This thesis is composed of two chapters. Chapter one reviews what is known about adaptive plasticity in response to predators, describes the physiological systems involved in such plasticity, and outlines the evolutionary consequences of adaptive plasticity. Chapter two describes a scientific experiment that investigates how malathion may impact adaptive plasticity in the Sierran Treefrog, Pseudacris sierra. Anuran tadpoles suffer high mortality rates due to predation. In response to strong selective forces relating to these high predation rates, tadpoles evolved the ability to adaptively respond to predators through morphological and behavioral plasticity. The morphological and behavioral responses are varied and depend on the hunting strategy of the predator, and the adaptive responses may be influenced by other biotic and abiotic factors. Tadpoles detect alarm cues released from tadpoles being eaten and kairomones that are released by predators. Tadpoles respond to these signals by changing tail and body shape along with a reduction of activity level, which enables tadpoles to escape predators more effectively. These changes in morphology can occur within a week, and behavioral changes can occur within 15 minutes. The adaptive responses are critical for increasing survival rates of tadpoles to metamorphosis and may have important evolutionary consequences for anurans. Amphibians are in decline worldwide, and pollutants are considered to be a major contributor to these declines. Every year 5.2 billion pounds of active ingredients of pesticides are applied worldwide, and these application rates have led to ubiquitous low-level contamination of aquatic ecosystems. How low-level contamination of pesticides directly and indirectly affect how tadpoles respond to their predators is poorly understood. One potential indirect effect of pesticides is the inhibition of adaptive plasticity. Pesticides have been shown to modulate corticosterone levels in tadpoles. Corticosterone is the most likely mediator of the physiological response that results in adaptive morphological change. If the physiological system of tadpoles relies on corticosterone as the mediator of adaptive response, and pesticides can modulate corticosteone levels, then pesticides may inhibit or negatively impact adaptive responses to important biotic factors, like predators. Pesticides have been shown to weaken immune systems, affect developmental and physiological pathways that lead to malformations, and cause direct mortality in anurans. Little research has investigated the effect of pesticides on adaptive morphological and behavioral plasticity in response to predators. Adaptive phenotypic responses to predators increase survival rates to metamorphosis and are important in stabilizing amphibian populations through time. If pesticides influence the ecological interactions of tadpoles and their predators, this could play a part in amphibian declines. In the experiment explained in Chapter two, I tested the hypothesis that malathion at a concentration of 0.1 mg/L inhibits anti-predator morphological and behavioral responses of Pseudacris sierra to the predatory dragonfly larvae Anax junius. The results of this experiment show that malathion alone caused the tail muscle depth to increase to the same magnitude as tadpoles that only experienced a predator’s presence. Malathion also caused a significant increase in tail depth, demonstrating that malathion directly causes morphological change. The experiment did not support the hypothesis that malathion inhibits adaptive plasticity, and malathion had no impact on behavioral plasticity. The results from this experiment give evidence that an ecologically relevant concentration of malathion can influence morphological components that are critical in escaping depredation events, which could affect predator-prey interactions.

No description available.

A laboratory study of the mobility of Malathion in the soil environment was conducted. / Master of Science

Made available in DSpace on 2014-06-11T19:22:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-03-05Bitstream added on 2014-06-13T20:10:13Z : No. of bitstreams: 1 bianchi_j_me_rcla.pdf: 700606 bytes, checksum: e0d730f76b54734539c18e1d9a52fbe4 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os agrotóxicos, substâncias químicas muito utilizadas para combater pragas na agricultura e nas residências, além de contribuírem para o aumento da produtividade e, conseqüentemente, o crescimento economia, também são responsáveis pela contaminação ambiental, quando utilizados indiscriminadamente, e por muitos casos de intoxicações e doenças genéticas nos seres humanos. Dependendo de sua composição química, degradabilidade e persistência no ambiente, os agrotóxicos podem comprometer a cadeia alimentar e afetar o ecossistema como um todo. Dentre os agrotóxicos bastante utilizados em todo o mundo, está o organofosforado malation. Pelo seu amplo uso, torna-se preocupante os possíveis danos que este inseticida possa promover no meio ambiente e nos organismos a ele expostos, pois alguns trabalhos já mostram esta sua ação detrimental. Desta forma, este trabalho investigou o potencial de indução de danos no DNA, para diferentes concentrações de malation, por meio das técnicas de aberrações cromossômicas e teste do micronúcleo em A. cepa e micronúcleo e ensaio do cometa em células HTC (hepatoma tissue culture). Nos testes com células meristemáticas e F1 de A. cepa, expostas por 24 e 48 horas ao malation, foram verificadas freqüências significativamente elevadas de AC e MN. Após 24 horas de exposição, não houve aumento na quantidade de AC, mas sim de MN. A análise das células F1 de A. cepa, juntamente com o teste de AC, forneceu dados importantes sobre a fixação dos danos genéticos induzidos pelo malation. Nenhum resultado relevante de citotoxicidade foi verificado em A. cepa. Após passarem pelo teste de recuperação, somente as células expostas às menores concentrações testadas apresentaram diminuição nos índices de AC e MN. / Pesticides, chemicals widely used to combat pest in agriculture and in homes, besides contribute to the increase productivity and the economy, are also responsible for environmental pollution and for many intoxications cases and genetic diseases in human beings. Depending on their chemical composition, degradability and degree of persistence in the environment, the pesticides can endanger the food chain and affect the entire ecosystem. Among the pesticides most used in the world is the organophosphate malathion. Due to its widespread use it has been worrying the possible damages that can promote this insecticide in the environment and in organisms exposed to it. This study investigated the induction potential of DNA damage with several malathion concentrations using chromosomic aberration (CA) techniques, micronucleus test (MN) in A. cepa and micronucleus and comet assay in hepatoma tissue culture (HTC). In A. cepa tests, meristematic and F1 cells exposed for 24 and 48 hours to malathion showed significative frequencies of CA and MN. After 24 hours of exposure, there was no increase in the CA amounts, but was observed an increase to frequency of MN. The F1 cells analysis, together with the CA tests, provided important data on the genetic damages fixation induced by malathion. No relevant results of citotoxicity were verified in A. cepa. After the recovery test, the cells exposed to the smaller tested concentrations of malathion showed reduction of AC and MN indices. In the HTC cells test, was verified by the comet assay, genotoxic effects for all the tested concentrations, after exposure for 24 hours to the insecticide, but by the micronucleus tests, no significative results were found. These data suggest that the DNA lesions were repaired and not elapsed in mutation. With the concomitant realization of different tests, the clastogenic action of malathion could be verified.

Tobacco SABP2 has been shown to display high affinity for salicylic acid (SA) and methylsalicylate (MeSA) and plays an important role in SAR signal development. Using biochemical approach, SABP2 has been shown to demonstrate strong esterase activity in converting MeSA to SA. Recent study shows that tetra fluoroacetophenone, a synthetic analog of SA, competitively inhibits SABP2 esterase activity as well as suppresses SAR signal development in tobacco mosaic virus (TMV)-infected tobacco plants. Not much has been studied on the effect of pesticides on plant defenses. Because both AChE and SABP2 are esterase-like proteins belonging to α/β hydroxylase superfamily, we hypothesize that pesticides may inhibit the MeSA esterase activity of SABP2 and block SAR development. Biochemical and molecular biology techniques were used to test this hypothesis. SAR in tobacco-TMV plant-pathogen system is measured by significant decrease in TMV-induced lesion sizes in secondarily inoculated distal leaves.

Der Hen´s Egg Test for Micronucleus-Induction (HET-MN) wurde bereits mehrfach als ein alternatives, versuchstierfreies Testsystem für in-vivo Mutagenitätstests am Nager unter Verwendung von angebrüteten Hühnereiern vorgestellt (Wolf and Luepke 1997; Wolf, Niehaus-Rolf et al. 2002; Wolf, Niehaus-Rolf et al. 2003; Wolf, Niehaus-Rolf et al. 2008). Während der häufig verwendete in-vivo Micronucleustest am Nager aufgrund seines komplexen in-vivo Stoffwechsel für die Bewertung von Mutagenen sehr gute Ergebnisse liefert (Wolf 2003), können Alternativen zur Testung am Tier, zum Beispiel in-vitro Micronucleustests, die Vorteile der in-vivo Testung nicht erreichen, weshalb sie häufig zu sensitiv sind. Gegenüber herkömmlichen in-vitro Tests stellt die Komplexität des angebrüteten Hühnereis, welches sowohl toxikodynamische als auch toxikokinetische Wirkungen der Prüfsubstanz aufzeigen kann, einen besonderen Vorteil dar, so dass der HET-MN unter anderem nicht nur direkte Mutagene sondern auch Promutagene detektieren kann. Mit Hilfe von zehn ausgewählten Substanzen wurde in dieser Arbeit die Prädiktivität des HET-MN weiter untersucht. Hierzu gehörten, neben den in-vitro und in-vivo negativen Stoffen Ephedrin-HCl und Ampicillin-Natrium und den in-vitro und in-vivo positiven Substanzen para-Chloranilin, Carbendazim und Vinorelbintartrat, auch die fünf so genannten irrelevant positiven Substanzen Isophoron, 2,4-Dichlorphenol, Phthalsäureanhydrid, Menthol und Malathion, für die uneinheitliche in-vivo und in-vitro Daten vorliegen, die aber aufgrund validierter Testverfahren als in-vivo negativ eingestuft werden. Die im HET-MN ermittelten Ergebnisse zeigen für alle zehn Substanzen, im Vergleich zu den gängigen Einstufungen auf Grundlage validierter Tests, eine vollständige Übereinstimmung und damit eine sehr hohe Prädiktivität des HET-MN. Die Ergebnisse stärken daher die Position des HET-MN als einen zuverlässigen in-vitro Test mit besonderen metabolischen Fähigkeiten, der darüber hinaus dazu beitragen kann, so genannte irrelevant positive Substanzen besser einzustufen.

The three insecticides of interest were lindane (an organochlorine), malathion (an organophosphate) and piperonyl butoxide (PBO; a synergist). Based on minimum cytotoxicity (> LC25), the following concentrations were chosen for the pesticide mixture studies: 70μM lindane (Lind), 50μM malathion (Mal) and 55μM PBO. In the AlamarBlue cytotoxicity assay, individual pesticide and mixtures of malathion/PBO (MP) and malathion/lindane (ML) prompted cytotoxicity with varying intensities (Mal 18.8%, Lind 20.4%, PBO 23.5%, ML 53.6% and MP 64.9%). Cytopathological analysis revealed apoptotic features in treated cells and the DNA Ladder Assay confirmed the presence of DNA fragments. The specific mode of cell death was examined via the 7-aminoactinomycin D (7-AAD) Staining Assay. Apoptosis was detected in each treatment (Mal 6.5%, Lind 12.0%, PBO 13.2%, ML 19.3% and MP 23.4%). Furthermore, 7-AAD staining in combination with fluorescent-labeled monoclonal antibodies, PE-CD45RB/220 and FITC-CD90, was performed. B-cells were more susceptible to Mal and PBO treatments than were T-cells. The pro-oxidant activity of the pesticides was monitored via the Dichlorofluorescin Diacetate assay. Exposure to pesticides for 15 minutes increased H2O2 production above the controls, Mal 21.1%; Lind 10.8%; PBO 25.9%; ML 26.8%; MP 37.8%. The activities of antioxidant enzymes, glutathione peroxidase (GSH-Px) and glutathione reductase (GR) were altered by these treatments. GR was significantly reduced for the pesticide mixtures only (control: 51.7; Mal: 48.2; Lind: 50; PBO: 52.3; ML: 40.5; MP: 42 Units/mg). GSH-Px activity was severely reduced for all the pesticide treatments (control: 44.9; Mal: 30.2; Lind: 30.6; PBO: 32.4; ML: 21.1; MP: 21.1 Units/mg). These results indicate that exposure to these pesticide and pesticide mixtures induces apoptosis and oxidative stress. / Master of Science