Evidence of selection in human gene functional categories

Eric Larson

Austin Hughes, mentor

The fist research project I worked on this summer looked for evidence of selection operating on different categories of SNPs in two human populations, those of African and European descendents, in order to find possible complex disease association candidates. I calculated the genetic diversity and distance between populations for 24,934 SNPs in 226 genes and compared them across six categories relating to type and location of the SNP, and across nine gene functional categories. Significant purifying selection was present in intolerant non-synonymous SNPs. Evidence of purifying selection was also found in tolerant non-synonymous and 5’UTR SNP categories. Immune response genes had an increased rate of amino acid evolution. Genes involved with signal transduction showed evidence of purifying selection. Biotic response genes showed diversifying selection. Gene functional groups also showed significantly different genetic distances giving evidence of possible past convergent and divergent selection.

The second project I worked on was investigating the evolution of the sodium pump. It is generally accepted that warm-blooded birds evolved from cold-blooded reptiles, and that the sodium pump plays a role in thermal regulation. We are trying to find how the sodium pump evolved and affected thermal regulation in birds. To do this, I made phylogenetic trees using sodium pump related protein sequences from many different species in the genbank database. I have also found the genes that code for these proteins and mapped them on their genomes when possible. This project is still a work in progress.

Patterns in Uca spp. Drove Movement

Raven Bier

Renae Brodie, mentor

Uca spp. have been observed in droves of dozens to thousands of individuals. This has especially been described of Uca pugilator in the southern United States. The U. pugilator droves comprised of thousands of individuals are typically moving away from the sandy habitats in which they burrow in order to find foraging areas of higher organic content. Based on observations made in Field Season 2004, the timing of a drove returning to its burrows may be partly a predator avoidance behavior because U. pugilator droves were observed to “anticipate” the incoming tide by returning to their burrows before the tide enters the foraging area. Additionally, when the tide did inundate the foraging area, any exposed Uca spp. were susceptible to predation by juvenile Callinectes sapidus that entered the flat with the tide. Previous studies have speculated that the away from tide (AFT) movement results from feeding inhibitory factor (FIF) release as a consequence of satiation. The purpose of this study was to determine if there were a relationship between the tidal cycle and peak AFT movement. Subsequently, foraging time of the drove was also examined in response to the satiation hypothesis. Observations were conducted on a mudflat at Baruch Marine Field Lab, South Carolina over one tidal cycle (July 2^nd to 17^th) from sunrise to sunset and for a 24 hour period on July 21^st. Data including number, direction, and sex of U. pugilator, U. pugnax and U. minax were recorded for 1 minute every 9 minutes beginning the first crab for that day passed through a 1 m. wide set of stakes placed perpendicular to the drove pathway. These data were recorded for two sets of stakes 1 m. apart and adjacent to each other. Observations were repeated until sunset arrived or the flooding tide hit the stakes. Additionally, a laboratory experiment was conducted for 7 days in which 10 male and 10 female U. pugilator individuals were each placed in a 4x4x10 cm Plexiglas box with a LED light sensor placed in the center of the box to record back and forth movement by the subject. Data from this experiment is not yet available, but will help to determine if AFT movement is the result of an endogenous biological clock rather than external cue. The observed drove did not show any nocturnal behavior. A correlation between peak AFT movement and the tidal cycle was found, and significantly consistent foraging duration was not apparent. This suggests that AFT movement may be a predator avoidance behavior and contrasts the idea that it is a result of satiation. To further relate the findings of this study with the satiation hypothesis, it will be necessary to determine if there is consistency in the levels of organic material and water sediment saturation for the duration of a tidal cycle.

Kushal Parikh, University of Notre Dame

Dr. Tim Mousseau, Mentor

Project 1: Acid Phosphatase Activity in Flight Muscle Histolysis

Histolysis occurs naturally in various species. Some cricket species are known to undergo histolysis for issues related to reproduction, survivals, etc. My project was to understand and predict acid phosphatase (AP) activity in flight muscle histolysis of Achetus domesticus, also known as house cricket. Achetus domersticus are known to undergo histolysis on 3^rd and at times, on 4^th day of their emergence. During this time, AP activity increases as a result of programmed cell deaths which are responsible for the muscle degeneration. Using flight muscles obtained from various crickets that were along different points of adulthood, we conducted AP assays using p-NP as a standard and p-NPP as a substrate. In doing so, we obtained a gradual process of AP activity that takes place leading up to histolysis in flight muscles. At the same time, we also determined the effects of juvenile hormone (JH), which may be the agent of increased AP activity. Though, JH had been previously linked to AP activity, current analysis provided first-hand experience of its effects and reinforced previous findings. Analysis revealed that the AP activity is highest at day 3 and day 4 after emergence in Achetus domesticus and also inhibition of JH circulation also impedes histolysis.

Project 2: Sex Ratio Distortion in Brachys tesselatus

Brachys tesselatus, a beetle species found in Southeastern America, shows great sex ratio distortion in nature. Populations have been found to present ratios of females to males ranging from approximately 2:1 to as high as 16:1. Through previous studies, it was inferred that this distortion was due to a rickettsia bacterium. My project included testing whether or not the sex ratio distortion was indeed stable and repetitive in nature by comparing this year’s results against previous years’. Thousands of beetles were sexed by inspection of their abdomens, which revealed a hairline if they were female. Further analysis also presented preliminary indications that infections may be positively correlated to higher weight and in turn, better fitness. Furthermore, it was also shown that sex ratio distortion was not dependent on geographic location as various in populations clumped together was just as large as between populations that were quite far from each other. Further analysis of other factors such as temperature, ecosystem, etc. would be necessary to reveal what outside factors affect this sex ratio distortion. In addition, a small percentage of females revealed capability for parthenogenesis, which could be tested to see whether it is a result of lack of males or whether a direct result of infection.

Visual and olfactory cues used by Manduca sexta to find and feed on flowers

Poppy Markwell

Robert Raguso, mentor

Experiments were performed in a wind tunnel to measure the extent that naive laboratory reared tobacco hornworm moths, Manduca sexta (Lepidoptera, Sphingidae), use olfactory and visual cues to locate flowers from a distance and feed from them. The nocturnal hawkmoths, Manduca sexta, were flown upwind to seven treatments with different combinations of visual and olfactory cues. We found that there is a threshold between 20-40 cm at which odor and visual sign are not regarded as a part of the same multimodal sensory cue; decreasing their potential as feeding behavior releasers. By decoupling flower scent from the visual stimulus, this experiment shows that both visual and olfactory cues are necessary for M. sexta to feed, whether odor is a plume or a cloud, and visual display is an array or a flower.

Latitudinal Size Clines and Phenotypic Plasticity in Crickets
Sarah Waggoner
Timothy Mousseau, mentor
 
 I studied the interactions latitudinal size clines and cell-size-plasticity in insects. Most animals exhibit size clines up and down latitudes. Some animals are larger and higher latitudes and some show the converse. At the same time, some studies have shown that cell size in some animals can be affected by the temperature at which the animal is reared. This begs the question: could cell-size plasticity be responsible for the effects that we observe as Bergmann's Rule? This is really two questions rolled into one: Is Bergmann's Rule adaptive, or the result of physical constraints? And are cell size and body size correlated? The data I collected showed no correlation between cell size and body size, implying an adaptive component to latitudinal size clines in insects.

Pygmy Sunfish (Genus Elassoma)...A New Species?

Stefanie Sedlack

Dr. Joseph Quattro, mentor

There are currently six identified species of pygmy sunfish (Genus Elassoma). Three known species of sunfish, E. evergladei, E. okefenokee, and E. zonatum are found in northern Florida. Morphological data of the sensory pores suggests that pygmy sunfish found in various sites around Franklin County, Florida may comprise a new seventh species. Prior research on pygmy sunfish used the 16S and d-loop loci of the mitochondrial DNA and the sixth intron of the lactate dehydrogenase A gene (LDHA6) of the nuclear DNA to determine phylogenetic relationships between the species. In order to determine if samples from Franklin County, Florida represent a new species, I amplified portions of the 16S, d-loop, and LDHA6 loci for 29 individuals using procedures outlined in previous research. A neighbor-joining phylogenetic tree was constructed based on original 16S data (530 bps) for Franklin County specimens, as well as previously published sequence data for all other currently recognized species of Elassoma. Franklin County specimens were monophyletic and distinct from all nominal species of Elassoma which suggests that this population represents an undescribed species. Research to obtain d-loop and LDHA6 sequences continues and will help to test the hypothesis that the Franklin County population represents an undescribed species.

The impact of uridine analogs on Leishmania 

Valerie Hilliard

Richard Showman, mentor

 My project this summer was to test uridine analogs for anti-leishmanial activity. The first step of the project was to optimize growth conditions for Leishmania tarentolae. Just as other students in the lab have struggled with growing the Leishmania, I also encountered several problems. After two weeks of trying to keep the cultures alive for more than a day, I began to do growth curves with the Leishmania and different supplemented media (such as with or without folic acid, etc.) Then I began having problems with contamination, even after systematically eliminating all possible sources by replacing all media and working in a hood. Finally, I switched to Leishmania major. The reason for starting with L. tarentolae was that it is not pathogenic, while L. major is. Soon I began testing the uridine analogs to see if they had an effect on the Leishmania. Trypanosomes have a unique RNA editing system that is not shared by humans. Uridines that are not encoded for are inserted and uridines that are encoded for are deleted. In some cases greater than fifty percent of the nucleotides that are present in mature mRNA have been generated by this unique RNA editing. Since humans do not have this system, it would be an excellent target for drugs. The idea was to use a live/ dead assay of Calcein AM and an ethidium dye on samples of Leishmania grown with the analogs. However, the dyes only showed fluorescence for the bacteria that were contaminating the samples. After trying various other dyes and still having no luck in finding one that would fluoresce with Leishmania, we decided I would determine if the cells were alive or dead by checking for movement under a microscope. I tested nine uridine analogs, doing cell counts several times a day for ten days. Although most analogs did not kill the majority of the L. major, in several cases they did slow down growth and after a week a couple appeared to have caused around a third of the cells to die. 

Analysis of mitochondrial DNA control regions from African population samples

Ransford Opong

Jacqueline Bui

Diana Rivers

Kenyatta Frazier

Bert Ely, mentor

The goal of our summer research project was to add African HVR1 sequences to the African DNA Roots Project database. We mostly worked on DNA samples Mali and Benin. The HVR1 region was amplified by PCR and analyzed by agarose gel electrophoresis. DNA samples giving rise to bright bands on the gel were sent to the Rexagen company for sequencing. The portion of the mtDNA that was sequenced was the HVR1 which consists of about 440 base pairs. After we received the sequence data, we aligned the sequences, identified haplotypic differences and sorted the sequences into haplogroups that had been previously defined. By the end of the summer, we were able to add more than 150 sequences to the database.