A diferenciação morfofisiológica entre rainhas e operárias de Apis mellifera decorre da alimentação recebida durante o desenvolvimento larval, que estimula o aumento da produção de Hormônio Juvenil naslarvas que originarão rainhas. Dentre as diversas diferenças morfológicas entre operárias e rainhas encontramse estruturas especializadas para a coleta de pólen e própolis, localizadas na região da tíbia e do basitarso das pernas posteriores de operárias. A diferenciação das pernas tem início entre o quarto e o quinto estágio do desenvolvimento larval. Utilizandose Microscopia Eletrônica de Varredura o presente trabalho relata a presença das cerdas formando as estruturas castaespecíficas na fase de pupa de olho marrom. A partir de estudos de hibridação de microarrays de cDNA com amostras de RNA de A. mellifera de diversas fases do desenvolvimento larval, foram encontrados 91 genes com ortólogos conhecidos em Drosophila, diferencialmente expressos entre rainhas e operárias no período crítico da diferenciação de castas. Destes, cinco estão relacionados com o desenvolvimento de apêndices: ataxin2 (atx2), cryptocephal (crc), dachshund (dac), grunge (gug) e Retinoic and fat acid Binding Protein (RfaBP). O perfil destes genes, e ainda, ultrabithorax (ubx), distalless(dll) e abdominalA (abdA) (estes porsuassuasfunções durante a diferenciação das pernas de insetos) foram analisados por RTPCR em Tempo Real em pernas posteriores de operárias e rainhas desde o quarto estágio larval até o estágio de pupa de olho branco. Apenas ubx e abdA foram encontrados mais expressos em operárias ao final do desenvolvimento larval e início do desenvolvimento pupal. Estudossimilares dos genes abdA, dac, dll e ubx nossegmentos das pernas de pupas de olho branco indicam a tíbia como domínio de expressão de dac. Imunolocalizações utilizando um anticorpo contra um epitopo conservado entre Ubx e AbdA, FP6.87, em pernas posteriores de prépupas de operárias e rainhasrevelam a presença destas proteínas na tíbia apenas de operárias e diferencialmente localizadas no basitarso de operárias e rainhas. Os dados acima apresentados apontam Ubx, um gene Hox, como pontochave na regulação da formação das estruturas castaespecíficas. / Diphenism in the honey bee, Apis mellifera,resultsfromdifferential feeding of female larvae. Among the morphological differences, the hind legs of workers have structures that is used for carrying pollen and propolis, e.g. the corbicula, while the queens hind legslack thisstructures. The corbicula is an expanded region of the tibia deprived of bristles, which has a single bristle in the middle that seems to have a sensorial function. Using scanning electronic microscopy, we found that the leg structures and bristles of the corbicula are already formed in browneyed pupa. Microarray analysis has demonstrated that five of 240 differentiallyexpressed genesin developing castes are potentially related to the caste differences in leg development (ataxin2, cryptocephal, dachshund, grunge and Retinoic and fat acid Binding Protein). Using qPCR, we analyzed the expression of abdominalA, ataxin2, cryptocephal, grunge, Retinoic and fat acid Binding Protein and ultrabithorax genes during hind leg development. cryptocephal, ataxin2, grunge and Retinoic and fat acid Binding Protein genes, which are involved in imaginal disc elongation and bristle formation and are inhibited by juvenile hormone, were not found to be differentially expressed. However, ultrabithorax and abdominalA are over expressed in workersin the early pupalstage. By using immunohistochemistry, Ubx was localized in the tibia and basitarsus of prepupae of workers and in the basitarsus of pre pupae of queens. The pattern of Ubx expression suggests that this Hox gene is a key player in leg structuresformation and caste differentiation in A.mellifera.

The Asian clam, Corbicula fluminea, is an invasive bivalve species that now occurs through most of the lower 48 United States. While a significant degree of salinity tolerance has been observed in C. fluminea, owing to its estuarine lineage, the physiological and behavioral responses to changes in salinity by these organisms are not completely understood. It was hypothesized that Corbicula would initially avoid elevated salinity levels (>1 g/L) behaviorally through valve closure, but would eventually have to open to dispel anaerobic waste products and deal with the salinity. To explore this, Corbicula were collected and put through a series of experiments at salinity exposures of 0, 2.5, and 5.0 g/L, with tissue water content and hemolymph osmolality being measured. After an initial 96-hour exposure, it was observed that the percent tissue water content of clams in 2.5 g/L and 5.0 g/L water dropped 3.29% and 4.18%, respectively, below that of the control groups in 0 g/L. After a 24-hour time-course experiment, this change in tissue water was found to largely occur within the first eight hours of exposure for the 2.5 g/L and 5.0 g/L groups. It was also noted that the hemolymph osmolality of both the 2.5 g/L and 5.0 g/L groups rose to approximately 78 mOsm/kg and 148 mOsm/kg, respectively, matching the osmolality of their exposure water in roughly the same time span and indicating that little behavioral avoidance of the elevated salinity was occurring. The osmolality of the control group did not match the osmolality of the 0 g/L water at 0.5 mOsm/kg, but was held at a constant level around 50 mOsm/kg. In a later experiment measuring the same variables for clams in 10.0 g/L, it was found that the tissue water and osmolality did not begin to change significantly until after 12 hours, indicating behavioral avoidance at this salinity level. A context study was also conducted comparing oxygen consumption and percent tissue water between various salinities in a light and dark exposure to determine if ambient light influenced siphoning of the clams and exposure to the salt. In this experiment, it was observed that clams held in salinities of 5.0 g/L for 24 hours consumed roughly 1.90 mg O2/L/g/h, whereas clams held in the control only consumed roughly 0.73 mg O2/L/g/h. These findings suggest that Corbicula osmoregulate in freshwater but osmoconform at salinities of 2.5 g/L and 5.0 g/L. The data from the context study also suggests that this conformation comes at a significant metabolic cost. Furthermore, and in contrast to the results of some previous studies, a significant level of behavioral avoidance of elevated salinity does not appear to commence until the clams are at a salinity above 5 g/L.

While a significant degree of salinity tolerance has been observed in the bivalve mollusk species Corbicula fluminea, the physiological and behavioral responses to changes in salinity by these organisms are not completely understood. It was hypothesized that Corbicula would initially avoid any salinity stress behaviorally through valve closure, but would eventually have to open to dispel anaerobic waste products and deal with the salinity. To explore this, Corbicula were collected and put through a series of experiments at salinity exposures of 0, 2.5, and 5.0ppt, with tissue water content and hemolymph osmolality being measured. After an initial 96-hour exposure period, it was observed that the tissue water content ratio of clams in 2.5ppt and 5.0ppt water dropped below that of the control groups in 0ppt. After a 24-hour time course experiment, it was observed that this change in tissue water largely occurred within the first eight hours of exposure for the 2.5ppt and 5.0ppt groups. It was also noted that the hemolymph osmolality of both the 2.5ppt and 5.0ppt groups rose to match the osmolality of the water in roughly the same time span. The osmolality of the control group did not match the osmolality of the 0ppt water, but was held at a constant level above it. In a later experiment measuring the same variables for clams in 10.0ppt, it was found that the tissue water and osmolality did not begin to change significantly until after 12 hours. The findings suggest that Corbicula osmoregulate at salinities lower than 2.5ppt, but osmoconform in salinities above that threshold. Furthermore, it seems that the clams are able and willing to tolerate conformation at 2.5ppt and 5.0ppt, but that they are reluctant to conform in 10.0ppt, behaviorally avoiding exposure for as long as possible.

A diferenciação morfofisiológica entre rainhas e operárias de Apis mellifera decorre da alimentação recebida durante o desenvolvimento larval, que estimula o aumento da produção de Hormônio Juvenil naslarvas que originarão rainhas. Dentre as diversas diferenças morfológicas entre operárias e rainhas encontramse estruturas especializadas para a coleta de pólen e própolis, localizadas na região da tíbia e do basitarso das pernas posteriores de operárias. A diferenciação das pernas tem início entre o quarto e o quinto estágio do desenvolvimento larval. Utilizandose Microscopia Eletrônica de Varredura o presente trabalho relata a presença das cerdas formando as estruturas castaespecíficas na fase de pupa de olho marrom. A partir de estudos de hibridação de microarrays de cDNA com amostras de RNA de A. mellifera de diversas fases do desenvolvimento larval, foram encontrados 91 genes com ortólogos conhecidos em Drosophila, diferencialmente expressos entre rainhas e operárias no período crítico da diferenciação de castas. Destes, cinco estão relacionados com o desenvolvimento de apêndices: ataxin2 (atx2), cryptocephal (crc), dachshund (dac), grunge (gug) e Retinoic and fat acid Binding Protein (RfaBP). O perfil destes genes, e ainda, ultrabithorax (ubx), distalless(dll) e abdominalA (abdA) (estes porsuassuasfunções durante a diferenciação das pernas de insetos) foram analisados por RTPCR em Tempo Real em pernas posteriores de operárias e rainhas desde o quarto estágio larval até o estágio de pupa de olho branco. Apenas ubx e abdA foram encontrados mais expressos em operárias ao final do desenvolvimento larval e início do desenvolvimento pupal. Estudossimilares dos genes abdA, dac, dll e ubx nossegmentos das pernas de pupas de olho branco indicam a tíbia como domínio de expressão de dac. Imunolocalizações utilizando um anticorpo contra um epitopo conservado entre Ubx e AbdA, FP6.87, em pernas posteriores de prépupas de operárias e rainhasrevelam a presença destas proteínas na tíbia apenas de operárias e diferencialmente localizadas no basitarso de operárias e rainhas. Os dados acima apresentados apontam Ubx, um gene Hox, como pontochave na regulação da formação das estruturas castaespecíficas. / Diphenism in the honey bee, Apis mellifera,resultsfromdifferential feeding of female larvae. Among the morphological differences, the hind legs of workers have structures that is used for carrying pollen and propolis, e.g. the corbicula, while the queens hind legslack thisstructures. The corbicula is an expanded region of the tibia deprived of bristles, which has a single bristle in the middle that seems to have a sensorial function. Using scanning electronic microscopy, we found that the leg structures and bristles of the corbicula are already formed in browneyed pupa. Microarray analysis has demonstrated that five of 240 differentiallyexpressed genesin developing castes are potentially related to the caste differences in leg development (ataxin2, cryptocephal, dachshund, grunge and Retinoic and fat acid Binding Protein). Using qPCR, we analyzed the expression of abdominalA, ataxin2, cryptocephal, grunge, Retinoic and fat acid Binding Protein and ultrabithorax genes during hind leg development. cryptocephal, ataxin2, grunge and Retinoic and fat acid Binding Protein genes, which are involved in imaginal disc elongation and bristle formation and are inhibited by juvenile hormone, were not found to be differentially expressed. However, ultrabithorax and abdominalA are over expressed in workersin the early pupalstage. By using immunohistochemistry, Ubx was localized in the tibia and basitarsus of prepupae of workers and in the basitarsus of pre pupae of queens. The pattern of Ubx expression suggests that this Hox gene is a key player in leg structuresformation and caste differentiation in A.mellifera.

American Electric Power's (AEP) coal-fired Clinch River Plant, a power-generating facility in Carbo, Russell County, Virginia (USA), has impaired Clinch River biota through toxic spills in 1967 and 1970, and effluent copper (Cu) concentrations that were reported to have exceeded water quality criteria from 1985-1989. These impacts have provided impetus for many research projects addressing the absence of bivalves, including federally protected species of native mussels (Unionoidea), from sites influenced by CRP effluent. Modifications in CRP effluent during 1987 and 1993 drastically reduced Cu levels and warranted the present study, which assessed long-term biological recovery in Clinch River biota near the CRP. In 2000-2001, surveys of benthic macroinvertebrate communities and instantaneous measures of effluent toxicity did not foretell significant reductions in survivorship and growth of field-caged Asian clams (Corbicula fluminea) at sites downstream of the CRP. More importantly, these results indicated renewed toxicity in CRP effluent. Additional transplant studies using two enclosure types were conducted to isolate effects attributable to CRP effluent from the potentially confounding effects of substrate variability among study sites. While it was found that mean growth of clams was greatest in the enclosure that minimized substrate variability (p=0.0157), both enclosure types clearly distinguished significant impairment of survivorship and growth at sites downstream of the CRP discharge, and strengthened the association between impairment and CRP effluent. An intensive field investigation was undertaken to determine whether impairment observed in transplant studies extended to resident bivalves. During 2001-2002, densities and age structures of C. fluminea and distributions of mussels suggested that impairment indeed extended to resident bivalves for a distance of 0.5 to 0.6 km downstream of the CRP discharge. Impairment of bivalves was less evident below (1) a fly ash landfill and (2) coal mining activities and low-volume leachate from a bottom ash settling pond. With respect to long-term recovery, modifications in CRP effluent treatment have reduced Cu concentrations from an average of 436 mg/L in 1985-1989 to 13 mg/L in 1991-2002. Subsequently, Cu body burdens of Asian clams (Corbicula fluminea) transplanted within CRP influence have decreased from 442% of levels accumulated at reference sites in 1986, to 163% of these levels in 2002. The reduction in effluent Cu largely explains recovery of most benthic macroinvertebrate community parameters (e.g., richness, diversity) at influenced sites from levels that were typically less than 70% of reference levels, to levels that frequently range from 80 to greater than 100% of reference levels. Nevertheless, bivalves remain impaired downstream of the CRP; survivorship and growth of C. fluminea transplanted to CRP-influenced sites have typically been less than 40 and 20% of reference values, respectively. Furthermore, C. fluminea has seldom been encountered within CRP influence for nearly two decades. Likewise, native mussels remain absent within CRP influence, but recent surveys suggest their downstream distributions are more proximate to the CRP discharge than has been reported previously. A preliminary assessment of factors potentially contributing to toxicity revealed that (1) water reclaimed from settling basins for discharge with CRP effluent significantly impaired fecundity of ceriodaphnids at concentrations of 50%, (2) LC50 values for industrial treatment chemicals were misrepresented on Material Safety Data Sheets and consequently, were subject to misapplication by operators, (3) Cu concentrations of 96 mg/L significantly impaired growth of Asian clams in artificial stream testing, and (4) effluent Al exceeded acute and chronic water quality criteria, suggesting this ion should receive further consideration in future studies. / Master of Science

The studies described herein focused on the use of intermittent halogenation to control biofouling of water intake systems by the zebra mussel, Dreissena polymorpha, the comparative response of zebra mussels and the Asian clam, Corbicula fluminea, to a surfactant -based chemical control agent, the nontarget impact associated with the control agent, and the use of the Asian clam as a biomonitor of the control agent. Effects of intermittent (2-4 hr/day) treatments with chlorine or bromine at levels of 0.5 and 1.0 mg/L (total residual oxidant) upon settling of zebra mussel veligers were examined in studies conducted in a field laboratory on western Lake Erie. Veliger densities in the water column at the field site peaked at 530/L, while mussel densities on settling monitors reached 147,083/m² over the course of the study period. Zebra mussel settling in test systems treated with the halogens was reduced by as much as 91 % in comparison with controls, although mussel densities of up to 6,044/m² still occurred. Treated mussels which remained settled had growth rates similar to controls, and reached 2-4 mm length over 30 days. The intermittent halogen treatments had no significant impact on either adult zebra mussels or Asian clams. The studies indicate that while the treatment regimes may reduce zebra mussel densities within intake systems, the threat of eventual fouling due to cumulative settling remains. / Ph. D.

Cellulolytic responses of the Asiatic clam, Corbicula fluminea and a snail, Mudalia dilatata, to selected constituents of power plant effluents (i.e., zinc, cadmium, acidic and alkaline pH, individually and paired) were investigated in 30-day exposures. Exposures were conducted in both laboratory and field-oriented artificial streams and then validated in the river receiving system of a power plant. Cellulolytic activity was reduced by laboratory and field exposures to cadmium and zinc at all levels tested from 0.012 to 0.10 mg cadmium/L and generally at 0.025 to 1.0 mg zinc/L. Clams detected acute lethal levels of metal and used valve closure as an avoidance mechanism for 14 days. Snails, however, did not effectively avoid exposures and were more sensitive to acute stress during all exposures. These behavioral responses were corroborated by both cellulolytic activity and metal accumulation. Measurements of cellulolytic activity for both test species in laboratory exposures differed from those in field artificial streams. Reduced enzyme activity in controls by day 30 was attributed to artificially induced stress associated with the laboratory environment. This factor precluded any analysis of laboratory responses for periods of exposure longer than 20 days as well as recovery analysis. Field oriented artificial streams provided a sufficient environment to adequately assess long-term stress and recovery as measured by cellulolytic activity and metal accumulation in both clams and snails. Enzyme activity responded to metal exposure with respect to both degree and duration of exposure. Cadmium and zinc combined exposures caused significantly reduced cellulolytic activity at the same concentration as those for cadmium alone. Reduced enzyme activity caused by cadmium and zinc addition at levels that were not detectable suggested that the cellulolytic index was sensitive to sublethal stressors. This was supported by metal uptake patterns in clams and snails. Cellulolytic activity responded to zinc addition at alkaline and acidic pH in a manner that supported pH optima for cellulases and bioavailability of metals. Effects seen in macroinvertebrate assemblages (diversity, richness, and similarity) were compared with cellulolytic activity of caged Corbicula from a site specific power plant discharge. Enzyme activity inhibition was the most sensitive indicator measured. Reductions in cellulolytic activity at stations monitored for total zine content were consistent with effects seen at comparable exposures to zine in field-located artificial streams. A zine concentration of 0.05 mg/L consistently caused the first significant reductions in cellulolytic activity. This concentration is comparable to the U.S. Environmental Protection Agency's Water Quality Criteria value (0.047 mg/L zinc) for protection of aquatic life. / Ph. D.

L’urbanisation croissante et la propagation des activités humaines entrainent l’augmentation de l’apport de nombreux micropolluants dans le milieu aquatique. Le compartiment aquatique est alors défini comme le réceptacle ultime de la pollution environnementale apportée par diverses sources : les effluents municipaux et industriels, les lixiviats des sites d’élimination des déchets solides, le ruissellement des eaux urbaines et des eaux des sites agricoles. Parmi les contaminants retrouvés dans l’eau, il y a les psychotropes (benzodiazépines, antidépresseurs,antiépileptiques, etc…) qui appartiennent à la grande famille des médicaments. Ce sont des molécules biologiquement actives, ubiquistes, qui sont principalement d’origine urbaine, qui sont apportées continuellement dans l’environnement aquatique et qui sont fortement consommées au niveau mondial. Depuis quelques années, les scientifiques commencent à s’intéresser à cette famille thérapeutique mais il existe encore un réel manque de connaissance sur l’intégralité du problème de diffusion des psychotropes,de leur passage en station d’épuration à leur présence dans l’environnement et enfin à leur impact sur les organismes aquatiques. Ces travaux de thèse cherchent donc à documenter ce point en se focalisant sur l’étude de la présence, du devenir et de l’impact des psychotropes dans la Garonne estuarienne (France) et le fleuve Saint Laurent (Québec, Canada). Dans un premier temps, une méthode analytique multi-résidus permettant l’analyse de 47 psychotropes dans les eaux usées et les eaux de surface a été développée et validée. Des échantillonneurs passifs de type POCIS (Polar Organic Chemical Integrative Sampler) ont été calibrés et validés afin de permettre le suivi intégratif et continu des psychotropes dans les eaux de surface.Dans un second temps, les effluents d’entrée et de sortie de station d’épuration des agglomérations de Bordeaux en France et de Repentigny et Montréal au Québec ont été caractérisés. Les résultats de cette étude montrent que les traitements épuratoires de chacune des stations d’épurations étudiées ont une efficacité limitée pour éliminer les psychotropes dans les eaux usées. Par la suite, le suivi effectué dans la Garonne estuarienne et dans le fleuve Saint Laurent a mis en évidence la présence de psychotropes dans l’environnement qui sont majoritairement apportés par l’amont des villes de Bordeaux et Montréal.La comparaison des résultats obtenus pour la France et le Québec montre qu’il existe bien des habitudes de prescriptions et de consommations différentes entre les deux pays. Même si les procédés d’épuration sont différents entre les deux pays (traitement secondaire en France et primaire au Québec),l’efficacité d’élimination sur la famille thérapeutique des psychotropes reste insuffisante. Enfin ila été observé que le débit du milieu récepteur jouait un rôle important dans la dilution et le devenir des composés.Dans un troisième et dernier temps, des études in vitro et in vivo ont montré que certains psychotropes pouvaient avoir un impact toxique sur les bivalves d’eau douce étudiés (Elliptio complanata et Corbiculafluminea) lors d’exposition de composés seuls ou en mélange à des concentrations de l’ordre du mg.L-1 (concentrations expérimentales) et même du ng.L-1 (concentrations environnementales). / Increasing urbanization and human activities cause an increase of micropollutant input in the aquatic environment. The water compartment is then defined as the ultimate receptacle of environmental pollution originating from various sources: municipal and industrial wastewaters, leachates from solidwaste disposal sites, waters of urban and agricultural runoff.Among the contaminants found in the water, there are psychotropic drugs (benzodiazepines, antidepressantsor antiepileptics) belonging to the family of pharmaceuticals. These are biologically active molecules, ubiquitous, which are mainly of urban origin, which are continuously introduced into the aquatic environment and are heavily consumed around the world.In recent years, scientists have begun to show interest for this therapeutic family, but there is still areal lack of knowledge about the full psychotropic diffusion problem; from their presence in wastewater treatment plant to their presence in the environment and finally to their impact on aquatic organisms.In this global perspective these thesis works seek to document the presence, fate and impact of psychotropic drugs in the Garonne estuarine (France) and the St. Lawrence River (Quebec, Canada).Firstly, a multi-residue analytical method for the analysis of 47 psychotropic drugs in wastewater and surface waters has been developed and validated. POCIS passive samplers were calibrated and validated for continuous integrative monitoring of psychotropic drugs in surface waters.Secondly, the treatment plant influents and effluents of Bordeaux in France and Repentigny and Montreal in Quebec have been characterized. The results of this study show that treatment processes ofstudied wastewater treatment plants have limited performances to remove psychotropic drugs in wastewaters. There after, the monitoring performed in the Garonne estuarine and in the St. Lawrence River has highlighted that the presence of psychotropic drug in the environment is mainly related toupstream input of Bordeaux and Montréal.Comparison of the results obtained in the two countries surveyed show that there are many different patterns of prescriptions and consumption between the two countries. Although the treatment processes are different between the two countries (secondary treatment in France and primary in Quebec), their removal efficiency is still insufficient for psychotropic drugs. Finally it was observed that theflow rate of the receiving environment plays an important role in the dilution and the fate of the compounds.In a third and final stage, in vitro and in vivo studies have shown that some psychotropic drugs could have a toxic impact on the studied freshwater bivalves (Elliptio complanata and Corbicula fluminea) upon exposure alone or in mixture with concentrations in the order of mg.L-1 (tests concentrations) and even ng.L-1 (environmental concentrations).

A study of Asian clam (Corbicula fluminea Müller) colonization in relation to changes in aquatic vegetation community as a result of management of Hydrilla verticillata (L. f.) Royle with grass carp was conducted at the Lewisville Aquatic Ecosystem Research Facility (LAERF), Lewisville, TX, from April 2015 through October 2016. Percent vegetation cover, C. fluminea abundance and water quality metrics (pH, turbidity, conductivity, DO, calcium, chlorophyll a) from 16 experimental subjects were analyzed. Treatments included four replicated grass carp stocking densities; 1-control with no fish stocked (n = 4), 2-low density of 40-43 fish per vegetated ha (n = 4), 3-medium density of 72-81 fish per vegetated ha (n = 4) and 4-high density of 110-129 fish per vegetated ha (n = 4). Data analysis showed statistical significance in the relation of C. fluminea abundance to percent vegetation cover (multiple linear regression, r2 = 0.820), grass carp stocking densities (two-way analysis of variance, p = <0.001) and chlorophyll a (multiple linear regression, r2 = 0.339). Findings of this research indicate the possibility that management of hydrilla had enabled establishment of secondary invasive species.

Dischargers to the Trinity River in North Texas were required to dechlorinate their effluents in 1990-91. Field surveys were conducted above and below an outfall to determine the response of resident immature insects and caged in situ juvenile Asiatic clams to chlorinated and dechlorinated effluent. Within six months after dechlorination began, insect community composition and C. fluminea survival significantly improved at stations below the outfall. Significantly lower clam growth within one mile below the dechlorinated effluent indicated the presence of non-chlorine toxicants. Effects from chlorinated and dechlorinated effluent exposure were comparable between Ceriodaphnia dubia lab tests and in situ C. fluminea.