. The goal of this dissertation was to have both an applied and basic research focus towards understanding resistance in phlebotomines. I began by comparing in vivo and in vitro methods for blood-feeding two species of sand flies, Phlebotomus papatasi and Lutzomyia longipalpis, in the laboratory, both of which are important leishmaniasis vectors. I investigated the susceptibility of both species to ten different insecticides by calculating lethal concentrations that caused varying levels of mortality. Based on these results, I determined diagnostic doses and diagnostic times for both species to the same ten insecticides using an accepted, but novel, assay for sand flies. Finally, I tested for known mechanisms of insecticide resistance in four artificially resistant-selected colonies of sand flies, as well as tested for novel resistance mechanisms. Through applied research, I developed methods for efficient sand fly rearing and for determination of population resistance to insecticides, tools that have worldwide applicability. Through basic research, I determined that laboratory populations of sand flies have sufficient standing genetic variation needed to survive sublethal doses of insecticides; however, I was unable to develop artificially-selected colonies resistant to these insecticides. My research has generated information to provide new insights into the evolution of insecticide resistance in natural sand fly populations. My results support that resistance development may be possible, but evolutionary challenging, an encouraging finding that may be exploited by vector biologists and public health officials to prevent or slow the development of resistance in sand flies to insecticide

Program and Proceedings: The Nebraska Academy of Sciences 1880-2023. 142th Anniversary Year. One Hundred-Thirty-Third Annual Meeting April 21, 2023. Hybrid Meeting: Nebraska Wesleyan University & Online, Lincoln, Nebraska

AERONAUTICS & SPACE SCIENCE Chairperson(s): Dr. Scott Tarry & Michaela Lucas HUMANS PAST AND PRESENT Chairperson(s): Phil R. Geib & Allegra Ward APPLIED SCIENCE & TECHNOLOGY SECTION Chairperson(s): Mary Ettel BIOLOGY Chairpersons: Lauren Gillespie, Steve Heinisch, and Paul Davis BIOMEDICAL SCIENCES Chairperson(s): Annemarie Shibata, Kimberly Carlson, Joseph Dolence, Alexis Hobbs, James Fletcher, Paul Denton CHEM Section Chairperson(s): Nathanael Fackler EARTH SCIENCES Chairpersons: Irina Filina, Jon Schueth, Ross Dixon, Michael Leite ENVIRONMENTAL SCIENCE Chairperson: Mark Hammer PHYSICS Chairperson(s): Dr. Adam Davis SCIENCE EDUCATION Chairperson: Christine Gustafson 2023 Maiben Lecturer: Jason Bartz 2023 FRIEND OF SCIENCE AWARD TO: Ray Ward and Jim Lewi