This study used rag1-/- mutant zebrafish, which lack functional T and B lymphocytes, to investigate whether innate immune cells from vaccinated mutant zebrafish demonstrate enhanced survival compared to phagocytes from naïve mutant fish. Edwardsiella ictaluri, an economically significant aquatic pathogen and the causative agent of enteric septicemia of catfish (ESC), was used for the trials. Quantification of live bacteria from sampled kidneys was accomplished via colony counts, luminescence readings, and differential DNA extractions using Ethidium Monoazide (EMA) and Propidium Monoazide (PMA) followed by qPCR. There was a general trend of less bacteria in vaccinated mutant fish. Additionally, the mortality in the vaccinated fish was less than the naïve group, suggesting that the vaccinated fish are better able to withstand the bacteria load. Giemsa-stained cytospins showed E. ictaluri exclusively within macrophages from sampled kidneys, suggesting that the macrophages are the critical site of pathogenesis in rag1-/- zebrafish.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Approximately 30% of patients with epilepsy do not achieve adequate seizure control through current anti-seizure drugs (ASD) and treatment methods. Therefore, a critical need exists to efficiently screen ASDs to enhance our ability to tailor treatment protocols and improve patient outcomes. The zebrafish pentylenetetrazol (PTZ) seizure model has become an increasingly popular screening paradigm for novel ASDs. Here, we present an optimized PTZ assay to improve reliability and reproducibility based on work in our laboratory. This optimized assay improves robustness in our screening of anti-seizure drugs (topiramate, lamotrigine, carbamazepine and GS967). These findings show that electroencephalogram (EEG) and calcium sensitive GFP from fusion protein (GCaMP) assays largely correlate with the behavioral findings, helping us connect physiological and behavioral responses to ASDs. Genetic epilepsy syndromes, like voltage gated sodium channel SCN2A and SCN8A pathogenic variants, are often poorly controlled by current medications. Our optimized assay relied on a fast and precise zebrafish seizure model using mRNA overexpression of hSCN2A and hSCN8A variants including: hSCN2A R1882Q and R853Q and hSCN8A R1872Q. All three pathogenic variants increased seizure activity, and the ASDs significantly decreased this seizure activity. This mRNA overexpression assay can be used to quickly evaluate seizure activity induced by pathogenic variants in voltage gated sodium channel genes and test ASDs to determine efficacy. In a separate study, we tested if the addition of the human SCN2A sodium channel could potentially rescue the loss of the zebrafish scn1Lab gene. Our GCaMP assay data indicates that this loss was successfully rescued. Cumulatively, these findings can be used to improve the screening of novel ASDs and treatments for patients with refractory epilepsy.

The vertebrate nervous system arises during embryogenesis from an epithelial sheet of cells called the neural plate that subsequently folds to become a rod of cells called the neural tube. Several signaling pathways act on the neural progenitors of the neural tube to give rise to the diverse set of neurons and glia that will make up the spinal cord and brain in adulthood. In vertebrates, Muscle segment homeobox (Msx) genes are expressed in the dorsal neural tube during development, and pattern dorsal neural progenitors to give rise to dorsal neuronal subtypes. Additionally, Msx genes are involved in the regulation of neurogenesis and proliferation in the neural tube. In zebrafish, three msx genes are expressed in the neural tube: msx1a, msx1b, and msx3. The Akimenko lab has identified a potential enhancer of msx3 called Fragment C that drives expression in the dorsal neural tube. We hypothesized that Fragment C is a bona fide enhancer of msx3 specifically in the neural tube, and that this enhancer contributes toward proper patterning and neurogenesis/proliferation in the developing neural tube of zebrafish. To test this hypothesis, I have generated zebrafish mutants with a deletion of the Fragment C enhancer using CRISPR/Cas9 that also have a transgenic Fragment C enhancer driving reporter expression of enhanced green fluorescent protein (Egfp). The deletion of Fragment C abolishes msx3 expression in the neural tube excluding the dorsal-most cells likely corresponding to the roof plate. The spatial domain in which msx3 is lost corresponds to where Fragment C drives expression in the neural tube, suggesting that Fragment C contains an enhancer of msx3. This domain of expression corresponds to where dorsal neural progenitors reside. Analysis of markers for the cells with Fragment C-driven Egfp expression shows that at least some of these cells are indeed neural progenitors, many of which give rise to neurons during embryonic and larval development. The deletion of Fragment C and loss of msx3 expression in neural progenitors did not affect the numbers of neurons or neural progenitors amongst cells with Fragment C-driven expression, nor did it affect the dorsoventral location of cells in the neural tube. Taken together, we conclude that Fragment C-driven msx3 expression does not contribute to the dorso-ventral position of neural progenitors nor the balance of proliferation and neurogenesis in the developing neural tube. However, a role for msx3 in regulating neural progenitor identity along the dorso-ventral axis without affecting progenitor position cannot be ruled out.

No description available.

No description available.

Zebrafish (Danio rerio) are ray-finned fish of the teleost class, whose fins consist of an exoskeletal domain and an endoskeletal domain. The exoskeletal domain of the fins contains the fin rays and originate from embryonic fin folds that elongate as the fins are growing. The elongation of the fin fold is supported by two parallel sets of rigid fibrils oriented along the proximal-distal axis, called actinotrichia. Actinotrichia fibrils are composed of two primary components, a collagenous component and actinodin proteins. The actinodin proteins are encoded for by the actinodin (and) family of genes which are found in the genomes of finned fish while absent in limbed tetrapods. CRISPR/Cas9 was used to create loss-of-function deletions in the and1 and and2 genes, resulting in the absence of actinotrichia in the zebrafish double mutants. We hypothesised that the loss of actinotrichia during zebrafish development would result in developmental defects leading to fin ray defects in the adult zebrafish. The and1/2 mutants that lack actinotrichia presented with fin fold and cell migration defects during development that persisted into adulthood and resulted in shorter fins, disturbed fin ray patterning, and a decrease in ray number. In addition, an unexpected fusion between the hypurals of the caudal fin endoskeleton revealed an additional function of the actinotrichia fibrils in caudal fin endoskeletal patterning. During zebrafish development, actinotrichia fibrils play a vital role in ensuring normal fin development, normal patterning and formation of the fin rays, and the normal development of the caudal fin endoskeleton.

Pharmaceuticals are emerging surface water contaminants, and are manufactured, used, and released into environment in considerable amounts. Concerns have been raised due to the inherent potency and bioactivity of these molecules, which makes effects at low concentrations more likely. The ubiquitous presence and stability of pharmaceuticals brings up concerns about the frequency and length of exposures. However, the distribution and fate of these compounds in surface water bodies is not clear. There is limited information about the potential effects in non-target, especially aquatic, species vulnerable to cumulative or lifelong exposures. Carbamazepine (CBZ) and gemfibrozil (GEM) are two of the most frequently detected pharmaceuticals in surface water. This thesis examined sub-lethal adverse reproductive effects of chronic direct exposure of CBZ and GEM to F0 zebrafish and several generations of unexposed offspring; the effects of exposure on testicular steroidogenesis were also examined. Chronic exposure of zebrafish to CBZ and GEM reduced ex vivo production of 11KT in testes. In vivo, CBZ decreased reproductive output, 11-ketotestosterone (11KT), male courtship and aggression behaviours, and sperm morphology in F0 parents. The F1, F2 and F3 offspring of CBZ exposed males had lower reproductive output, altered courtship, aggression, sperm morphology and lower 11KT compared to fish from the unexposed lineage. The adverse effects persisted into the F3 generation which suggested transgenerational paternal effects. GEM decreased reproductive output in F0 parents and a reduction in 11KT, altered male courtship, aggression and sperm morphology. Unexposed F1 male offspring, but not other generations, had sub-lethal toxic effects from parental exposure. We therefore suggest that CBZ and GEM act as endocrine disruptors in fish and that chronic exposure may reduce male reproductive fitness. / Dissertation / Doctor of Philosophy (PhD) / Human pharmaceuticals reach aquatic environments through municipal wastewater. The bioactivity of pharmaceuticals at low concentrations has raised concerns about undesired effects in aquatic species like fish, which can experience chronic exposures. This thesis examined adverse reproductive effects of direct chronic exposure of carbamazepine and gemfibrozil to parental zebrafish and their un-exposed offspring for multiple generations. Exposure to both compounds reduced androgens and reproduction and altered behaviour, and sperm quality in males. Effects persisted in the unexposed offspring. Parental carbamazepine exposure impacted multiple generations. We suggest that carbamazepine and gemfibrozil may reduce male reproductive fitness by reducing male sex steroids.

O Kraft 36EC® (i.a. abamectina) e o Score 250EC® (i.a. difenoconazol) são agrotóxicos intensamente utilizados nas culturas de morango em regiões de clima tropical, embora sejam compostos classificados como extremamente tóxicos e muito perigosos ao ambiente. Misturas de agrotóxicos são aplicadas nas culturas agrícolas e a presença destes compostos na água pode potencializar os efeitos tóxicos a organismos aquáticos não alvos. A ocorrência do escorrimento superficial (runoff) contaminado com agrotóxico e a contaminação por aspersão direta (spray drift) em ecossistemas tropicais próximo aos corpos hídricos também podem comprometer o ecossistema aquático local devido à toxicidade desses compostos a organismos não alvos. Considerando os riscos ecológicos inerentes ao uso destes agrotóxicos, o objetivo principal desta pesquisa foi avaliar o efeito ambiental dos praguicidas Kraft 36EC® e Score 250EC® e de seus princípios ativos, abamectina e difenoconazol, respectivamente. Foram realizados testes de toxicidade aguda em laboratório a partir de exposições dos compostos isolados e em misturas utilizando como organismo teste embriões e adultos de Danio rerio. Experimentos in situ (mesocosmos) foram realizados a fim de avaliar os efeitos do runoff e spray drift contaminados com os agrotóxicos Kraft 36EC® e Score 250EC®, isolados e em misturas. Tanto nos mesocosmos quanto em experimentos de laboratório analisou-se, além da mortalidade, biomarcadores bioquímicos. Pelos resultados obtidos verifica-se que não foram observadas diferenças significativas entre a toxicidade isolada dos ingredientes ativos e suas respectivas formulações comerciais em testes agudos. Nos testes de toxicidade com as misturas dos ingredientes ativos, observou-se que a mistura de abamectina + difenoconazol promoveu um efeito tóxico sinérgico a adultos de Danio rerio em exposições agudas. Observou-se também a ausência do mesmo efeito nas exposições com as misturas das formulações comerciais neste mesmo estágio de vida. A exposição dos embriões às misturas de Kraft 36EC® e Score 250EC® mostrou um efeito antagônico nas baixas concentrações e efeito sinérgico nas mais altas. Verificou-se um aumento significativo na atividade da enzima de biotransformação 7-etóxiresorufina-0-deetilase (EROD) e nos níveis de malondialdeído MDA nas brânquias de peixes expostos à formulação comercial da abamectina. Houve um aumento na atividade da glutationa-S-transferase (GST), glutationa peroxidase (GPx) e níveis de MDA, e diminuição da glutationa redutase (GR) nas brânquias dos peixes expostos ao difenoconazol e sua formulação comercial. Também foi observado um aumento nas respostas dos biomarcadores analisados nas brânquias dos organismos expostos às misturas tanto dos princípios ativos quanto das formulações comerciais. A exposição ao Kraft 36EC® spray drift em campo promoveu os maiores efeitos deletérios no metabolismo de Danio rerio, seguido das exposições ao runoff contaminado com Kraft 36EC® e Score 250EC®. As formulações comerciais Kraft 36EC® e Score 250EC® e as misturas das mesmas promoveram alterações significativas no metabolismo de detoxificação, e causam estresse oxidativo em peixes. Diferenças no padrão de respostas dos biomarcadores entre os experimentos realizados em mesocosmos e em laboratório ficam evidentes devido à influência das concentrações utilizadas, das interações ecológicas entre as comunidades presentes no meio e das variáveis ambientais nos experimentos em mesocosmos. A avaliação da toxicidade de agrotóxicos, especialmente em países tropicais, e indicações para futuras pesquisas são discutidos. / Kraft 36EC® (a.s. abamectin) and Score 250EC® (a.s. difenoconazole) are intensely used pesticides in strawberry crops in tropical regions, even though they are classified as extremely toxic and very dangerous to the environment. Mixtures of agrochemicals are applied to agricultural crops and the presence of these compounds in water may potentiate toxic effects to non-target aquatic organisms. The contamination of edge-of-field water bodies through runoff and spray drift may compromise the local aquatic ecosystem due to the toxicity of these compounds to non-target organisms. Considering the potential ecological risks related with the use of these pesticides, the main objective of this research was to evaluate the environmental effects of the pesticides Kraft 36EC® and Score 250EC® and its active ingredients abamectin and difenoconazole, respectively. Acute toxicity tests were performed in the laboratory with the isolated compounds and their mixtures using zebrafish (Danio rerio) embryos and adults as test organism. A mesocosm experiment was also performed to evaluate the effects of simulated runoff and spray drift containing Kraft 36EC® and Score 250EC® individually and in mixtures. Besides lethality, selected biomarkers were also evaluated in the fish from the mesocosm and laboratory experiments. No significant differences were observed in effects of the active ingredients and their respective commercial formulations in acute fish tests after single exposure to each test compound. The acute mixture toxicity tests with the active ingredients showed that this mixture exerts a synergistic toxic effect to adult fish. The absence of the synergistic effect on exposures with the commercial formulations mixtures was also observed. Embryo exposure to Kraft 36EC® and Score 250EC® mixtures showed an antagonistic effect at low concentrations and synergistic effect at the highest. A significant increase was observed in the activity of the EROD biotransformation enzyme and in the levels of MDA in fish gills after exposure to Kraft 36EC®. An increase in GST activity, GPx and MDA levels, and a decrease of GR in fish gills was noted after fish exposure to difenoconazole and its commercial formulation. An increase in the responses of the biomarkers analyzed in the gills of the organisms exposed to the mixtures of both the active ingredients and the commercial formulations as compared to single-compound exposure was also observed. Exposure to simulated spray drift of Kraft 36EC® in the mesocosms promotes the greatest deleterious effects on metabolism of Danio rerio followed by exposures to the runoff contaminated with a Kraft 36EC® and Score 250EC® mixture. The commercial formulations Kraft 36EC® and Score 250EC® and their mixtures lead to significant changes in the detoxification metabolism resulting in oxidative stress to fish. Differences in biomarkers responses between the experiments performed in the mesocosms and the laboratory were noted and are due to the different concentrations tested; the ecological interactions between the communities present in the mesocosms and differences in environmental variables. The need and indications for future research related with the evaluation of pesticide toxicity, especially in tropical countries, are discussed.

Os éteres de difenilas polibromados (PBDEs) são largamente usados como retardantes de chama e têm sido detectados em diversas amostras biológicas, devido às suas características físico-químicas e bioacumuladoras, as quais fazem com que estes compostos tenham uma elevada persistência no meio ambiente. A exposição a estes compostos pode ser nociva ao homem e aos organismos vivos, uma vez que muitos estudos têm relatado que este composto possui atividade citotóxica. Desta forma, no presente estudo, investigamos o mecanismo de ação dos BDE-47, BDE-99, BDE-154 e BDE-209 sobre o processo autofágico de células HepG2, pois além de ser um processo de reciclagem de moléculas intracelular, a autofagia atua ainda na citoproteção celular. Foram realizados ensaios de imunocitoquímica para detecção de alterações no processo autofágico, no qual foi avaliado, sob microscopia de fluorescência, o padrão de distribuição das proteínas LC3-I e LC3-II; paralelamente, foi realizada a técnica de western blotting para a quantificação de três proteínas associadas ao processo de autofagia - LC3, Beclin-1 e p62. Em nossas análises de imunocitoquímica pode-se observar que os quatro PBDEs em estudo foram capazes desencadear o processo de autofagia, sendo que a magnitude do efeito tóxico foi dependente do tempo de exposição (24 e 48 horas), da concentração utilizada (0,1; 1; 5; 25 ?mol/L) e variável de acordo com o congênere em questão, sendo os BDE-47 e BDE-99 os que apresentaram maior potencial de ativação da autofagia. Além disso, esta indução de autofagia ainda pode ser detectada nos ensaios de western blotting, pois os PBDEs alteraram os níveis de LC3-II, Beclin-1 e p62, havendo um padrão de toxicidade semelhante ao visualizado nos ensaios de imunoistoquímica. Adicionalmente, devido ao fato dos PBDEs poderem se acumular no meio ambiente, principalmente em organismos aquáticos, uma análise dos efeitos destes compostos sobre um modelo ecotoxicológico foi realizada. O zebrafish (D. rerio) foi utilizado como organismoteste, a fim de avaliar a toxicidade destes contaminantes ambientais sobre seus estágios embrionários e larvais. Foram analisados endpoints de letalidade, subletalidade e teratogenicidade após a exposição aos PBDEs em estudo. Sendo que os compostos BDE-47, BDE-99 e BDE-209 (na presença de luz) apresentaram alterações em pelo menos um dos indicadores avaliados, sendo que o parâmetro mais afetado foi a inflação das bexigas natatórias, indicando uma possível teratogenicidade causada por estes compostos. Por outro lado, os BDE-154 e BDE- 209 (na ausência de luz) não causaram malformações no organismo-teste. Diante dos resultados obtidos, pode-se concluir que estes compostos podem causar alterações na homeostase celular desencadeando o processo autofágico, sendo que se absorvidos por organismos, podem afetar o desenvolvimento de espécies como o zebrafish. / Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants and they have been detected in several biological samples due to their physicochemical and bioaccumulating characteristics, which make these compounds to have a high persistence in the environment. The exposure to these compounds can be harmful to man and other living organisms, since many studies have reported that this compound has cytotoxic activity. Thus, in the present study, we investigated the mechanism of action of the BDE-47, the BDE-99, the BDE-154 and the BDE-209 on the autophagic process of HepG2 cells, because beyond being an intracellular molecule recycling process, autophagy acts also in the cell cytoprotection. Immunocytochemistry assays were performed to detect changes in the autophagic process, in which the distribution pattern of the LC3-I and LC3-II proteins was evaluated under fluorescence microscopy. In parallel, the western blotting technique was used to quantify three proteins associated with the autophagy process - LC3, Beclin and p62. In our analyzes of immunocytochemistry it can be observed that the four PBDEs in the study were able to trigger the autophagy process, and the magnitude of the toxic effect was dependent on the exposure time (24 and 48 hours), on the used concentration (0.1; 1; 5; 25 ?mol/L), and it varies according to the congener. The BDE-47 and BDE-99 are the ones with the highest potential for autophagy activation. In addition, this autophagy induction can still be detected in the western blotting assays, because the PBDEs altered the levels of LC3-II, Beclin-1 and p62, with a similar pattern of toxicity as seen in the immunohistochemical assays. In addition, due to the fact that PBDEs may accumulate in the environment, mainly in aquatic organisms, an analysis of the effects of these compounds on an ecotoxicological model was performed. The Zebrafish (D. rerio) was used as a test organism in order to evaluate the toxicity of these environmental contaminants on their embryonic and larval stages. In this study, lethality, sublethality and teratogenicity endpoints were analyzed after exposure to the PBDEs. The BDE-47, BDE-99 and BDE-209 compounds (in the presence of light) presented alterations in at least one of the indicators evaluated, and the most affected parameter was the swim bladder inflation, indicating a possible teratogenicity caused by these compounds. On the other hand, the BDE-154 and the BDE-209 (in the absence of light) did not cause malformations in the test organism. Considering the results, it can be concluded that these compounds are able to cause changes in cellular homeostasis triggering the autophagic process, and whether absorbed by organisms, they can affect the development of species such as zebrafish.