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ItemUSING ASCORBIC ACID AND HIGH SALINITY TO EXTEND THE VIABILITY OF PROALES SIMILIS (ROTIFERA) DIAPAUSING EGGS(Georgia Institute of Technology, 2021-05) Brashear, JillianRotifers are sensitive indicators of environmental conditions and serve as model organisms for assessing toxicity. It is understood that rotifer cysts (diapausing eggs) are convenient for toxicity assessment because they remove the need to maintain animal cultures, reduce variability in tests, and can be stored for on-demand use. Indeed, cyst-based toxicity tests for the rotifer Proales similis have helped to fill a need for an additional marine animal model to the rotifer Brachionus plicatilis in ecotoxicology. A challenge to implementing tests is the need for readily- available P. similis diapausing eggs, which need to be reliably preserved and hatched on demand. This study explores preservation methods to extend the viability of P. similis eggs. We explore factors including storage temperature, salinity, and the addition of ascorbic acid to measure their effects independently and combined. We found that storing the diapausing eggs at approximately 4℃ in the presence of 20µM ascorbic acid and a 220ppt salinity is effective for extending egg viability. With longer viability, P. similis diapausing eggs are more readily available and thereby valuable as a tool in toxicity assessments.
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ItemExpanding test methods for marine ecotoxicology testing using Proales similis(Georgia Institute of Technology, 2020-05) Park, NancyThis study is the first inquiry into the suitability of the rotifer Proales similis as a test animal for researchers studying marine pollution. It also introduces a novel toxicity test based on inhibition of hatching rates of P. similis diapausing embryos. We exposed P. similis to various concentrations of cadmium, copper, and mercury and then quantified effects on survival, hatching, reproduction, and ingestion. We found that mortality and cyst hatching endpoints were more robust to heavy metal toxicity than reproduction or ingestion. Overall, we confirmed that P. similis is a suitable tool for convenient and quick ecotoxicology assessments, and we established cyst inhibition as a novel, reliable endpoint for observing effects of toxicity on rotifer populations.
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ItemRotifer Growth Under Astaxanthin Enrichment(Georgia Institute of Technology, 2017-12) Siegfried, EmmaRotifers and astaxanthin both play an important part in the aquaculture industry. Rotifers are used as a substitute for copepods, the main source of food for larval fish in natural systems, due to the ease with which they can be cultured. Astaxanthin is a carotenoid and antioxidant which brightens the coloring of fish and improves fish health. Rotifers are believed to be a method through which astaxanthin can be bioencapsulated and vectored to larval fish. As a result, it is important to understand the effect of astaxanthin on rotifers themselves. This experiment uses a multitude of different protocols to determine how different astaxanthin compounds effects rotifers on both the individual and population levels. Reproductive tables and fluorescent imaging were used to assess the health of individual rotifers; population density measurements in mass cultures were used to assess rotifer population health. The reproductive ability of rotifers was significantly different from control under multiple astaxanthin treatments. Astaxanthin enrichment also created a higher stable population density in the mass cultures. The fluorescent imaging showed that the rotifers reached peak astaxanthin concentration within the rotifer gut after 3 hours, and but concentration returned to control levels within 24 hours of removal from astaxanthin. These results all point to the fact that astaxanthin helps to increase rotifer health and fitness, and that these rotifers could be used as a vector for astaxanthin to larval fish.
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ItemEstablishing a Working Protocol for Plasmid Cloning and shRNA Design in Endogenous Brachionus manjavacas Gene TRP7(Georgia Institute of Technology, 2016-07-18) Krishnappan, SharadhaCurrent transfection protocol in rotifers only allows for temporary transfection within rotifers and does not allow for the continuous knockdown of endogenous genes, thereby inhibiting the possibility of observing long-term biological effects in response to specific perpetual gene knockdowns. This study aims to address this particular issue by establishing a working protocol for plasmid cloning and shRNA design within an endogenous gene of B. manjavacas with known biological effects, allowing for the exploration into the optimization of a transfection protocol and demonstration of RNAi knockdown of the known gene within the rotifers as subsequent studies. Manipulation of gene expression in rotifers could occur through plasmid vector insertions, which induce silencing of a gene’s expression with short hairpin RNA (shRNA), via RNAi. This would effectively stimulate gene knockdown, allowing for the observation of biological effects such as changes in fecundity and lifespan. With the establishment of a working protocol for plasmid cloning and shRNA design, as a result of this study, the optimization of a transfection protocol for rotifers is explored. With increased efficiency in the transfection of rotifers, populations of rotifers expressing the plasmid can be amassed, allowing for experimental design that examine the varying aging mechanisms and effects that are stimulated due to permanent changes in target gene expression through RNAi. This, in turn, could give rise to the identification of evolutionarily conserved genes that regulate organismal aging, which could lead to further implications in the field of pharmacological intervention in mammalian aging as well as in the field of biogerontology overall.