Volatile constituents of bark from the two species of pine hosts of the mountain pine beetle, (Dendroctonus ponderosae Hopkins) were investigated as postulated factors in host tree discovery and selective attack by the insect. The anemo-olfactory, klinokinetic and klinotactic responses of pedestrian adult flown females to odors of different aged pines of the species Pinus ponderosa Laws, (ponderosa pine) and Pinus monticola Dougl. (western white pine) were used as criteria to test for evidence as to possible involvement of host tree volatiles in host selection. Volatile substances from pine bark were collected through sublimation under vacuum from frozen state. The volatile extracts were analyzed by Gas-Liquid Chromatography. Total volatile extracts and their ether soluble fraction from mature pine trees were "attractive" to beetles, whereas those from saplings were "repellent." No qualitative differences were found in the ether-soluble fraction of mature trees and saplings respectively, but proportions of individual constituents in the extracts differed. Ethanol at various concentrations caused arrestance of the anemo-olfactory response of pedestrian beetles. As a constituent of trees under stress ethanol may thus play an important role in the programme of responses which lead ultimately to attack. Problems encountered during the storage of tree samples and their extracts have been discussed. / Forestry, Faculty of / Graduate

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Malaria (vector: Anopheles gambiae) is a major infectious disease that kills about 1 million people each year. For the improvement of its treatment and vector control during the past decades, several issues such as high medicine cost, insecticide resistance, and lack of an effective vaccine have prevented adequate control of malaria. Additionally, the low selectivity of malaria vector insecticides also presents a public health problem. The purpose of developing novel carbamate insecticides in our laboratory is to offer effective and selective insecticide options to achieve the ultimate goal of malaria control. First, 50% inhibition concentration (IC50) data was collected from three mammalian AChEs with eight commercial carbamate insecticides by using the Ellman assay. The IC50 values varied from 57 nM to 7358 nM. The AChE sensitivity pattern and level were shown to be similar between the recombinant mouse and ICR male mouse brain cortex homogenate (slope = 0.99, R2 = 0.96). Then eight novel carbamate insecticides that are possible malaria vector control agents were selected for further neurotoxicity testing in non-target organisms. For commercial carbamate insecticides, the IC50 varied from 9.1 nM to 2,094 nM. For the novel carbamate insecticides, it varied from 58 nM to 388,800 nM. Based on IC50 data from previous work on A. gambiae, the selectivity index (IC50 of non-target species / IC50 A. gambiae) ranged from 0.17 to 5.64 and from 0.47 to 19,587 for commercial and novel carbamate insecticides, respectively. Subsequently, the AChE protein sequence alignment comparison and cladogram were used to compare the genetic and evolutionary relationship among five different organisms. The alignment score ranged from 88 for mouse vs. human to 54 for hen vs. T. californica. The evolutionary relationships among species was obtained from the cladogram. Recombinant mouse vs. recombinant human was shown to have the most similar inhibitor potency profiles (alignment score = 88, closest taxa position on cladogram, similar AChE sensitivity pattern [R2 = 0.81] and level [P > 0.05] to the novel carbamates). Neurotoxic esterase (NTE) assay showed that the novel carbamates did not significantly inhibit NTE, inhibition of which underlies a significant hazard for anticholinesterases, especially organophosphates, in several nontarget vertebrate organisms. The NTE activity in the presence of novel carbamate insecticides ranged from 93% to 116% of the control, while in the commercial group, bendiocarb significantly inhibited NTE, leaving only 76.5% of the initial reactivity at 1 mM inhibitor concentration. Further in vivo bioassay using Daphnia magna was conducted to compare the aquatic toxicity of commercial and novel carbamates. The data showed that except for PRC331 (3-tert-butylphenylmethylcarbamate), all novel carbamates were of similar potency as bendiocarb (LC50 = 611 nM) for aquatic toxicity, and their LC50 values ranged from 172 nM (PRC331) to 1109 nM. In conclusion, the novel carbamate insecticides would appear to be an improvement over commercial carbamate insecticides because of greater selectivity, negligible NTE inhibition capacity, but in some cases with potent in vivo toxicity to Daphnia magna. However, since the envisioned usage of these compounds is in bednets or as indoor residual sprays (IRS), any environmental exposures to nontarget aquatic organisms are expected to be minimal. / Master of Science

Tableau d'honneur de la Faculté des études supérieures et postdoctorales, 2016-2017. / Les canaux sodiques dépendants du voltage (NaV) participent à la genèse et la transmission de l'influx nerveux via l'initiation du potentiel d'action. Chez l'insecte, ces protéines sont la cible de nombreux insecticides neurotoxiques. Étant donné le déclin des populations d'insectes pollinisateurs observé récemment, il devient désormais important d'avoir des méthodes permettant le développement d'insecticides qui ne ciblent pas les pollinisateurs. L'objectif général de ma thèse a donc été de mettre au point des méthodes in-vitro et insilico pouvant être appliquées à grande échelle dans le but d'évaluer le risque potentiel que certains insecticides pourraient représenter pour l'abeille. Pour ce faire, nous avons procédé à la caractérisation biophysique et pharmacologique des canaux sodiques NaV1 et NaV2 d'abeille. Nous avons aussi créé un modèle moléculaire du canal NaV1 d'abeille permettant la conception rationnelle d'insecticides. De plus, nous avons caractérisé le canal NaV1 de Varroa destructor, un parasite important de l'abeille. Les études insérées dans cette thèse ont permis de démontrer que le canal NaV2 n'est pas une cible secondaire des insecticides pyréthroïdes. De plus, nous avons pu mesurer les effets et l'affinité d'insecticides sur leur cible moléculaire chez l'abeille suite à l'expression du canal NaV1 d'abeille en ovocyte de grenouille. Cela permet le criblage de composé à haut débit afin de sélectionner des composés représentant un faible risque pour l'abeille. De même, l'expression du canal NaV1 de Varroa destructor en système d'expression hétérologue permettrait l'utilisation de la méthode de criblage de composés à haut débit dans le but d'identifier des insecticides qui représentent un risque important pour cette peste. D'ailleurs, nous avons déterminé que le fluvalinate, un insecticide pyréthroïde utilisé pour contrôler les populations de Varroa destructor dans les ruches d'abeille, a une affinité différente pour le canal NaV1 d'abeille que pour celui de Varroa destructor. Cela indique une différence dans les sites de liaisons du composé qui pourrait éventuellement être exploitée. Mes travaux ouvrent donc la voie au développement de composés ciblant davantage des animaux nuisibles comme le Varroa destructor que des animaux utiles comme l'abeille. De plus, les caractérisations effectuées pourraient être utiles pour des études centrées sur les rôles et l'évolution des canaux ioniques appartenant aux familles NaV et CaV. / Voltage-gated sodium channels (NaV) are implicated in the genesis and transmission of action potentials. In insects, these proteins are the target of a number of neurotoxic insecticides. In the background of the pollinator decline observed recently, it has become necessary to develop insecticides which do not target beneficial insects such as bees. The main objective of my thesis was to develop in-vitro and in-silico methods which could be used on a large scale to evaluate the risk associated with the use of certain compounds for bees. To do so, we assessed the biophysical and pharmacological properties of the honeybee's NaV1 and NaV2 channels. We also created a molecular model for the NaV1 channel which enables the rational design of insecticides. Furthermore, we have characterized the Varroa destructor NaV1 channels. The investigations featured in this thesis demonstrate that the NaV2 channel is not a secondary target of pyrethroïd insecticides. However, following expression in frog oocytes, it is possible to quantify the effects and affinity of those insecticides for their molecular target in the honeybee, the NaV1 channel. This makes possible the use of high throughput screening technologies for the selection of insecticides which would represent a small risk for bees. Moreover, the expression of Varroa destructor NaV1 channels in frog oocytes enables the use of medium throughput screening technologies to identify compounds which could be deleterious for this pest. Indeed, we determined that fluvalinate, a pyrethroïd insecticide used to control Varroa population in honeybee hives, has an affinity for the honeybee NaV1 channel that is different than that for the Varroa channel. This indicates that the binding site of this compound on the Varroa channel would differ from the binding site on the bee channel. This difference could be exploited to improve the specificity of fluvalinate. The work presented here represents a first step in the development of methods which could be used to decrease the toxicity of insecticides for bees while increasing their specificity of against pests such as Varroa destructor. Furthermore, the characterizations performed provide new insights on topics such as the roles and the evolution of NaV et CaV channels.

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