Graduate Students

Name

Title

Abstract

Fiifi Agyabeng-Dadzie

Completion Time:

36 Months

Faculty Advisor:

Dr. John F. Elder

Developing Restriction Associated DNA Tags for the Ion Torrent Personal Genome Machine

The identification and classification of similar cryptic species such as Kryptolebias marmoratus and Kryptolebias ocellatus, as distinct species has always been problematic within the biological sciences. Though Kryptolebias marmoratus and Kryptolebias ocellatus are both known to be self-fertilizing hermaphrodites, out crossing has been observed resulting in heterozygous offspring. Recently, a viable hybrid (Gitmo) between K. marmoratus and K. ocellatus has been isolated in the Valdosta State University aquatic lab. This hybrid questions the proposed species status of these fishes.

Classifications of organisms are usually based on both morphological and genotypic studies. Based on morphological studies, both fishes were thought to be synonymous because of very similar anatomical and physiological characteristics. Mitochondrial studies (genotypic analysis) of these fishes have suggested otherwise. Unfortunately, thorough whole genome studies are very expensive and labour intensive. Methods such as Restriction Fragment Length Polymorphisms (RFLP), Amplified Fragment Length Polymorphisms (AFLP) and microsatellite analysis are more commonly used. These methods, though helpful, make analyses based on the length polymorphisms of the DNA fragments and only a small portion of the genome. Furthermore, surveys yield a very limited amount of genetic information.  

A more reliable method of analyzing large numbers of these genotypic markers concurrently would be extremely valuable for taxonomic questions. Developing Restriction Associated DNA Tag (RADTag) genome sequencing using methods for the Ion Torrent Personal Genome Machine (PGM) provides a way not only to cut down the cost of genomic sequencing but also provides a method that performs thousands of Single Nucleotide Polymorphisms (SNP) analyses at once, without the use of a reference sequence. Analysis of RADTag sequences on public domains “Stacks” software displayed thousands of markers present in the Hon9 (Kryptolebias marmoratus) genome and confirms the Hon9 strain to be highly homozygous (0.999) across all loci. This achievement paves the way for further genomic studies with high throughputs and accuracy. This RADtag genome will provide the information needed for resolving the questionable relationship between K. marmoratus and the presumptive K. ocellatus cryptic species.

Key words: (RAD-Tags, SNP analysis, AFLP, RFLP, cryptic species)

Arjun

Arjun Adhikar

Completion Time:

30 Months

Faculty Directors

Drs. Michael Smith and James A. Nienow

 

“Impact Assessment of Deepwater Horizon Oil Spill and BP Dispersants on Diatoms at Perdido Bay and Pensacola Bay”.

A primarily taxonomical study was conducted to assess the distribution of diatoms after the Deepwater Horizon oil rig explosion in the Gulf of Mexico, April 20, 2010.  The study is focused on the possible effects of the Deepwater Horizon oil spill on diatoms.  Sampling was conducted on bimonthly basis for six months at Perdido Bay and Pensacola Bay, starting 24 September, 2010, and ending 10 May, 2011.  Samples were collected from the surface as net plankton, sediment, and as whole water.  Global Positioning System (GPS) was used to keep a record of exact locations of sampling.  Physical characteristics such as water temperature, dissolved oxygen (DO), light intensity, salinity, and turbidity were taken at the time of sampling.  Samples were preserved in Lugol’s iodine to avoid further growth and were transported back to the lab for further testing.  The primary objectives of the study was diatom community characterization, comparing datasets with previous studies, and recording the number of deformed diatom valves possibly attributed to the Gulf of Mexico oil spill.  A total of 524 species were encountered contributing to 90 genera.  Species diversity was measured using the Shannon-Wiener index.  Relative abundance of species was also calculated by counting at-least 400 valves in on transect per slide for each sample.  To characterize the community structure, PRIMER-E ver.  5.0 was used to assess the Bray-Curtis similarity index and to perform multidimensional scaling, and principal component analysis.  The result showed a high diversity and variability in the Perdido/Pensacola Bay ecosystem and an insignificant number of the deformed valves (<<0.01%).

Kier Ancona

Kier Ancona

Completion Time:

30 months

Faculty Advisor:

Dr. William J. Loughry

"Time Budget Analyses of Wild Nine-banded Armadillos"

Nine-banded armadillos exhibit obligate polyembryony, whereby they produce litters of genetically identical quadruplets by repeated twinning of a single fertilized egg.  High levels of altruism have been predicted among these clonal littermates, yet intensive long-term field studies have revealed no evidence of this.  The "time constraints" hypothesis attempts to explain these findings by arguing that armadillos are precluded from evolving complex social interactions, such as altruism, because of their evolutionary history.  That is, armadillos have very low metabolic rates, eat low quality, widely scattered prey, and have very short active periods, so they may not have the time to be social.  I collected data relevant to this hypothesis from May-July of 2007 and 2008 at Yazoo National Wildlife Refuge, Hollandale, Mississippi.  Focal animal observations lasting up to 10 min were obtained from marked armadillos during two 7 h time periods: 16:00-23:00 and 23:00-06:00.  Supplementary scan data were collected at first sighting of animals during the first of these time periods.  I present data describing the general pattern of armadillo time budgets, as well as sex, age, temporal and environmental influences on time allocation.  A comparative analysis of time budgets in other mammals was also performed to determine where armadillos fall relative to other species.  My findings showed that nine-banded armadillos spent almost all of their active time feeding with little variation in time budgets.  My study represents the first detailed description of armadillo time budgets and should shed light on the validity of the time constraints hypothesis. 

Jason

Michael J. Bland

Completion Time:

36 months

Faculty Advisor:

Dr.Brian C. Ring

 

“The Development of a piggyBac-based Transformation Vector Useful in Fish Species.”

The transposable element piggyBac is a highly versatile and efficient method of transforming DNA sequences into cells or organisms. My work demonstrates the first step towards transformation of piggyBac-mediated DNA sequences into the germ-line of the zebrafish Danio rerio, an important and widely used model organism, and the first piscine species to be transformed by this technique. A piggyBac construct along with the piggyBac transposase supplied in the form of mRNA was microinjected into early-stage zebrafish embryos. The piggyBac construct contains DNA sequences arranged into three selection cassettes. The KanR cassette allows for antibiotic selection in prokaryotes while the HygR cassette allows for eukaryotic selection. The third cassette contains a dominant cytoskeletal actin promoter upstream of the green fluorescent protein that causes many cells in the fish to glow green when expressed. Establishing piggyBac’s utility in zebrafish raises the possibility of its use in other fish species, specifically the self-fertilizing hermaphrodite mangrove killifish, Kryptolebias marmoratus. Genetic factors controlling hermaphroditism in this fish are not well understood, and genetic transformation allowing gene over-expression or insertional mutagenesis is important for understanding genetic mechanisms controlling this unique mode of reproductive biology. To establish K. marmoratus as a model organism capable of genetic transformation aquaculture techniques were established to maximize embryo output at the earliest possible stage of development. Thirty-two different fish were surveyed for overall embryo production and developmental stage of eggs laid, resulting in 1,814 embryos observed and staged. These fish were found to increase egg production under a high-feed diet versus a low-feed diet. Differences in fecundity and embryonic stage were also observed among different clonal genotypes.

Matthew Cannister

Matthew Cannister

Completion Time:

24 Months

Faculty Advisor

Dr. David. Bechler

"A Survey of the Fish Fauna of the Withlacoochee River in South Georgia"

A 45 site ichthyofaunal survey of the Withlacoochee River was conducted in 2009 and 2010 employing seining, gill nets, fish traps, trot lines, fishing poles, creel surveys, and searches of existing ichthyology collections for data on the Withlacoochee River drainage. The survey yielded 51 fish species, of which 3 (Alosa alabamae, Micropterus notius, Pteronotropis metallicus) were labeled species of concern in Georgia, and 2 of those species (Alosa alabamae and Micropterus notius) are currently listed on the IUCN (International Union for Conservation of Nature) red list of threatened species, but insufficient information is available on these species to classify them federally through the Endangered Species Act. The first known sympatric populations of Elassoma evergladei and the recently described Elassoma gilberti were found at one location. Other species of a conservation concern found in the drainage basin were the invasive Lepomis cyanellus and a putative hybrid between Cyprinella venusta and possibly Cyprinella lutrensis. Multidimensional scaling using PRIMER 6 and statistical analyses demonstrated differences in species composition as a function of stream order.

C. Chessler

Christine Chessler

Completion Time:

22 Months

Faculty Advisor:

Dr. J.M. Lockhart

Gopher Tortoise Hatchling Demography and Adult Burrow Thermal Profiling at Reed Bingham State Park, Cook County, Georgia

Research into the early life stages of the gopher tortoise has been minimal due mainly to high mortality rates of tortoise eggs and hatchlings. Over an 18-month period, hatchling dispersal and variability were studied. Temperature fluctuations within nest aprons and burrows were monitored to determine potential effects on embryo survival and tortoise thermoregulation, respectively. Surgically implanted 134.2 kHz, 8.5 x 2.12 mm PIT tags were placed into hatchlings (N = 174) that were artificially brooded during the 2008 and 2009 breeding season for identification upon recapture. Burrow temperature ecology was monitored through the placement of Thermocron iButtons connected to PVC-tubing, which is presented as a novel method for data collection. Thermal profiles showed temperature fluctuations to be greatest at the mouth of the burrow when compared to the relatively stable temperature of the deepest part of the burrow during all months observed. Furthermore, temperature fluctuations of natural nests typically were marginal when compared to that of artificial nest fluctuations in this study. Through limited recapture observations, it was found that hatchlings had no clear preference for refuge, directionality, or overall distance when dispersing from their natal burrows. Most recaptures (63%) took place during the afternoon hours (12:00-17:00), and during the months of March and October. These results coincide with optimal foraging and basking hours, as well as seasonality associated with post-hatching and post-over wintering events. Significant size and measurement variability was observed between the 2008 and 2009 hatchlings, which may be a consequence of climatic variability between years. In summary, this work provides demography data, as well as burrow and nesting ecology concentrated on a life stage of the gopher tortoise often under represented. The results and methodology of this project have the potential to be useful in various conservation programs directed at managing this threatened species.

Adarsh Gopinath

Adarsh Gopinath

Completion Time:

40 Months

Dr. John Elder

Relationship Between Cannabinoid Receptor 1 Gene Variation and Behavioral Differences Among Kryptolebias marmoratus Laboratory Isogenic Lines

The endocannabinoid system (ECS) is a cellular signaling system composed of molecules that include endogenous cannabinoids (endocannabinoids), membrane bound receptors called cannabinoid receptors and the enzymes that regulate the concentration of endocannabinoids. In human beings and other vertebrate models the ECS is implicated in the regulation of several bodily functions from appetite control to processing rewards. Cannabinoid receptor 1 is a G-protein coupled receptor encoded by cnr1a gene. It forms an integral part of the ECS. Polymorphism in the cnr1 gene in humans is associated with substance addiction, depression, anorexia and several other mental disorders. Kryptolebias marmoratus is a neotropical synchronous self-fertilizing hermaphroditic teleost that has been documented to produce clonal progeny in isolation. A behavioral study has managed to separate isogenic K. marmoratus labstrains across a shy-bold continuum. In descending order of boldness, K. marmoratus labstrains Vol, Rhl, 50.91 and Hon 9 were chosen to have the cnr1 gene sequenced from archived genomic DNA samples and analyzed for polymorphism. The coding region of cnr1 was found to be monomorphic among all four labstrains tested and highly conserved across several vertebrate species. Although no association could be established between the coding region of cnr1 and variation in boldness, further studies characterizing genetic control elements, rates of transcription and tests for the presence of post-transcriptional modification in cnr1 mRNA among the labstrains need to be performed to better understand the relationship between this gene and the boldness trait in K. marmoratus.

John Griner

John Griner

Completion Time:

48 Months

Faculty Advisor:

Dr. J. Nienow

A Study on the Diversity of Subaerial Algae in Southern Georgia:  Two Possible New Species of Coelastrella (Chlorophyta, Chlorophyceae) 

Studies on the Diversity of Subaerial Algae in Southern Georgia: Two Possible New Species of Coelastrella (Chlorophyceae, Chlorophyta) -Subaerial algae are a diverse group of photosynthetic microorganisms defined by their ability to grow on surfaces exposed directly to the air.  They are especially common in humid regions, including parts of southern Georgia.  We have studied a number of strains isolated from southern Georgia maintained in unialgal culture as part of a larger effort aimed at a) determining the diversity of subaerial algae and b) elucidating the mechanisms enabling them to thrive in an environment with limited access to liquid water.  Genetic and morphological investigations of two previously unidentified strains, VSU 102 and VSU 114, indicate that they may represent unknown species of green algae.  An analysis of the nuclear 18s rRNA gene commonly used in phylogenetic studies placed one of the strains (referred to as VSU 114) in close proximity to Coelastrella saipanensis (Chlorophyta, Scenedesmaceae) and Coelastrella vacuolata (Chlorophyta, Scenedesmaceae).  Morphological data, including the presence, number, and structure of meridional ribs in the cell walls, suggest that these taxa are distinct and should possibly be considered separate species.  Analysis of the 18s rRNA gene in strain VSU 102 placed this strain in close proximity to Coelastrella multistriata (Chlorophyta, Scenedesmaceae).  Although Coelastrella multistriata and VSU 102 differ genetically by their 18s rRNA content, no morphological differences could be determined.  Thus, VSU 102 may represent a cryptic species of Coelastrella multistriata. 

Philip Hightower

Philip Wayne Hightower

Part-time Student

Completion Time:

39 Months

Faculty Advisor:

Dr. D. Bechler

"The Life History of the Crayfish Procambarus spiculifer in the Alapahoochee River "

A fifteen month life history analysis was conducted on a population of Procambarus spiculifer in the Alapahoochee River. The crayfish P. spiculifer inhabits lotic waters in portions of Georgia, Florida, and Alabama. Cephalothorax lengths ranged from 6.07 mm to 59.80 mm and weights varied from 0.02 g to 53.37 g. The mean sizes of adults were 45.90 mm for form I males, 42.77 mm for form II males, and 38.43 mm for mature females. Form I males and mature females were collected year round. Relationships of cephalothorax lengths with body weights, chelae lengths, cephalothorax widths, ovarian eggs, and abdominal eggs were examined. Cephalothorax width and chelae width comparisons among sex classes were also performed. Peak juvenile introductions into the population occurred in June, late fall, and early winter. A maximum life span of two to three years was determined.

Reproductive maturity occurs around the ages of 12 to 15 months. The mean for ovarian egg counts was 602 eggs (n = 61) and the mean for abdominal egg counts in the laboratory was 464 eggs (n = 6). Abdominal egg diameters had a mean of 1.74 mm (n = 120) with a range of 1.04 mm to 2.03 mm. Females have the ability to reproduce more than once and new ova begin development immediately after a clutch of eggs is laid. The right vas deferens is probably the only functional one in P. spiculifer. GSIs were determined for mature females, immature females, form I males, and form II males. A gastric stomach analysis revealed that both males (n = 20) and females (n = 20) consume more vegetal matter than animal matter in their diet.

Dallas Ingram

Dallas Ingram

Part-time Student

Completetion Time:

47 Months

Faculty Advisor:

Dr. J. M. Lockhart

Effects of Commercial Poultry Operations on Diseases in Wild Turkeys (Meleagris gallopavo) in South Georgia

Wild turkeys are susceptible to most of the same diseases that affect domestic poultry. In 2004, a large commercial poultry company announced that it would be building a chicken processing plant and hatchery in Cook and Colquitt Counties in Georgia. Hunter killed and sick or dead wild turkeys were submitted to the Veterinary Diagnostic and Investigational Laboratory in Tifton, Georgia from 2005 through 2008. Turkeys were examined grossly for lesions and tested for parasites, bacteria and viruses to determine if the introduction of commercial poultry production to south Georgia would adversely affect the health of the wild turkey population, and, if so, how widespread this effect would be. A significant difference was seen between years for eastern equine encephalitis serum neutralization test, West Nile polymerase chain reaction (PCR), St. Louis PCR, number of protozoans, degree of parasite infection, Newcastle disease virus serology, health score and serology health score. Significant change was also noted between the control group (Madison County) and the test group with the number of nematodes, the degree of parasite infection, and the health score. When comparing locations, a significant difference was noted for Salmonella sp. bacterial culture, nematodes, and parasite degree of infection. This is the first report of avian encephalomyelitis virus antibodies and parvovirus in wild turkeys.

 

 

Jarvis

Taylor Jarvis

Completion Time:

23 Months

Faculty Advisor:

Dr. Gretchen Bielmyer

   

Gerard J

Gerard Johnson

Completion Time:

44 months

Faculty Advisor:

Dr. J. Elder

"Phylogeny of the freshwater crayfish sub-famly Cambarinae based in 16S RDNA gene analysis."

Freshwater crayfish have been a mainstay in biological experiments as a model species ever since Huxley’s (1880)seminal publication The Crayfish. Crayfish have been used in research ranging from vision pigment studies to neural physiology. Non-native species have been introduced on four continents due to their immense economic value. Although crayfish taxonomy is reasonably well resolved at the highest levels there are some problems at the levels of genus and species. New exploration, technology and methodology have lead to the discovery of not only new species but a phylogenetic complexity that would have not been imagined in Huxley’s era. This complexity is caused by the conservatism of some morphological characters, high intraspecific diversity and convergence. The ambiguity of crayfish taxonomy is visibly evident for species native to South Georgia and North Florida, which are centers of crayfish diversity. Molecular phylogenetic analyses were employed to provide insight into three aspects of crayfish phylogeny. Using data from the 16s ribosomal gene, we determined (1) the evolutionary relationships of a previously unanalyzed species, Procambarus spiculifer, (2) relationships within the genus, Procambarus, and (3) the phylogeny of the entire sub-family Cambarinae. The resulting trees of the Neighbor Joining, Maximum Parsimony, and Minimum Evolution analyses are all consistent with the hypothesis that their phylogenies are significantly different than the traditional systematic representation of relationships within the subfamily. The subfamily Cambarinae is not divided into the three distinct clades according to their genus of Procambarus, Cambarus, and Orconectes in any of the analyses.

T.A. Key

Travis Key

Completion Time:

28 Months

Faculty Advisor:

Dr. David Bechler

Juvenile Fish Usage of Limulus Breeding Depressions in an Intertidal Mudflat

Horseshoe crab (Limulus polyphemus) breeding behavior results in the presence of sediment depressions in intertidal zones. Water exits the intertidal at low tide and juvenile fish inhabit these water-filled depressions which retain water until the subsequent high tide. Depressions vary in size, presence of vegetation (eelgrass, Zostera marina), and prey (zooplankton and meiobenthos). The purpose of this research was to analyze fish composition as species and number in depressions and to determine if depression occupation was based on fish being stranded in the depressions on the intertidal or if depressions were being selected based on depression characteristics. In Summer 2008, data collection in Port St. Joseph Bay, Florida included species of fish, number of fish, sediment surface area, water volume, vegetation, zooplankton and meiobenthos for each depression sampled. AIC was used to establish models with fish number and mean fish size as dependent variables and surface area, volume, vegetation, zooplankton, and meiobenthos as independent variables. These models were applied at assemblage, population and individual ecological levels. The most abundant species were Cyprinodon variegatus and Fundulus similis, which represented 61.1% of all fish. The mean standard length of each of these two species fluctuated over time reflecting possible breeding events. The composition of fish in depressions compared to depressions characteristics indicated that occupation is behavior specific for each species. Selection was based on depression size, vegetation presence or absence, and prey types. Selection factors varied based on mean standard length of fish and species. This research indicates the importance for Limulus presence in intertidal ecosystems as their breeding behavior may increase the survival of juvenile fish.

J A Kirkley

Joseph (Ashley) Kirkley

Part-time Student

Completion Time:

96 Months

Faculty Advisor:

Dr. David L. Bechler

Biogeography and Population Genetic Structure of the Water Moccassin, Agkistrodon piscivourus, in the Alapahoochee Watershed

This study examines the biology of the Water Moccasin, Agkistrodon piscivorus conanti, in the Alapahoochee watershed, Lowndes and Echols Counties, Georgia.  Included is a biogeographical investigation incorporating population genetics, a diet survey, a length/weight relationships analysis, sex ratio analysis and observations on reproductive ecology.  Collections occurred over a two year period in 2008 and 2009. Data recorded in the field and laboratory included location, sex, snout-vent length, weight, gut contents and condition of the snake at time of collection. DNA was extracted and analyzed based on published microsatellite primer pairs for Crotalus contortix and cross amplified in Agkistrodon piscivorus that were tested for use in our study.  Overall sex ratios did not deviate from the expected in this study but, were definitely skewed relative to the year collected and location from which they were collected.  The majority of guts examined had no contents, and the remainder had small meals suggesting snakes were foraging at the time of capture. Length-weight relationships were comparable for both males and females.  Presence of eggs and embryos showed that ovulation occurs in May followed by a 3 to 4 month gestation period with birth occurring by end of August.  The genetic analyses in this study involve only three microsatellite loci and limited numbers of individuals per population. One locus, CH4B, was highly divergent between populations indicating that the populations sampled may be exhibiting restricted gene flow.

Ashley

Ashlie Kuliy

Completion Time:

27 Months

Faculty Advisor:

Dr. Jonghoon Kang

Statistical Analysis of Sequence Characteristics of Single Transmembrane Cluster of Differentiation Proteins: A Study of Functional Relevance

Cluster of Differentiation (CD) proteins are human white blood cell markers involved in immune reactions. They are used as targets for immunological studies, diagnostic techniques of disease states as well as utilized in therapeutic applications for cancers and other maladies of the body. Analysis of sequence characteristics of CD proteins has enabled the identification of functional trends that may be useful in understanding their roles within the immune system. Through statistical analysis, Principal components analysis (PCA) and K-means clustering, 36 Type II CD proteins were analyzed by one hundred twenty-four biochemical and biophysical properties derived from amino acid sequences alone. The analysis revealed these 36 CD proteins can effectively be grouped into two distinct known functional groups. In addition, possible unknown functional properties of CD71 have been identified specifically. This research shows the potential for a quantitative approach in proteomics to unlock predictive capabilities. It is believed that upon the basis of this analytical technique, functional properties may be predicted of currently undiscovered CD proteins as well as known, poorly characterized CD proteins.

Landon Lasseter

Landon T. Lassenter

Completion Time:

36 Months

Faculty Advisor:

Dr. John Elder

Computational Studies of the Cu-taxol Complex: An Analysis of Solubility and Stability

The natural product taxol is one of the most significant cancer drugs to date. Taxol is the sole member of a class of drugs with a unique mechanism of action. It is an effective treatment for numerous types of cancer, but one limitation to the compound is low solubility in an aqueous environment. In this computational study, taxol is bound with copper in a variety of configurations to create the Cu-taxol complex and tested using Spartan Molecular Modeling software and the semi-empirical PM3 method. The configurations generated involved either a single Cu-N bond and five Cu-O bonds (non-hydrated Cu-taxol); or a single Cu-N bond, four Cu-O bonds, and a single Cu-H2O bond (monohydrated Cu-taxol-H2O). Of the 252 configurations calculated, 4 likely non-hydrated Cu-taxol configurations and 16 likely monohydrated Cu-taxol-H2O configurations were generated for a total of 20. These resulting complexes were analyzed using the modified Aqueous Stability Factor to determine their relative solubility and their stability was found to be superior to taxol. The results also show that shifting the Cu-O bonds can drastically change the polarity of the complex, leading to the conclusion that it is a polarity adaptive molecule.

Kelly Luke

Kelly Luke

Completion Time:

27 Months

Faculty Advisor:

Dr. David Bechler

"The role of dyadic interactions in the mixed mating strategies of the mangrove killifish, Kryptolebias marmoratus"

Kryptolebias marmoratus is a small cyprinodont fish native to tropical and subtropical waters of Florida, Brazil, and the Caribbean.  It is the only known self-fertilizing, hermaphroditic vertebrate and the only vertebrate to display androdioecy, a complex system of reproduction in which hermaphrodites and males are present. This study describes the repertoires of reproductive and agonistic behaviors displayed by the mangrove killifish, K. marmoratus, and examines the roles played by hermaphrodites and males in a complex mating system.   The term construct is used to refer to specific pairings based on whether they involved males or hermaphrodites.  90-minute behavior observations were conducted in the laboratory on dyadic pairs of similar size K. marmoratus  for two possible mating pairs and one construct involving male-male pairings:  (1) hermaphrodite-male, (2) hermaphrodite-hermaphrodite, and (3) male-male.  This allowed observation of behaviors that could not be observed in the field.  Kryptolebias marmoratus exhibited a total of 25 distinctive acts or behaviors.  Acts were divided into four categories based on the type of behavior that was performed: aggressive, submissive, neutral, and reproductive. My observations of the different pair constructs of K. marmoratus reflect on the interactions that are taking place in a community or assemblage of fish where both males and hermaphrodites occur.  Leading and follow acts play an important role in the behavior repertoire of these fish.  In the herm-male construct, males initiate the reproductive process entirely and actively pursue the hermaphrodite.   In the herm-herm construct, there was no evidence that hermaphrodites behave like other simultaneous hermaphrodites and alternate sexual roles. Hermaphrodites are extremely aggressive towards one another and the aggressor establishes dominance immediately.  The male-male construct was subdivided into two subconstructs based on the presence or absence of the caudal ocellus.  It appears the presence of the caudal ocellus signals to males the possibility of a potential mating partner.  However, when no ocellus occurred on either male in the construct, their behavior was similar to the herm-herm construct, in that both members of the pair were aggressive towards one another. 

Heera

Heera Malik

Completion Time:

30 Months

Faculty Advisors:

Drs. Smith and Nienow

 

To investigate the community structure of diatoms of Choctawhatchee Bay in response to the BP oil spill

 

The primary objective of this research was to characterize the structure of the diatom community in Choctawhatchee Bay in response to BP oil spill. Three types of samples, net plankton, sediment and whole water, were collected on a bimonthly basis for six months. Diatoms in the samples were identified at the species level whenever possible by both light microscopy (LM) and scanning electron microscopy (SEM). A total of 87 genera and 437 species were recorded; this represents approximately 40% of species previously recorded in the Gulf of Mexico. Net plankton samples were compared with historical data to determine if a significant shift in the community structure had occurred; the absence of similar data did not permit a comparison of sediment communities. Current and historical plankton associations were only 28 % similar. This low similarity could be result of natural variation in the composition of the phytoplankton, differences in sampling techniques used, differences in the application of taxonomic principles, or changes brought about by the BP oil spill. At this point we cannot distinguish which of these is the most important factor.  Moreover the number of abnormal valves observed, much less than 0.1% of the total number of valves argues against a major impact of BP oil spill on the diatom community.

 

J Ryce

J. Ryce Martin

Completion Time:

46 Months

Faculty Advisor:

Dr. John Elder

Population Genetics of Isolated Cyprinid Minnow, Pearl Dace (Margariscus margarita), on Isle Royale, MI

In 1949, Hubbs and Lagler described morphological variation among populations of cyprinid minnows (pearl dace, Margariscus margarita) of inland lakes on the Isle Royale National Wilderness Area. For one of these lakes in particular, Harvey Lake, Hubbs and Lagler’s described a population of Pearl Dace that were morphologically distinct and argued this was sufficient to warrant subspecific status of the Harvey Lake pearl dace population. Harvey Lake is approximately 168 feet above Lake Superior’s current water level and has been isolated by elevation since the last time Lake Superior’s water level was high enough to reach Harvey Lake, approximately 10 to 15 thousand years ago when the last glaciation receded into Canada. Hubbs and Lagler argued that the morphological variation of the Harvey Lake pearl dace population is due to the isolation of Harvey Lake resulting in an allopatric genetic differentiation of the population from those found in lower elevation Isle Royale lakes and ponds. Recently the National Park Service has become concerned about the conservation of the fishes of Isle Royale. The need for genetic analyses has recently arisen to assess the extent of gene flow, if any exists, among the Harvey Lake population and the inland lakes adjacent to it. Statistical analyses of a limited battery of microsatellite loci data yields some preliminary suggestions of genetic differentiation among all populations of Isle Royale Pearl Dace inclusive of both Harvey Lake and other lowland Isle Royale populations.

Mercer

Elizabeth Mercer

Completion Time:

21 Months

Faculty Advisor:

Dr. Catherine Bush

A worldwide Phylogenetic Analysis of Scutellaria (Lamiaceae)  The plant genus Scutellaria L. (Lamiaceae) is comprised of approximately 350 cosmopolitan species.  Twenty-four species are found in the southeastern United States.  The genus has been used medicinally on multiple continents, predominately for its anxiolytic, anti-inflammatory and analgesic properties.  Recently, certain species in the genus have been investigated for their ability to inhabit cancer cell growth.  Despite the vast interest in the group, a molecular phylogeny has never been performed on the genus as a whole.  My project involves a preliminary phylogenetic analysis of Scutellaria with representative from every continent except Antarctica.  DNA from field, herbaria (MO, NY, VSC) and lab specimens were extracted for a total of 30 species including one outgroup taxon, Holmskioldia sangunea.   Gene regions sequenced include matK and the trnL intron + trnL-F spacer.  The resulting phylogeny will provide preliminary scaffolding on which a more targeted approach to medicinal screening within the genus can be based.

 

Jennifer Newsome Pic

Jennifer Newsome

Completion Time:

23 Months

Faculty Advisor

Dr. Brian C. Ring

Funded by NIH Grant # 1R15HD060017

 

Mechanisms of Ovotestis Development in the Mangrove Killifish

The mangrove killifish is one of the few synchronous hermaphroditic vertebrates in the world. This fish has a gonad containing both ovarian and testicular tissue, known as an ovotestis, that enables the fish to self-fertilize. The objective of this study was to use a forward genetic screen to cause mutations that disrupted ovotestis development. In this three generation screen N-ethyl-N-nitrosourea (ENU) was applied at varying concentrations to a parental generation (P) of mangrove killifish. Mortality, egg production, and fertility of the P fish were recorded over a 10-week period and compared to pre-treatment values. ENU exposures ranging from 2.8 to 3.8mM caused an initial decrease in fertility which recovered by the seventh week post-treatment (~90%). A histological examination was carried out on neoplasia found in 2 of these P fish. The first generation progeny of these fish, (F1) were screened for fertility. From 7,350 F1 embryos collected over the first 10 weeks, 1,334 F1 fish hatched and 284 were screened and allowed to self-propagate. Seventy-three of the offspring in the second generation (F2) progeny exhibited heritable zygotic mutations. A pilot screen was also conducted in which 7 F1 fish with observable mutant phenotypes were chosen to be propagated into the third generation to screen for sterility. Sterile mutants were identified by the absence of viable progeny. Two maternal-effect sterile mutants were identified as well as 1 non-egg laying sterile mutant. This study established the utility of the mangrove killifish as an effective model for embryological and early developmental studies, with the potential to investigate the genetic regulation of ovotestis development.

Tatyana

Tatyana Nienow

Completion Time:

35 Months

Facutly Advisor:

Dr. Theresa J. Grove

 

Functional Characterization of Calesequestrin from the Killfish Fundulus heteroclitus

 

 

Calsequestrin is a calcium-binding protein found in muscle that undergoes dramatic conformational changes during muscle contraction and relaxation.  In my project I will express and purify calsequestrin from the small intertidal fish F. heteroclitus in order to characterize its thermodynamic and calcium-binding parameters in a fish adapted to variable temperatures.

Antonio Ortiz

Antonio Ortiz

Completion Time:

23 Months

Facutly Advisor:

Dr. Cristina Calestani

Transcriptional Regulation of the Pigment Cell Differentiation Gene Polyketide Synthase in the Sea Urchin, Strongylocentrotus purpuratus

Embryogenesis is a remarkably dynamic and precisely regulated process, and the exact genetic programming that controls embryo development from fertilization to a complete organism is not fully understood. In order to understand the process of cell differentiation during embryo development we utilize the purple sea urchin, Strongylocentrotus purpuratus, as our animal model. Our objective is to gain further understanding of the gene transcriptional regulation, particularly of the Gene Regulatory Networks (GRNs) playing a role in the development of larval pigment cells, which are part of the larval immune system (Hibino et al., 2006). In order to reconstruct the pigment cells GRN we will study the cis-regulatory elements, more specifically those controlling the expression of the differentiation gene polyketide synthase (Sp-Pks) in pigment cells. Sp-Pks encodes a protein essential for biosynthesis of the S. purpuratus pigment known as echinochrome (Calestani et al., 2003). Comparative genomics was performed utilizing the software FamilyRelationsII, MEME, and SANN in order to predict cis-regulatory modules and putative DNA-binding sites in the -3kb region of the Sp-Pks promoter. The SANN and MEME analyses were done utilizing five co-expressed pigment cell genes: Sp-Flavin-monoxidase 1, 2, 3, Sp-Sulfotransferase, and Sp-Pks. Furthermore, we compared them in two additional sea urchin species: Allocentrotus fragilis and Strongylocentrotus franciscanus. From these analyses we selected putative sites to test via site-directed mutagenesis within the -3kb promoter. We then utilized the Yeast-One-Hybrid system to identify putative transcription factors that bind to the Sp-Pks promoter. The characterization of all the regulatory elements that control the transcription of Sp-Pks both spatially and temporally will allow for further understanding of the developmental gene regulatory network upstream of this terminal differentiation gene. 

Lance

Jacob L. Perry

Completion Time:

35 Months

Faculty Advisor:

Dr. Brian C. Ring

While employed full-time at Landis International

Funded by NIH Grant # 1R15HD060017

Cloning, Characterization, and Expression Analysis of the Vasa Gene form Kryptolebias marmoratus  Kryptolebias marmoratus, formerly known as the Mangrove Rivulus, has been studied in the laboratory setting since the late 1950’s. K. marmoratus is a selfing hermaphrodite with a unique gonad, termed the ovotesties, consisting of both ovarian and sperm tissue.  One particular gene of interest to the development of this gonad is the vasa gene.  Vasa  is an ATP-dependent RNA helicase, which has been studied in many different organisms such as: Drosophila melanogaster (fruit fly), Homo Sapiens (humans), Ambystoma mexicanum (salamander), Xenopus laevis (frog), Danio rerio (Zebrafish), Oryzias latipes (medaka), and Strongylocentrotus purpuratus (sea urchin).  The vasa gene was cloned and sequenced from K. marmoratus.  The gene consist of 2,273 nucleotiedes, a predicted amino acid sequenced of 641base pairs, and a 3’ untranslanted region (UTR) of 234 base pairs in length. BLASTp and phylogenetic analysis revealed that the kmvasa gene is closely related to T. oreintalis (Pacific blue fin tuna) and  has a close relationship to O. latipes (medaka).  The ATP-dependent RNA helicase vasa protein was detected in only the gonad region of K. marmoratus hermaphrodites and males.  In other species, the vasa gene is also expressed at similar levels throughout embryogenesis.
Key words: (ovotesties, vasa ATP-dependentRNA helicase, UTR, phylogenetic analysis,and QPCR)

Salter John

John Salter

Completion Time:

31 Months

Faculty Advisor:

Dr. David Bechler

 

Fish Assemblage Structure in Lentic Vegetated Microhabitat in South Georgia  Aquatic vegetation provides multiple resources such as shelter, food, and breeding habitats for many fish species.  Fishes that occupy habitats with similar ecological characteristics are described as fish assemblages.  However, not all vegetation offers the same set of resources.  Therefore, I hypothesize not all fish assemblages that occupy aquatic vegetation are identical.  Based on vegetated structure complexity in the water column, I predicted that submergent vegetation would contain the most fish diversity.  This study involved an analysis of fish assemblages at 18 vegetated lentic sites in south Georgia.  Total area, percent vegetated surface area coverage, water volume, and major plant species as well as other physicochemical data were recorded for each locality.  Comparative analysis of each location was conducted using, one-way ANOVA, Freidman test, Principal Component Alalysis (PCA) and linear regression analysis.  Thirty-two fish species were collected across all sites, and significant differences in fish assemblages existed between sites.  No defining factores related to assemblage structure were identified.  PCA identified Gambusia holbrooki, Leptolucania ommata, Elassoma okefenokee, and Lepomis macrochirus as principal species defining fish assemblage structure.  From these results, three fish subguilds of aquatic vegetation were identified.

Anna Sanford

Anna Sanford

Completion Time:

23 Months

Faculty Advisor:

Dr. Robert L. Gannon

“Characterization of Serotonin Receptors in the Hamster Circadian System”

Knowledge of the circadian system and how the projection of serotonergic (5-HT) fibers throughout the system can modulate it is very important in further understanding how certain neurological disorders can affect the circadian rhythms of patients. Very little is actually known of the location of the receptors within the circadian system. The purpose of this research is to complete the anatomical gaps in 5-HT receptor distribution within the Syrian hamster circadian system. Immunohistochemistry technique was performed to visualize previously undescribed 5-HT receptor subtypes in the suprachiasmatic nucleus (SCN), the intergeniculate leaflet of the thalamus (IGL) and the raphe nucleus, and western blotting was used to identify the molecular weight of the protein being labeled. In the 5-HT1 family, including 5-HT1A, 5-HT1B, 5-HT1D, and 5-HT1F, receptor protein was observed in the SCN, IGL and raphe nucleus except for the 5-HT1A receptor which showed no protein in the SCN. The 5-HT1F receptor displayed differences in day-night expression only within the SCN. For the 5-HT2 family, 5-HT2A, 5-HT2B, and 5-HT2C receptors displayed moderate protein staining only in the raphe nucleus. The 5-HT2C receptor had a differential expression during the day and night. The 5-HT3, 5-HT4 and 5-HT5B receptors also had weak protein staining only in the raphe nucleus. The 5-HT6 receptor had moderate protein staining in the SCN and the raphe nucleus, and weak protein staining of the IGL. A difference in the day-night expression was observed in the SCN with this receptor. These findings complete the gaps in knowledge of protein expression for each 5-HT receptor subtype in the hamster circadian system and provide new possible target avenues for circadian pharmacology studies.

Sofia Sucar

Sofia Sucar

Completion Time:

23 Months

Faculty Advisor:

Dr. Brian C. Ring

Funded by NIH Grant # 1R15HD060017

A Simultaneous Genetic Screen for Zygotic and Sterile Mutants in a Hermaphroditic Vertebrate

The mangrove killifish (Kryptolebias marmoratus) is unique among vertebrates due to its self-fertilizing mode of reproduction involving an ovotestis, analogous to the nematode Caenorhabditis elegans. It develops externally, is easy to maintain, and reaches sexual maturity in about 100 days, making it a desirable vertebrate model organism for developmental genetics. As a continuation of an initial pilot screen (Moore, Sucar, Newsome, Ard, Bernhardt, Bland, & Ring, 2012) in which 73 out of 284 F1 fish were found to display zygotic defects in their F2 offspring (25%), a three generation genetic screen was performed using N-ethyl-N-nitrosourea as a mutagen in order to a) confirm zygotic mutant allele heritability and b) simultaneously score for homozygous recessive mutant sterile F2 fish. One to ten F2 fish per family were set up and their progeny screened. From a total of 307 F2 fish screened, 10 were found to be 10 males and 16 were found to be sterile. From the remaining 281 fish, 92 showed a wild type phenotype and 189 were carriers of zygotic recessive alleles. These carriers produced 25% progeny exhibiting several zygotic phenotypes previously described in zebrafish (Mullins et al., 1994) as well as in a K. marmoratus pilot screen. Also, new phenotypes such as golden yolk, no trunk, and short tail, were discovered. In order to confirm inheritance of sterility, fish were grown from the F3 generation from siblings of sterile F2 mutants. From 284 F3 fish belonging to 10 previously identified sterile families, 12 were 10 males. From the remaining 272 fish, 69 were wild type, 83 were sterile and 120 were classified as */+ (either wild type or carriers) because their genotypes were undefined. This screen provides proof of principle that K. marmoratus is a powerful vertebrate model for developmental genetics and can be used to identify mutations affecting fertility.

Katie Vernon

Katie Vernon

Completion Time:

52 Months

Faculty Advisor:

Dr. Jenifer Turco

 

Determination of Rickettsial Viability and Characterization of Macrophage Death in Cultures of Gamma-Interferon-Treated Macrophages Infected with Rickettsia prowazekii

Rickettsia prowazekii is an obligate intracellular, Gram-negative bacterium that is transmitted via body lice and is the causative agent of epidemic typhus.  During an R. prowazekii infection, the bacteria target the endothelial cells lining small blood vessels, as well as macrophages.  When cultured macrophage-like cells are pre-treated with gamma interferon  (IFN-g) and then infected with R. prowazekii, the macrophages die within a few hours (Turco & Winkler, 1984; Turco & Winkler, 1994).  It is unknown if the rickettsiae are killed as the macrophages die and the mechanism of cell death seen in IFN-g-treated, infected RAW264.7 cells has yet to be characterized.  The research presented here had two objectives: to determine if the rickettsiae are viable after infection and death of IFN-g-pre-treated, macrophage-like RAW264.7 cells and to determine if the osmotic lysis inhibitor polyethylene glycol (PEG) has the ability to decrease cell death in RAW264.7 cells treated with IFN-g.  Rickettsial viability was assessed by determining the ability of the bacteria to grow in untreated Vero cells after their release from dead IFN-g-treated, infected RAW264.7 cells.  The rickettsiae released from the dead IFN-g-treated RAW264.7 cells were able to grow in Vero cells over 49 hours, indicating that they are viable after the death of their host cells.  Sufficiently large PEGs can inhibit osmotic lysis, which contributes to macrophage death during oncosis and/or caspase-1- dependent pyroptosis.  At 30 mM, PEGs ≥ 2,000 daltons (nominal average molecular weight) have a protective effect against cell death in IFN-g-treated, infected RAW264.7 cells, while            PEGs ≤ 1,450 daltons do not have an effect on RAW264.7 cell death.

Lisa Wilson

Lisa Wilson

Completion Time:

24 Months

Faculty Advisor:

Dr. David Bechler

 

Diet, Critical Thermal Minimum and Occurrence of Batrachochytrium dendrobatidis in Cuban Treefrogs (Osteopilus septentrionalis)

Amphibians have undergone global population declines attributed to a variety of causes including climate change, pathogens, and introduced species.  In Florida, introduced species have affected native amphibians through adult and larval competition and predation.  One species, the Cuban treefrog (Osteopilus septentrionalis) has been reported to replace native treefrogs.  The exact mechanism of replacement is unknown.  This study, which focused on populations in northern Florida, examined diet, cold tolerance, and the presence of a fungal pathogen, Batrachochytrium dendrobratidis (Bd). Native anurans represent a small portion of Cuban treefrogs diet, although the potential for predation exist.  Data on cold tolerance indicates the potential for continued northward expansion into Georgia and costal South Carolina and westward expansion into Gulf Costal states.  The success of the Cuban treefrog as an invader is likely to decline in colder areas, although continued climate change and possible adaption may expand the area in which they are successful.   There was no evidence of Bd.  Due to their history characteristics and lack of susceptibility, Cuban treefrogs are unlikely to act as reservoirs or vectors of Bd and are unlikely to test positive.

Charles Wright

Charles Wright

Completion Time:

36 Months

Faculty Advisor:

Dr. David Bechler

 

An Assessment of the Anthropogenic Affect of Bridges on Fish and Macroinvertebrate Assemblages

Anthropogenic impacts such as bridge sites can greatly alter established streambed morphology and associated ecology. At bridge sites, streams are often channelized approaching the site and deep pools are created at the bridge site causing ecological disturbances of fish and invertebrate assemblages. However, restoring channels and reducing negative construction practices allows the return of natural habitats that are likely to include more sensitive species. Recent conservation studies have suggested that sites of anthropogenic origin may serve as potential habitats for reestablishment of populations following a drought event. This study examined fish and macroinvertebrate assemblages, and physiochemical factors associated with these assemblages at 14 bridge sites involving first through fourth order streams in the Suwannee River Basin of south Georgia. Fish assemblages were least diverse upstream of bridge sites, most diverse at bridge sites, and intermediate downstream of bridge sites.  Macroinvertebrate assemblages did not exhibit as distinctive a pattern as did fish assemblages. Upstream macroinvertebrate assemblages were less diverse than bridge site and downstream assemblages, a pattern that was disrupted for the bridge site by third order stream data. The results from this study suggest that bridge sites, if properly engineered, can serve as valuable refuges for reestablishing fish and macroinvertebrate assemblages up and down stream after events such as the severe drought that impacted south Georgia in 2011.