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.

Matthew Cannister

Completion Time:

24 Months

Faculty Advisor

Dr. D. Bechler

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.

John Griner

Completion Time:

Months

Faculty Advisor:

Dr. J. Nienow

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 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.

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.

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. 

Kuliy, Ashlie

Completion Time:

27 Months

Faculty Advisor:

Dr. Jonghoon Kang

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.

Jennifer Newsome

Completion Time:

23 Months

Faculty Advisor

Dr. Brian Ring

Funded by NIH Grant # 1R15HD060017

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.

Anna Sanford

Completion Time:

23 Months

Faculty Advisor:

Dr. R. L. Gannon

Lisa Wilson

Completion Time:

24 Months

Faculty Advisor:

Dr. David Bechler

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.