Brown marmorated stink bug, Halyomorpha halys (Stål), is a crossover pest impacting agriculture and invading urban environments. Studies were conducted to better understand the management of the bug through its physiology, behavior and susceptibility to insecticidal intervention. Halyomopha halys exhibit great variability in overwintering site selection with choices including manmade structures and tree bark. Because of these diverse sites, the bug must have the ability to withstand varying conditions throughout the overwintering cycle. We were able to determine that H. halys is chill intolerant and capable of adapting its tolerance to temperatures by season, sex, and location of acclimation. The mean supercooling point (± SEM) in the winter in Minnesota was -17.06°C ± 0.13° and in Virginia was -13.90°C ± 0.09°. Laboratory experiments conducted in Blacksburg, VA were able to determine baseline lethal high temperatures over time against H. halys adults. To achieve 100% mortality, temperatures fell between 45°C and 50°C, 40°C and 45°C, and 42°C and 45°C, over 15-min, 1-h, and 4-h, respectively. Moving forward, we were able to utilize this information to develop heat treatment guidelines for export shipping cargo infested with overwintering H. halys. In a controlled field experiment, we determined that exposing the coldest areas of an infested vehicle to temperatures greater than 50°C for a minimum of 15 minutes resulted in 100% mortality of overwintering BMSB adults. In 2012 and 2013, citizen scientists were recruited through Virginia Cooperative Extension to assist in evaluating several in home light traps designed to help eradicate overwintering H. halys adults in homes. Over the course of the two year study, fourteen houses participated in the study with 72% of those houses having stink bug activity. It was found that the most effective trap was an aluminum foil pan trap. In 2013, the trap was 19 times more effective at catching stink bug adults than any other trap tested. In September of 2014, a near-field experiment was conducted to determine the residual efficacy of several recommended and labeled insecticides for treatment of homes against invading H. halys adults. This study used constructed window screen bags that were dipped in insecticide solution. After the initial treatment, bugs were exposed to the bags for 24h weekly, up to 54 days after treatment (DAT). It was determined that 2 DAT all insecticides had activity except for indoxacarb. All insecticides lost efficacy after 29 DAT except for lambda-cyhalothrin, beta-cyfluthrin, beta-cyfluthrin + imidacloprid, lambda-cyhalothrin + thiamethoxam, and dinotefuran, which had some measureable activity even after 40 DAT. Each of these insecticides contained a pyrethroid alone or in combination with a neonicotinoid. Laboratory bioassays were conducted to determine the LC50 values of clothianidin, dinotefuran, imidacloprid, and thiamethoxam against H. halys nymphs using a systemic application method. Those LC50 values were found to be 0.077, 0.013, 0.068, and 0.018 ppm, respectively. Field experiments conducted in Virginia in 2012 and 2013 showed a significant reduction of stink bug damage using two soil applications of neonicotinoid insecticides in pepper and tomato. In North Carolina, a single drip irrigation application significantly reduced stink bug damage in 2012 and 2014 using dinotefuran or imidacloprid. / Ph. D.

L’utilisation intensive des insecticides pour lutter contre les insectes ravageurs de culture et vecteurs demaladies, a conduit à des polémiques sur le mode d’action des insecticides. Ces polémiques sont liéesau fait que le mode d’action des insecticides, notamment des néonicotinoïdes est mal connu. Ils agissentprincipalement sur les récepteurs à l’acétylcholine (ACh) de type nicotinique (nAChR) qui jouent un rôlefondamental dans la transmission synaptique cholinergique. Bien que ces récepteurs soient bien décritschez les mammifères, très peu d’études ont évalué l’effet des néoniotinoïdes sur un récepteur natifd’insecte.Au cours de cette thèse, nous avons pour la première fois exprimé en ovocytes de xénope un récepteurhomomérique ⍺7 de blatte et étudié ces propriétés pharmacologiques vis-à-vis des néonicotinoïdes,comparativement au récepteur a7 de rat. Nos résultats révèlent un récepteur atypique qui est insensibleà l’⍺-bungarotoxine et qui n’est pas activé par les néonicotinoïdes. Ainsi, bien que les gènes codantpour les sous-unités α7 de blatte et de rat forment un groupe monophylétique distinct des autres sousunitésd’insectes et de mammifères, les récepteurs homomériques obtenus semblent avoir despropriétés pharmacologiques différentes. Parallèlement, nous avons étudié les propriétéspharmacologiques des nAChR natifs et notamment l’effet modulateur d’un pyréthrinoïde, la permethrine,sur les courants induits par le dinotefurane. Ce travail a permis d’évaluer le mode d’action d’unantiparasitaire, le Vectra 3D. Enfin, nous avons également entrepris de développer la techniqued’extraction membranaire afin de l’utiliser comme alternative pour étudier le mode d’action desinsecticides.Pour conclure, cette thèse a permis une avancée sur l’étude de la caractérisation des propriétéspharmacologiques des récepteurs nicotiniques neuronaux des insectes et sur l’étude du mode d’actiondes insecticides néonicotinoïdes. / The intensive use of insecticides against crop pests and vectors of human and animal leads to several polemics about their mode of action. All these controversies are related to the fact that the mode of action of insecticides in insects is poorly unknown, in particular neonicotinoids which act on nicotinic acetylcholine (ACh) receptors (nAChR).During this PhD thesis, we characterized for the first time the pharmacological properties of a cockroach ⍺7 homomeric receptor in a xenopus oocyte. Our results revealed that cockroach ⍺7 in an atypical receptor that is insensitive to ⍺-bungarotoxin and not activated by neonicotinoids. Cockroach and rat ⍺7 receptors which are included in the same cluster have distinct pharmacological properties. We then studied the pharmacological properties of native receptors, in particular, the modulatory effect of permethrin on dinotefuran-induced currents. This work was included in the study of Vectra 3D. We also evaluated the use of insect central nervous system membrane extraction as a strategy to study the pharmacological properties of insect native nAChRs.To conclude, this PhD contribute to the study of the pharmacological properties of insect nAChRs and the study of the mode of action of neonicotinoids insecticides.

Bumble bees and other wild pollinators are crucial to the support of both natural and agricultural ecosystems. However, unprecedented declines of pollinator populations have been observed all over the world, raising concerns of a looming threat to both the human food supply, as well as sustainability of the biodiversity in local ecological niches. Though declines are well described, the cause behind these still evades scientists. Proposed contributors include anthropogenic-mediated environmental stress, including application of xenobiotics for pest control, and increase of pathogen diversity and abundance due to the shipment of infection human-managed colonies. This research examined these theories and attempted to quantify the threats they may pose. Through development of a chronic, oral toxicity experiment, susceptibility of all Bombus impatiens castes to clothianidin exposure was examined. This exposed a substantial increase in vulnerability of male bumble bees to realistic concentrations of neonicotinoid pesticides, highlighting the crucial need to examine all members of wild bumble bee life cycles before determining pesticide regulations. Additionally, sublethal effects on fitness-related foraging behaviors in Bombus impatiens were examined through development of a voluntary task switching assay. The results of this experiment suggest humoral immune stimulation, through pathogenic infection, leads to significant impairment of cognitive flexibility. Taken together, this data suggests that pesticides and pathogens are capable of causing severe detrimental effects, both lethally and sublethally, in wild bumble bees. I hope this data will eventually contribute to reassessment of environmental regulations and establishment of effective conservation strategies in order to sustain the critical populations of wild bumble bees.

Public awareness of declining pollinator populations has increased interest in creating “bee–friendly” urban landscapes. I quantified bee visitation and assemblages of 72 species of flowering woody plants common in urban landscapes. I found strong plant species effects and variation in seasonal activity of particular bee taxa but no overall differences in bee visitation or genus diversity between native versus nonnative species or trees versus shrubs. Analysis of pollen from a subset of these plants revealed small but statistically significant differences in total and essential amino acids between native and nonnative species and trees and shrubs, although each group had species with high quality pollen. Uptake and dissipation of soil–applied imidacloprid and dinotefuran was measured in nectar and leaves of two woody plant species, Ilex × attenuata and Clethra alnifolia to assess concentrations to which pollinators might be exposed in landscape settings. Three application timings were evaluated. Residues in nectar and tissue were analyzed by HPLC–MS/MS in two successive years. Residues in nectar following autumn or spring applications exceed concentrations shown to adversely affect individual and colony–level traits of bees. Summer application mitigated concentrations of imidacloprid (8–31 ng/g), but not dinotefuran (235–1191 ng/g), in nectar.

Durant leur cycle de vie, les insectes doivent faire face à différents perturbateurs pour réussir à survivre et à se reproduire. L’utilisation de plus en plus répandue des insecticides néonicotinoïdes, en raison de leur grande efficacité, a conduit à l’accumulation de résidus dans l’environnement. Ceux-ci ont certainement un effet additif toxique sur les insectes cibles. Cependant il a été montré que ces résidus peuvent aussi avoir un effet positif non désiré sur certains traits de vie des insectes ravageurs.Dans ma thèse, j’ai étudié les effets d’un insecticide néonicotinoïde sur le système olfactif d’un insecte ravageur, la noctuelle Agrotis ipsilon. Nos résultats montrent que l’exposition aigüe des mâles à des faibles doses de clothianidine modifie leurs réponses comportementales à la phéromone sexuelle: une baisse est observée à la dose 0,25 ng/insecte (<DL0) alors que la doses de 10 ng (DL20) induit une augmentation de la réponse chez les adultes naïfs ou pré-exposés à la phéromone. Cet effet biphasique à faible et très faible dose s’apparente à un effet hormétique et les modifications observées sont corrélées avec des changements de sensibilité du système olfactif central et non périphérique. Nous émettons l’hypothèse que la clothianidine agirait sur l’expression des sous-unités des récepteurs nicotiniques pour lesquels elle joue le rôle d’agoniste, changeant leur affinité pour l’acétylcholine et perturbant ou améliorant la transmission synaptique des signaux sensoriels selon la dose. Nos résultats montrent que la prise en compte d’effets de doses sublétales d’insecticides est essentielle non seulement pour les insectes non cibles, mais aussi des insectes cibles. / Insects face a multitude of environmental stresses, which they have to bypass in order to survive and reproduce. The extensive use of neonicotinoid insecticides, because of their high efficiency, leads to the accumulation of residues in the environment, which can have an additive toxic effect on target insects. However, such residues can also have unwanted positive effects on certain life traits of pest insects. In my thesis I studied the effects of a neonicotinoid insecticide on the olfactory system of the pest insect Agrotis ipsilon. Our results show that acute oral treatments of males with low doses of clothianidin modify their behavioural responses to the sex pheromone: a treatment with 0.25 ng/moth (<LD0) induces a decrease of pheromone responses whereas intoxication with 10 ng/moth (LD20) leads to an increase in the capacity of naive and pre-exposed males to locate a pheromone source as compared to controls. We propose that this biphasic effect, with low dose stimulation and very low dose inhibition is an hormeticlike effect, that is correlated with sensitivity changes within the central, but not the peripheral olfactory system. We hypothesize that the observed modifications might be due to a differential effect of clothianidin on the expression of different subunits of nicotinic acetylcholine receptors, which might change the affinity of the receptors for acetylcholine, and thus disturb or improve synaptic transmission of sensory signals as a function of the insecticide dose. Our results show that effects of sublethal doses of insecticides need to be taken into account not only for non-target, but also for target insects when evaluating pest management strategies.

Les abeilles domestiques subissent des pertes de colonies sans précédents, ce qui pourrait à terme avoir un fort impact économique compte tenu de leur apport à l’agriculture en service de pollinisation. Ces pertes sont définies comme causées par des stress multifactoriels. Cependant, l’impact de ces stress est en général testé séparément sur les individus ou les colonies. Les colonies sont connues pour concentrer les pesticides provenant de l’environnement à travers le pollen et le nectar récoltés mais aussi les pathogènes. L’étude de la co-exposition entre le thiaméthoxam, un insecticide néonicotinoïde, et deux virus de l’abeille, le DWV et le CBPV, a mis en évidence l’existence d’interactions entre chacun de ces virus et le pesticide. Diverses méthodes de transmission des virus ont été testées afin de s’approcher au mieux des conditions naturelles et une méthode de transmission du CBPV par contact répétable a été développée. Une cinétique de métabolisation du thiamethoxam a été effectuée pour la première fois, renseignant sur la forte probabilité d’excrétion du métabolite en conditions naturelles. Lors d’expérimentations en cagettes la co-exposition entre le CBPV et le thiaméthoxam à fortes doses chroniques a causé des mortalités synergiques, ou une augmentation des charges virales atteignant le seuil en nombre de copies virales connu pour déclencher des infections apparentes chez les abeilles. Les différences de résultats entre des abeilles provenant de colonies dans diverse conditions a souligné un effet de tolérance aux stress encore non étudié chez l’abeille domestique. En parallèle, la co-exposition au sein de la colonie entre le thiaméthoxam à une dose sublétale aigüe et le DWV a causé des premières sorties de butinage extrêmement précoces chez les abeilles, ainsi que de fortes proportions de premiers vols sans retour, ce qui pourrait à terme avoir un effet dévastateur sur les colonies. L’étude de la transcription de gènes suggère que les effets obtenus sur les charges virales en CBPV seraient dus à un effet du thiaméthoxam sur dorsal-1a, un facteur de l’immunité, et les sorties précoces à une baisse significative de la transcription de la vittelogénine, entrant en jeu dans les mécanismes de vieillissement, due au DWV. Le développement de clones moléculaires du DWV a été amorcé, ce qui pourra à terme permettre des études de génétique inverse sur ce virus, qui pourront expliquer ses modes de transmission et d’infections encore inconnus à ce jour. / Domestic honeybees are suffering from unprecedented colony losses, which could at term have a strong economic impact, considering their part in crop pollination, notably. These losses are defined as caused by multifactorial stresses. However, the impact of these stresses is usually measured separately, on individuals or colonies. Colonies are known to concentrate pesticides from their environment through collection of pollen and nectar, but they also concentrate pathogens. The study of a co-exposure between thiamethoxam, a neonicotinoid insecticide, and two honeybee viruses, DWV and CBPV, has shed light on the existence of interactions between each of these viruses and the pesticide. Different virus transmission methods have been tested to be as close as natural conditions, and a repeatable CBPV transmission method through contact has been developed. Metabolisation kinetics of thiamethoxam have been obtained for the first time, underlining the strong probability for excretion of the metabolite in natural conditions. During caged experiments, the co-exposure experiments between thiamethoxam at high chronic doses and CBPV cause synergistic mortalities, or an increase in viral loads which reached the number of viral copies threshold associated with clinical signs in bees. The varying results between bees from colonies kept in different conditions underlined a stress tolerance effect yet to be studied in the honeybee. In parallel, co-exposure in colonies between sublethal doses of thiamethoxam and DWV caused extremely precocious first foraging trips in bees, as well as large proportions of bees never returning to the hive after their firs exit, which both could in the end have a devastating effect on the colonies. The study of gene transcripts suggests that the obtained effects on CBPV viral loads could be due to a negative effect of thiamethoxam on dorsal-1a, an immune factor, and the precocious trips to a significant down-regulation of vitellogenin, which takes part in honeybee aging, cause by DWV. The development of molecular clones of DWV and a DWV recombinant was initiated, which will in the future allow for reverse genetics studies on this virus, which will help explain their transmission and infection mechanisms that are still unknown to this day.

Since their emergence in the early 1990s, neonicotinoid use has increased exponentially to make them the world's most prevalent insecticides. Although there is considerable research concerning the lethality of neonicotinoids, their sub-lethal and developmental effects are still being explored, especially with regards to non-mammalian species. The goal of this research was to investigate the effects of the neonicotinoid imidacloprid on the morphological and physiological development of northern bobwhite quail (Colinus virginianus). Bobwhite eggs (n = 650) were injected with imidacloprid concentrations of 0 (sham), 10, 50, 100 and 150 grams per kilogram of egg mass, which was administered at day 0 (pre-incubation), 3, 6, 9, or 12 of growth. Embryos were dissected on day 19 when they were weighed, staged, and examined for any overt structural deformities. Embryonic heart, liver, lungs and kidneys were also weighed and preserved for future use. Treated embryos exhibited increased frequency of severely deformed beaks and legs, as well as larger hearts and smaller lungs at the higher dosing concentrations. Some impacts are more pronounced in specific dosing periods, implying that there may be critical windows of development when embryos are highly susceptible to neonicotinoid exposure. This investigation suggests that imidacloprid could play a significant role in chick survival and declining quail populations in treated regions of the country.

Orientador: Osmar Malaspina / Resumo: As abelhas Apis mellifera africanizadas são consideradas importantes polinizadores no Brasil, uma vez que muitas culturas de alimentos dependem da polinização promovida por esses insetos. No entanto, com o crescimento da produtividade agrícola houve aumento do uso de agrotóxicos para o controle de pragas, os quais podem atingir também insetos não-alvo. Com a ação dos inseticidas como uma das possíveis causadoras da morte massiva desses polinizadores, pesquisas sobre o impacto dos agrotóxicos em abelhas receberam destaque. Diante do exposto, o presente estudo propôs investigar os efeitos de uma dose subletal de tiametoxam (TMX) (0,0227 ng de ingrediente ativo/μl de alimento), importante inseticida da classe dos neonicotinóides, no cérebro e no intestino de Apis mellifera africanizadas, por meio da avaliação da atividade de biomarcadores enzimáticos de exposição e de estresse oxidativo e pela ocorrência de peroxidação lipídica. O nível de estresse celular também foi investigado pela imunomarcação das proteínas de choque térmico HSP70 e HSP90 em conjunto com a detecção de morte celular pelo ensaio de TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling). Os dados mostraram que, no cérebro, o TMX aumentou a atividade de acetilcolinesterase (AChE) em 1, 3 e 5 dias de exposição, enquanto a carboxilesterase (CaE) diminuiu no primeiro dia e a glutationa s-transferase (GST) aumentou no quinto dia. Por sua vez, as enzimas antioxidantes foram menos atuantes no cérebro, se... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Africanized Apis mellifera bees are considered important pollinators in Brazil, since many food crops depend on the pollination by these insects. However, with the growth of agricultural productivity there has been an increase in the use of insecticides for pest control, which also can reach non-target insects. With the action of insecticides as one of the possible causes of the massive death of these pollinators, the studies of the effects of pesticides on bees were highlighted. Given the context, the present study aimed to investigate the effects of a sub lethal dose of thiamethoxam - TMX (0.0227 ng of active ingredient / μl of food), a insecticide of the class of neonicotinoids, in the brain and intestine of Africanized Apis mellifera, through the evaluation of the activity of enzymatic biomarkers of exposure and of oxidative stress and by the occurrence of lipid peroxidation. The level of cell stress was also investigated by the immunostaining of the HSP70 and HSP90 proteins together with the detection of cell death by the TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. The data showed that, in the brain, TMX increased acetylcholinesterase activity (AChE) at 1, 3 and 5 days of exposure, while carboxylesterase (CaE) decreased on the first day and glutathione s-transferase (GST) increased in the fifth day. On the other hand, antioxidant enzymes were less active in the brain, and only glutathione peroxidase (GPX) showed increased activity on the f... (Complete abstract click electronic access below) / Doutor