Insights into vector control through the modulation of An. gambiae G protein-coupled receptors

Thesis advisor: Marc A.T. MuskavitchMalaria is a life-threatening infectious disease caused by inoculation of the apicomplexan Plasmodium parasite into vertebrate hosts. Transmission of the parasite is mediated by the Anopheles mosquito, which has the capacity to efficiently transmit the parasite from host to host, as the disease vector. There are many factors that make anopheline mosquitoes competent vectors for disease transmission. The hematophagous (blood-feeding) behavior of the female mosquito is one of most fundamental factors in physical transmission of parasites, because the ingestion of blood from an infected host allows parasite entry into the mosquito and the completion of parasite sexual reproduction. In addition to this blood-feeding behavior, there are a host of biological (i.e., parasite replication) and behavioral factors (i.e., mosquito chemosensation, host preference) that contribute to the high vectorial capacity of these vector species. There are over four hundred Anopheles species worldwide, approximately forty of which are considered epidemiologically critical human malaria vectors. Anopheles gambiae, the primary vector in malaria-endemic sub-Saharan Africa, is responsible for the largest number of malaria cases in the world and is therefore one of the most important vectors to study and target with control measures. Currently, vector-targeted control strategies remain our most effective tools for reduction of malaria transmission and incidence. Although control efforts based on the deployment of insecticides have proven successful in the past and are still widely used, the threat and continuing increases of insecticide resistance motivate the discovery of novel insecticides. In this thesis, I provide evidence that G protein-coupled receptors (GPCRs) may serve as “druggable” targets for the development of new insecticides, through the modulation of developmental and sensory processes. In Chapter II, “A critical role for the Drosophila dopamine 1-like receptor Dop1R2 at the onset of metamorphosis,” I provide evidence supporting an essential role for this receptor in Drosophila melanogaster metamorphosis via transgenic RNA interference and pharmacological methods. In An. gambiae, we find that the receptor encoded by the mosquito ortholog GPRDOP2 can be inhibited in vitro using pharmacological antagonists, and that in vivo inhibition with such antagonists produces pre-adult lethality. These findings support the inference that this An. gambiae dopamine receptor may serve as a novel target for the development of vector-targeted larvicides. In Chapter III, “RNAi trigger delivery into Anopheles gambiae pupae,” I describe the development of a method for injection directly into the hemolymph of double strand RNA (dsRNA) during the pupal stage, and I demonstrate that knockdown of the translational product of the SRPN2 gene occurs efficiently, based on reductions in the levels of SRPN2 protein and formation of melanized pseudo-tumors, in SRPN2 knockdown mosquitoes. This method was developed for rapid knockdown of target genes, using a dye-labeled injection technique that allows for easy visualization of injection quality. This technique is further utilized in Chapter IV, “Uncovering the Role of an Anopheles gambiae G Protein-Coupled Receptor, GPRGR2, in the Detection of Noxious Compounds,” where the role for GPRGR2 in the detection of multiple noxious compounds is elucidated. We find that pupal stage knockdown of this receptor decreases the ability of adult Anopheles gambiae to identify multiple noxious compounds. While these findings provide a strong link between GPRGR2 and a very interesting mosquito behavior, they may also provide opportunities to develop better field-based strategies (i.e., insecticides baited traps) for vector control. The goal of this thesis is to understand the functional roles of selected mosquito GPCRs that may serve as targets for the development of new vector-targeted control strategies. Exploiting these GPCRs genetically and pharmacologically may provide insights into novel vector control targets that can be manipulated so as to decrease the vectorial capacity of An. gambiae and other malaria vectors in the field, and thereby decrease the burden of human malaria.Thesis (PhD) — Boston College, 2015.Submitted to: Boston College. Graduate School of Arts and Sciences.Discipline: Biology

Plasmodium falciparum: linkage disequilibrium between loci in chromosomes 7 and 5 and chloroquine selective pressure in Northern Nigeria.

In view of the recent discovery (Molecular Cell 6, 861-871) of a (Lys76Thr) codon change in gene pfcrt on chromosome 7 which determines in vitro chloroquine resistance in Plasmodium falciparum, we have re-examined samples taken before treatment in our study in Zaria, Northern Nigeria (Parasitology, 119, 343-348). Drug resistance was present in 5/5 cases where the pfcrt 76Thr codon change was seen (100% positive predictive value). Drug sensitivity was found in 26/28 cases where the change was absent (93% negative predictive value). Allele pfcrt 76Thr showed strong linkage disequilibrium with pfmdr1 Tyr86 on chromosome 5, more complete than that between pfcrt and cg2 alleles situated between recombination cross-over points on chromosome 7. Physical linkage of cg2 with pfcrt may account for linkage disequilibrium between their alleles but in the case of genes pfmdr1 and pfcrt, on different chromosomes, it is likely that this is maintained epistatically through the selective pressure of chloroquine

Brandt's East-Policy and Czechoslowakia 1969-1974

Die politische Fühgrung der ČSSR begann die Vorbereitungen der Verhandlungen mit der BRD auf Grund einer Weisung von Leonid Breschnew auf der Tagung des Politischen Ausschußes des Warschauer Paktes vom 20. August 1970. Bis dahin war die ČSSR einem wirksamen Druck der SED-Parteiführung ausgesetzt, die der Regierung Brandt feindlich gegenüberstand und von der Brandts Ostpolitik ständig warnte. Die Vertragsverhandlungen begannen zunächst mit Vorgesprächen, die von tschechoslowakischer Seite durch den Stellvertretenden Außenminister Milan Klusák (ab September 1971 den Stellvertretenden Außenminister Jiří Götz) und von deutscher Seite durch den Staatssekretär Paul Frank geführt wurden. Von März 1971 bis April 1973 fanden insgesammt sechs Vorgesprächsrunden statt, auf Drängen der tschechoslowakischen Seite wurde vor allem die Frage der Ungültigkeit des Münchener Abkommens zum Verhandlungsgegenstand. Der tschechoslowakische Standpunkt war, daß das Münchener Abkommen der Tschechoslowakischen Republik "unter Androhung von Angriffskrieg und Gewaltanwendung aufgezwungen wurde, daß es Bestandteil einer verbrecherischen Verschwörung Nazi-Deutschlands gegen den Frieden sei und eine schwere Verletzung der Grundregeln des Völkerrechts darstelle, und daß es deshalb von Anfang an ungültig sei, mit allen sich daraus..

Additional file 14: Table S1. of A critical role for the Drosophila dopamine D1-like receptor Dop1R2 at the onset of metamorphosis

Primer sequences. (DOC 44 kb

Additional file 2: Figure S2. of A critical role for the Drosophila dopamine D1-like receptor Dop1R2 at the onset of metamorphosis

dsDop1R2 knockdown-induced lethality is recapitulated with alternate RNAi constructs. (A) and (B) Ubiquitous knockdown of Dop1R2. (A) VDRC 3391-GD (genotype: w1118;UAS-dsDop1R2/+;Act5C-Gal4/+) results in 98 % lethality at 29 °C (n = 58) and 94 % lethality at 25 °C, or viability (n = 44). (B) VDRC 105324-KK (genotype: w1118;TM6B/+;UAS-dsDop1R2/+) results in 97 % lethality at 29 °C (n = 59), compared to control balancer siblings (genotypes: w1118;CyO/+;UAS-dsDop1R2/+ and w1118;CyO/UAS-dsDop1R2, respectively). All male escaper flies (n = 4) obtained when using the VDRC 3391-GD RNAi construct exhibited the hypomelanization phenotype (described in Fig. 7). (C) Salivary gland/amnioserosa targeted knockdown of Dop1R2 VDRC 3391-GD results in 68 % lethality at 29 °C in experimental flies (genotype: w1118;UAS-dsDop1R2/+;P{GawB}c729-GAL4/+), compared to controls (genotype: w1118;P{GawB}c729-Gal4/+) (n = 53). VDRC Dop1R2 knockdown stocks: 3391-GD (FBst0460369) and 105324-KK (FBst0477151). Driver stocks: Act5C-GAL4 (FBst0003954), P{GawB}17A-GAL4 (FBst0008474) and P{GawB}c729-GAL4 (FBst0006983). (PDF 230 kb

Additional file 11: Figure S9. of A critical role for the Drosophila dopamine D1-like receptor Dop1R2 at the onset of metamorphosis

Dop1R2 is stimulated by dopamine and antagonized by two known small molecules in vitro. Human Embryonic Kidney cells (HEK293 cells) were transiently co-transfected in a 96-well plate assay with plasmids encoding Drosophila Dop1R2 receptor and a luciferase reporter gene. (A) Increasing concentrations of dopamine activates the receptor (EC50 = 2.7 × 10−7 M). (B) Stimulation of the Dop1R2 receptor by dopamine (100 μM) is inhibited with increasing concentrations of either flupenthixol dihydrochloride (IC50 = 2.6 × 10−7 M) or butaclamol (IC50 = 21.6 × 10−7 M). Data represent the mean ± the Standard Error of the Mean (SEM) from three independent experiments, each performed in triplicate. Methods for this assay are as previously described [119]. (PDF 227 kb

Santa Clara

An archive of the Santa Clara University student newspaper from Santa Clara University in Californi

Insecticide resistance phenotypes from dry (blue) and wet (red) seasons with and without the synergist PBO.

<p>Bars are mean mortalities from four replicate bioassays (N = 25 each), with 95% binomial confidence limits. Odds ratios are shown above bars and represent the odds of mortality with PBO pre-exposure, compared to the odds of mortality with insecticide alone (data from the two seasons are pooled). *P≪0.001; ^NSnot significant (ײ-test).</p