Kier Ancona

Completion Time:

30 months

Faculty Advisor:

Dr. J. Loughry

Michael J. Bland

Completion Time:

36 months

Faculty Advisor:

Dr.B. 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.

Philip Wayne Hightower

Part-time Student

Completion Time:

39 Months

Faculty Advisor:

Dr. D. Bechler

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

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.

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.

Kelly Luke

Completion Time:

27 Months

Faculty Advisor:

Dr. D. Bechler

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. 

Anna Sanford

Completion Time:

23 Months

Faculty Advisor:

Dr. R. 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.