Browsing by Author "Turner, Bruce J."
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- Analysis of factors that affect parvovirus expressionBraddon, Virginia Rendall (Virginia Tech, 1994-05-05)The positions of sequences necessary for transcription from the promoter located at map unit 4 of the bovine parvovirus (BPV) genome were determined. The autonomous parvoviruses, of which BPV is a member, contain two transcriptional units with promoters active in temporal order during infection. BPV proteins also appear in the same temporal order; the nonstructural (NS) proteins are produced before the capsid proteins. Northern blot analysis of BPV RNAs suggest that, like human parvovirus B19, all transcripts of BPV are initiated from a single promoter. A reporter construct was created by cloning the sequences from BPV containing the TAT A box located at nucleotides (nt) 250 - 254 upstream of the luciferase gene. A series of mutants were generated by deletion of restriction endonuclease fragments. Expression was assayed by transient expression of the constructs in transfected bovine fetal lung (BFL) cells, derived from the natural host. The data indicates that expression can be directed from the sequences containing the TAT A box. Analysis of expression of the deletion clones show that sequences from nt 120 - 170 of the BPV genome are also required for transcription. A search of these sequences reveals at least two consensus binding sites for cellular transcription factors. These are AP-I and the major late transcription factor (MLTF). MLTF has been shown to induce transcription from the early promoter of the dependovirus adena-associated virus (AA V). The presence of viral proteins provided in trans decreases expression from all constructs, with one exception. Expression, when nt 0 - 50 are deleted, is increased in the presence of mutant BPV NS-I. Feedback inhibition of P4 expression by NS-I, the P4 gene product, is seen in H-l, a rat parvovirus. The expression of BPV proteins in synchronized HeLa cells, which are not a natural tissue culture host was examined to determine the effect of a non-permissive host on BPV transcription. Expression of viral proteins in some parvoviruses is blocked in non-permissive cells. A block in transcription is seen during infection of non-permissive cells by B 19. AA V P5 expression is negatively regulated without co-infection of a helper virus, a non-permissive state. Luciferase activity was 570/0, compared to BFL cells. and a similar decrease in expression in the presence of viral proteins was observed. Viral nonstructural and capsid proteins could be detected by immunofluorescence, but only in the cytoplasm, suggesting that expression of viral proteins necessary for replication was not the block to a productive infection, but rather their translocation to the nucleus, as seen during restrictive H-l infection of transformed cells. BPV proteins have been observed localized to nuclear foci of transfected, synchronized BFL cells. The subcellular localization of viral proteins was detected by indirect immunofluorescence labeling using antiserum that recognizes both nonstructural and capsid proteins. Punctate nuclear staining has not been observed routinely during BPV infection or transfection of actively dividing cells. AA V proteins expressed in either BFL and HeLa cells, synchronized by hydroxyurea, were also observed in distinct nuclear foci. The same pattern of localization has been observed during co-infection of cells with AA V and adeno-virus, and during H-I infection. Gel mobility shift assays show that a cellular protein from BFL cells synchronized in S-phase recognizes and binds the right terminus of BPV. The right terminus, in hairpin and double-stranded linear forms, is an effective competitor, indicating the complex is specific, and suggesting that sequence, rather than structure may be the recognition signal for this cellular protein. The left terminus of BPV, in the hairpin conformation is also an efficient competitor for complex formation. It has been shown that a cellular protein forms a complex with the left terminus and that the right terminus is an efficient competitor for complex formation. This data taken together suggests the same cellular factor may recognize both termini, and correlates with the observation that both can act as origins of replication, and could be recognized by similar proteins. The heterologous terminus of AAV in the hairpin conformation, is not an efficient competitor for complex formation between the right terminus of BPV and a BFL cell protein. The lack of competition may support the indication that the recognition signal is a specific sequence rather than a particular secondary structure.
- A biosystematic study of the rare plant Paronychia virginica Sprengel (Caryophyllaceae) employing morphometric and allozyme analysesRohrer, Wendy L. (Virginia Tech, 1997-09-18)Paronychia virginica Spreng. (Caryophyllaceae) is a perennial evergreen herb of exposed, relatively xeric habitats. Approximately 10 mid-Appalachian populations remain in Virginia, West Virginia, and Maryland and are disjunct from populations located primarily in Texas, Oklahoma, and Arkansas. A study was conducted to test the hypothesis that eastern and western populations differ significantly and, therefore, represent at least two distinct taxa. Statistical analyses of 8 qualitative and 24 quantitative morphological characters indicated very highly significant (P < 0.001) variation between eastern and western populations of P. virginica. Characters differing most significantly included sepal pubescence, awn length, awn pubescence, awn curvature, length-width ratio of leaves, and shape of leaf apices. Starch gel electrophoresis was performed and six enzyme systems/nine loci (EST-2, EST-3, LAP, MDH-1, MDH-2, PGI, PGM-1, PGM-2, and SKDH) were identified as being consistently scorable and informative. Although gene flow between populations of P. virginica was shown to be restricted (mean FST = 0.353), populations are maintaining relatively high levels of genetic diversity. Genetic variability was quantified for each population and mean values for number of alleles per locus (A), percent loci polymorphic (P), and expected heterozygosity (HEXP) were found to be 1.95, 47.22%, and 0.204, respectively, exceeding those values reported for seed plants, widespread species, and endemic species. Hierarchical F statistics suggest higher levels of genetic variability within individual populations than among populations, regardless of geographic location. All statistically significant (P < 0.05) deviations from Hardy-Weinberg equilibrium indicated a deficiency in heterozygotes at the respective loci. Considering results from both the morphometric and allozyme analyses, the current author suggests recognizing two distinct subspecies, P. virginica subsp. virginica in the eastern U.S. and P. virginica subsp. scoparia in the south-central U.S. Conservation efforts should be focused on the maintenance of existing populations in both eastern and western regions of the U.S. in order to preserve the genetic and evolutionary potential of these taxa.
- Cellular factors and viral elements for parvovirus replicationDeville, Catherine Michele (Virginia Tech, 1994-04-15)Autonomous parvoviruses, such as bovine parvovirus (BPV), need a factor present at the S-phase of the cell cycle for a productive infection, while dependent parvoviruses, the adenoassociated viruses (AAVs), require a helper virus to complete an infectious cycle. However, AAV can replicate autonomously in synchronized cells, suggesting that an S-phase factor substitutes for the helper virus. To investigate the nature of the cellular S-phase factor, we performed DNA retardation assays with uninfected nuclear extracts of S-phase cells, synchronized by hydroxyurea pretreatment, and radiolabeled parvoviral termini in their hairpinned conformation. We observed that proteins in HeLa cells, a tissue culture host for AAV, specifically interacted with the terminal sequences of this virus, which act as origins of replication (oris). These assays also showed specific binding between S-phase cellular proteins and termini (oris) of heterologous parvoviruses, for which the cells are not a natural host. For example, proteins from bovine fetal lung (BFL) cells, a tissue culture host for BPV, were able to bind to an AAV terminus and HeLa cell proteins interacted with both termini of the BPV genome. All DNA-protein complexes investigated appeared to be specific for S-phase synchronized cells. In order to begin to characterize the protein(s) involved in the complex formation, we performed SDS-PAGE electrophoresis of some retarded complexes. We report that a 54 kd protein was contained in the complex formed with the BPV left terminus and BFL cell extract. [Binding of BFL cell proteins to a BPV left terminus has been reported earlier]. Using a similar technique, we observed that two phosphoproteins of 55 and 90 kd were present in the retarded complex formed between a BPV left terminus and HeLa cell extract. An antibody directed against human p53, an anti-oncoprotein, was shown to compete binding of BFL cell extract and HeLa cell extract to the BPV left terminus. This antibody also competed the binding of HeLa cell extract to the AAV terminus. Our data suggest that proteins with similar characteristics, most probably among which is p53, are involved in the ori-binding complexes, possibly exerting a role as positive regulator of parvoviral replication. The secondary structure of the viral ends is remarkably conserved among parvoviruses. Of particular interest is the presence of mismatched/unpaired nucleotides, forming a bubble, in the stem of the left hairpin of almost all autonomous parvoviruses. To analyze the possible role of these unpaired/mismatched nucleotides in the BPV life cycle, two mutants clones lacking the bubble region were constructed and their replicative properties were analyzed after electroporation in permissive cells. Infectivity of the mutant clones was determined by three techniques: observation of cytopathic effect, detection of virally-coded proteins by indirect immunofluorescence, and transient DNA replication assays. We report that the mutant clone containing duplicate sequences of the (mismatched) nucleotides numbered 46 to 57 (BLOP) was defective for replication. The other bubbleless clone (BLOM), that contains duplicate sequences of the (mismatched) nucleotides 99 to 105, was able to replicate. The later clone produced monomer-length viral DNA at about 20% of the level of the infectious genomic clone of BPV, when electroporated as a linear excised sequence. This clone was infectious since it could be propagated by subsequent passage. Expression of viral structural proteins was seen by an indirect immunofluorescence assay using anti-capsid antibodies. Our results suggest that the bubble in the left hairpin of BPV is not required for the viral life cycle, but that specific sequences within the mismatched/unpaired region are necessary for viral replication.
- Chromosomal complements and variation in some Venezuelan annual killifishes (cyprinodontidae)Elder, John Franklin (Virginia Tech, 1988-05-05)Karyotypes of nine species of Venezuelan annual killifishes were compared. Karyotypic differences were found between species and between some genera. A chromosomal sexual dimorphism was found in Pterolebias hoignei (2N = 46). Males of this species possess a single large "Y" chromosome. No chromosomal divergence was detected among conspecific populations from different localities. Pterolebias hoignei and Pterolebias zonatus were found to differ both in diploid number (46 and 42 respectively) and in metacentric chromosome number (6 and 12 respectively). All Rachoviaspecies were found to have a diploid number of 44. Rachovia maculipinnisand R. brevis were found to differ from one another in metacentric chromosome number (20 and 12 respectively). Rachovia hummelincki and R. pyropunctata both possess metacentric chromosome counts of 10. No karyotypic differences were found between Austrofundulus transilis and Austrofundulus limnaeus (2N = 44, metacentrics = 12). Several populations of the latter although divergent in male color patterns, did not differ karyotypically. All species examined, except Rivulus stellifer, departed from the presumptive ancestral teleost diploid number of 48. The degree of chromosomal variation appears not to be as high among the New World as among the Old World annuals. This difference among lineages with similar life histories and reproductive strategies suggests that stochastic events have not played a major role in mediating chromosomal divergence in annual fishes.
- Evolutionary relationships in Oryza inferred from the gene that encodes the 10 kDa prolamin (seed storage protein) polypeptideMullins, Irene M. (Virginia Tech, 1997-12-01)Cereals represent one of the most important food crops in the world. Rice (Oryza sativa) is one of the most commonly consumed cereal grains, and as a result, has considerable economic and agricultural importance, despite their potential as a source of genetic material. The phylogenetic relationships among rice and its wild species are not well understood. The objectives of this study are to evaluate the rate and type of molecular variation present in the gene that encodes the 10 kDa prolamin polypeptide in Oryza (Poaceae), and use this information in understanding the evolution of the Oryza genus. Our principle hypothesis is that genetic diversity exists at the molecular level in wild species of Oryza, and that this diversity can provide useful information regarding the phylogenetic relationships among rice species and permit a more theoretical examination of the evolutionary processes, such as concerted evolution, within Oryza and its nine genomes. A phylogeny of Oryza is presented, and modes of evolution are discussed.
- Extra-pair paternity within the female-defense polygyny of the lizard, Anolis carolinensis: Evidence of alternative mating strategiesPassek, Kelly Marie (Virginia Tech, 2002-05-07)Mate competition is a prominent component of sexual selection theory. Typically, males attempt to mate with the most females possible and females attempt to mate with the highest quality males possible. In the polygynous female-defense mating system of Anolis carolinensis, males compete directly for females through territorial behavior. Inter-male competition is intense due to an average polygyny ratio of 1 male to 3 females despite a 1:1 adult sex ratio. Through high levels of territorial behavior (e.g., 100 displays/h, 27 m patrol distances/h, 70% of day in defense-related activities), males attempt to exclude other males from resident females who, in turn, both store sperm and ovulate a single-egg clutch at weekly intervals over a 4-month breeding season. Paternity of hatchlings in 16 naturally occurring breeding groups was analyzed to determine the extent to which the territorial resident male was able to prevent other males from fathering offspring of his resident females. Lizards residing in or neighboring a resident male's territory were collected and RAPD-PCR was used to determine the paternity of hatchlings. Of the 48 hatchlings from 26 females, resident territorial males fathered 52% of hatchlings; 15% were fathered by a male whose territory bordered that of the resident male and 21% were fathered by a smaller male living covertly within the resident male's territory. Paternity for the remaining 12% of hatchlings belonged to an unsampled male. Given that females mated with multiple males, laboratory-based controlled matings were conducted where females were sequentially paired with two males and RAPD-PCR was used to analyze which of the two males fathered the subsequent hatchlings to determine the mechanism of sperm precedence. Regardless of mating order, only one male of the pair fertilized the eggs. Male A. carolinensis have reproductive strategies present in addition to defending resident females and female A. carolinensis have options in addition to simply mating with the resident male. While sperm precedence is present in this species, it is not based on mating order, but may involve both the number of sperm deposited in the female's tract as well as the quality of those sperm.
- Gene Flow and Dispersal Among Populations of the Stonefly Peltoperla tarteri (Plecoptera: Peltoperlidae) in the Southern AppalachiansSchultheis, Alicia Slater (Virginia Tech, 2000-11-14)Despite a number of recent studies focused on the issue, patterns of stream insect dispersal in temperate streams of North America remain poorly understood. Movement of benthic invertebrates is notoriously difficult to measure directly using traditional means; however, genetic markers provide an ideal method for estimating stream insect dispersal. In this study, the control region of mitochondrial DNA was used to study gene flow and dispersal among populations of the stonefly Peltoperla tarteri in the Southern Appalachians. The control region of P. tarteri is approximately 1270 base pairs (bp) in length, 81% AT-rich, and contains variable numbers of a 74 bp tandem repeat containing the sequence motif "5'-GGGGGC-3". Many stream insects have long life cycles that result in the simultaneous existence of multiple cohorts throughout the larval development period. If larval development is fixed, genetic isolation among cohorts may confound genetic estimates of dispersal. Although larval head width data indicated that P. tarteri is semivoltine in southwestern Virginia, low levels of genetic differentiation among cohorts suggested that larval development of P. tarteri is not fixed and that some individuals complete development in one or three years rather than two. This 'cohort splitting' would result in individuals from distinct cohorts maturing at the same time and mating with one another. Such developmental plasticity may allow some individuals to avoid adverse environmental conditions. The extent and likely mechanism of dispersal was determined by comparing levels of population genetic differentiation within drainages to that among drainages. While there was no consistent relationship between genetic and geographic distance, genetic differentiation was generally high within and among drainages. Nested clade analysis indicates that historical fragmentation and range expansion coupled with contemporary gene flow explain the present day pattern of genetic variation in P. tarteri. In order for these historical patterns to have such a strong influence on present day genetic structure, both larval and adult dispersal must be restricted. However, the genetic data suggest that larval dispersal is more frequent than adult dispersal.
- Genetic marker frequency differences among strains of goldfish, Carassius auratusBorkholder, Brian D. (Virginia Tech, 1993-02-02)Starch gel electrophoresis and DNA fingerprinting were used to assess molecular genetic variation within and between strains of goldfish, Carassius auratus. Genetic variation observed both within and between strains was low using both methods. using isozyme markers, mean heterozygosity (0.0% - 4.4%) and percent polymorphic loci (0.0% - 30.6%) within strains were low compared to other vertebrate species. Analysis of the isozyme data using Chi-square tests against Hardy-Weinberg genotype expectations and wright's (1943, 1951) F statistics indicated modest heterogeneity in genotype frequencies between strains. DNA fingerprint band sharing values were determined both within and between strains of goldfish. Mean band sharing values within strains were high (68.0% -97.6%), comparable to values observed in highly inbred populations of other vertebrates. Levels of band sharing between strains were determined using a DNA mixing procedure. Band sharing values between strains (57.7% - 100%) were higher than those observed between strains of other domesticated species. Analysis of DNA fingerprint data using ANOVA indicated no significant differences between strains, suggesting that the fancy strains did not exhibit significant differentiation as manifested in DNA fingerprint phenotypes.
- The lethal and sublethal effects of aldicarb on the estuarine grass shrimp, Palaemonetes pugioDvorak-Grantz, Andrea Lynn (Virginia Tech, 1996-11-05)Estuaries, an important facet of coastal regions, are highly productive natural systems, frequently acting as drainage basins for various pollutants such as agricultural runoff. The estuarine grass shrimp, Palaemonetes pugio, has been shown to be a sensitive indicator of pesticide exposure. In this study, emphasis was placed on the quantification of the lethal and sublethal effects of aldicarb on three different life stages of P. pugio. Acute 96-h toxicity tests were conducted with newly hatched larvae, 22-d old larvae and adult grass shrimp to determine lethal toxicant ranges. LC₅₀ values were 85.0 ug/L for newly hatched larvae, 70.7 ug/L for 22-d old larvae and 125.4 ug/L for adults. The impact of aldicarb on specific neurological functions in the grass shrimp was examined using an acetylcholinesterase assay. Acetylcholinesterase (AChE) activity was reduced in the larvae after exposure to acute concentrations of aldicarb. Mean whole-body AChE activity for the newly hatched and 22-d larvae was significantly lower from the controls (P=0.009). Conversely, mean whole body AChE activity in the adult shrimp was not significantly different from the controls at any concentration (P=0.401), although there was a trend towards reduced activity at 50 and 100 ug/L exposures. A behavioral study was conducted to examine the ability of adult grass shrimp to detect and avoid aldicarb-treated seawater. Behavioral responses were measured in a modified steep gradient chamber. A partition divided the chamber, creating three distinct areas: 1) seawater 2) aldicarb-treated seawater and 3) mixing. There were significant differences between the control and exposed adult shrimp in the amount of time spent in the mixing area (P<0.05). Aldicarb exposed shrimp spent, on the average, 20% more time in the mixing area than the controls. Additionally, exposed shrimp spent significantly more time facing downstream, away from the toxicant source (P<0.05). Exposed shrimp displayed increasing hyperactivity and attempted to jump out of the chamber.
- Low Temperature Induction of Males and Other Developmental Anomalies in a Self-Fertilizing Hermaphroditic Fish SpeciesFisher, Michael T. IV (Virginia Tech, 1997-12-12)Rivulus marmoratus (Cyprinodont; Aplochelidae) is the only vertebrate known to reproduce by obligate internal self-fertilization. Selfing is the ultimate form of inbreeding and leads to virtual homozygosity in approximately ten generations, so that this mode of reproduction is effectively clonal. Populations of this species consist of arrays of homozygous clones, with high clonal diversity and low representation of each clone. Males occur at low frequency (<1%) in most populations, with a few notable exceptions. Females have never been recorded.High frequencies of males have been reported on two occasions; high numbers of males were reported on the island of Curacao in the 1970's, and males comprised up to 24% of the populations sampled on several Belize Cays during 1990-91. Hermaphrodites collected from one island, Twin Cays, proved to be heterozygous at mini-satellite loci when progeny tested and DNA fingerprinted. This was evidence that recent outcrossing had occurred.Earlier experiments with Floridian clonal lines had suggested that sex in this species was thermolabile; incubation of embryos at low temperatures (19° C)) produced up to 100% males. It was suggested that this might be part of an environmental sex determination (ESD) system in this species.If the induction of males is indeed part of and ESD in this species, then it may be that the temperature at which males are induced in a particular clone will be related to the temperature regime that clone encounters in nature. Therefore, it was hypothesized that the temperature at which males are induced would be related to the geographic origin of each clone, so that clones from the cooler extremes of the range would produce males at a lower threshold temperature than clones from the more equatorial center of the range. This study used laboratory reared descendants of clones from Vero Beach, Florida, Rio de Janiero, Brazil (the extreme north and south of the range), and from several Belize barrier islands and the Belize mainland (near the center of the range) in an effort to detect differences in the production of males between clonal lines based on their geographic origin. Eggs were collected from these hermaphrodites and incubated at 26° C)C, 22.5° C), or 19 ° C), representing normal, and below average temperatures for this species. These embryos were hatched and reared to sexual maturity at which time their sex was evaluated.Incubation at 26° C) resulted in 2-17% males, incubation at 22.5° C) resulted in 6-53% males, and incubation at 19° C) resulted in 47-74% males. However, the variation in male induction between clonal lines did not correspond to a geographic hypothesis.Also, significant numbers of deformed individuals were noted, particularly among individuals incubated at the lower two temperatures. Incubation at 26° C) resulted in 0-24% deformed, incubation at 22.5° C) resulted in 6-66% deformed, and incubation at 19° C) resulted in 36-87% deformed. It is clear that development at these lower temperatures is difficult for this species, and it may be that low temperature developmental stress is not a general challenge to this species.
- Molecular drive and population differentiation of satellite DNAs in Cyprinodon variegatusElder, John Franklin (Virginia Tech, 1993-02-05)The genome of the Atlantic sheepshead minnow, Cyprinodon variecratus (cyprinodontidae) contains at least two highly repetitive satellite DNA sequences. The major satellite sequence is present at roughly 106 copies per haploid genome. The basic repeating unit averages 170 bp in length, tandemly arranged, and is bracketed by a Hind III restriction site. The Hind III satellite has little or no sequence similarity with other fish satellite DNAs reported in the 1iterature. Comparative hybridization experiments detect related sequences in the genomes of several species of pupfish of the genus Cyprinodon as well as in some cyprinodontid species of the genera Jordanella and Floridichthys. No significant hybridization was detected with the New World species Cualac tesselatus and Megupsilon aporus or within species of the Old World genera Valencia and Aphanius.
- Morphometric Characterization of a Mercenaria spp. (Bivalvia) Hybrid Zone: Paleontological and Evolutionary ImplicationsPowell, Matthew G. (Virginia Tech, 2000-05-12)Paleontological documentation of hybridization events has the potential to address a multitude of evolutionary and paleobiological issues unanswerable by purely biological means. However, previous studies of modern hybrids suggest that their morphology is often insufficient for their reliable discrimination. This study analyzes the morphology of an extant, genetically-identified Mercenaria spp. (Bivalvia: Veneridae) hybrid zone using Bookstein coordinates and multivariate methods to answer two questions: (1) can hybrid Mercenaria spp. individuals be identified based on morphology alone, and (2) would a Mercenaria spp. hybrid zone be recognizable in the fossil record? Multivariate statistical procedures (principal components analysis, canonical variate analysis, etc.) using Bookstein coordinates demonstrate that, within the hybrid zone, hybrid individuals cannot be identified due to extreme overlap with the parental taxa. The hybrid zone as a whole, however, can be identified by comparison with pure-species populations sampled from outside the hybrid zone. Hybrid zones occupy parental species morphospace plus intermediate morphospace. The technique of using multiple pure-species populations to establish species morphospace is introduced to control for processes that may also result in morphological intermediates at ecological time scales (dimorphism, ecophenotypy, and geographic variation). Four alternative causal explanations of morphological intermediates through geological time (primary intergradation, uncoupled genetic and morphological divergence, time-averaged evolving populations, and developmentally instable populations) are evaluated. A literature survey strongly suggests that neither time-averaging nor developmental instability is occurring at the beginning of a lineage's evolutionary history, and that hybridization may be much more extensive than paleontological data suggest.
- Multiple paternity and the breeding biology of the red-eyed treefrog, Agalychnis callidryasd'Orgeix, Christian A. (Virginia Tech, 1996-05-05)External fertilization makes male anurans susceptible to direct intrasexual competition for fertilization opportunities at the egg mass. The red-eyed treefrog, Agalychnis callidryas, is one species in which pairs of males appear to simultaneously fertilize the clutch of a Single female. DNA fingerprinting revealed the presence of multiple paternity in two egg clutches examined from two matings involving a female with two males. The breeding biology of females and the potential costs and benefits of mating with multiple males were examined. Females were found to decrease the number of eggs in matings with multiple males. In addition, amplexed females moving toward Oviposition sites avoided secondary males by moving when approached by secondary males. Mortality to the eggs as a result of multiple males attempting to amplex females is suggested as the reason females avoid multiple males. Males were found to exhibit calling site defense from other males. Males used a combination of auditory and a visual behavior in defending calling sites. The call types are described and the contexts within which calls occur is discussed. Density of frogs was found to be a better indicator of the occurrence of matings involving multiple males than the operational sex ratio (number of males/number of females).
- Natural history note - A novel terrestrial fish habitat inside emergent logsTaylor, D. S.; Turner, Bruce J.; Davis, W. P.; Chapman, B. B. (University of Chicago Press, 2008-02)Reports of new habitats for a major group of organisms are rare. Fishes display diverse adaptations for temporary (amphibious) existence on land, but to our knowledge, none have ever been reported regularly living inside emergent logs. Here, we show that the mangrove killifish, Kryptolebias marmoratus, a species previously known to emerse (leave the water) regularly, is now known to emerse and aggregate in large numbers inside decaying mangrove logs that have been "galleried" by terrestrial insects. This behavior has now been documented in both Belize, Central America, and Florida, U.S.A., populations and represents the first known case of fishes entering terrestrial woody material. The dense packing of fish in the narrow log galleries may imply a novel social context in which intraspecific aggressive behaviors are reduced, possibly mediated by the physiological limitations imposed within this restrictive habitat.
- Nymphaea odorata (Water-lily, Nymphaeaceae): Analyses of molecular and morphological studiesWoods, Kristi Yvonne (Virginia Tech, 2003-02-06)Molecular and morphologic studies were used to determine the evolution, classification and differentiation of Nymphaea odorata. Molecular analyses of the nuclear internal transcribed spacer (ITS) region, the chloroplast trnL-F region, and inter-simple sequence repeat (ISSR) markers determined the variation present between and within two species of Nymphaea. The ITS region resulted in a phylogeny depicting strong separation between species (N. mexicana and N. odorata) and some separation between N. odorata's subspecies. The ITS region contained polymorphisms, which upon SAHN clustering and principle coordinate (PCOA) and minimum spanning tree (MST) analyses produced groups similar to the clades in the ITS phylogeny. Sixteen accessions were chosen for trnL-F analysis, where a subspecies-specific molecular marker was found. In most accessions the marker confirmed the original subspecies classification. Molecular analyses using ISSRs characterized among population variation in N. odorata and N. mexicana using five primers. ISSR markers among populations were highly variable within a species and were used in UPGMA, PCOA and MST analysis, which resulted in separation between the subspecies. Both univariate and multivariate analyses were performed on quantitative and qualitative morphological characters. An analysis of variance resulted in six morphological characteristics that were statistically significant (P< 0.05), the majority being leaf blade characteristics. Multivariate statistics of principle component analysis and discriminate analysis resulted in groups for each subspecies, both emphasized the importance of quantitative leaf blade characteristics. Overall, both morphology and molecular characteristics supported the classification of subspecies for ssp. odorata and ssp. tuberosa, due a lack of strong segregation of characteristics.
- Ontogenetic Changes and Environmental Hypoxia: Responses of Two Fish Species to Low Oxygen Concentrations at Early Life StagesBalfour, David Leigh (Virginia Tech, 1999-12-01)Hypoxia refers to any condition in which the water is less than fully saturated with oxygen. Although it is generally accepted that adults are more tolerant of hypoxic conditions than larval stages, there is little information to support this assumption. To determine whether reduced concentrations of dissolved oxygen (DO) affect fishes differently during various early life stages, I examined the responses of two species of fish (fathead minnows (Pimephales promelas) and rainbow trout (Oncorhynchus mykiss)) exposed to low dissolved oxygen concentrations at different ages during the first 100 days post-hatch. The changes in oxygen requirements and respiratory patterns that occur during ontogeny and exposure to hypoxia were observed. The results of this study suggest that the early larval stages appear to be at least as tolerant of short-term exposure to low dissolved oxygen concentrations as the older, more developed stages. Fathead minnows underwent a gradual transition from being metabolic conformers to regulators during development. Hemoglobin appeared to be playing a larger role in oxygen supply in the early post-hatch trout than in the minnows. Fathead minnow larvae produced relatively low concentrations of lactate upon exposure to hypoxia. Conversely, rainbow trout larvae exhibited significant increases in lactate concentration under similar conditions. This implies that there is a threshold oxygen concentration below which trout larvae utilize anaerobic metabolism to provide additional energy. Lactate dehydrogenase activity increased as the rainbow trout larvae aged, suggesting that they develop an anaerobic capacity which could be used to provide additional energy during hypoxia. The minnows did not exhibit this increase in activity. The ability of larval fishes to detect and avoid hypoxic conditions was also examined. The overall trends suggest that throughout this period of development, both fish species gradually leave an area as the dissolved oxygen concentration decline. Both species appeared to leave the hypoxic areas with deliberate motions, indicating that a directed sensor system allowed them to detect oxygen gradients. The results suggest that a combination of physiological, biochemical, and behavioral mechanisms may allow fishes to cope with hypoxia.
- Phylogeographic structure of the Atlantic pupfish, Cyprinodon variegatus (Cyprinodontidae), along the eastern coast of North America: Evidence from mitochondrial nucleotide sequencesFinne, Katherine Lee (Virginia Tech, 2001-02-13)Cyprinodon variegatus is a pupfish that inhabits the Atlantic coast of North America, nearly continuously, from Massachusetts to Belize. This research attempts to resolve the phlylogeography of C. variegatus by investigating the genetic sequence structure of the mitochondrial control region (D-loop, non-coding origin of replication) and cytochrome-b gene for evidence of northern and southern subspecies within the Atlantic Coast of the eastern United States. Additionally, it will be may be possible to determine if secondary hybrid zones developed as a result of the last retreat of ice from North America during the Pleistocene, about 17,000 years ago. A definitive monophyletic northern clade was found using parsimony, distance, and maximum likelihood phylogenetic methods to analyze the control region data. The cytochrome-b sequence data supported this monophyletic northern clade, although their utility for this analysis is marginal. Little evidence was found for the existence of a hybrid zone. More thorough sampling will be needed to make more accurate determinations regarding the existence of such a zone.
- Population Genetics of Death Valley Pupfishes (Cyprinodontidae:Cyprinodon Spp.) and the Identification of a New Retrotransposable Element FamilyDuvernell, David D. II (Virginia Tech, 1998-03-24)Study of the genetic relationships and evolutionary histories of pupfish populations (Cyprinodontidae: Cyprinodon spp.) from the remnant aquatic habitats of Death Valley was approached by exploring the genetic structure and divergence within and among populations using mitochondrial and nuclear DNA markers. The findings of these studies illustrate the influences of population size and isolation time in the divergence of small, fragmented populations largely via genetic drift. The information revealed in this study has implications for assessing priorities in the conservation of the unique evolutionary heritage among populations of the Death Valley pupfishes. A new retrotransposable element family was identified and characterized. This family of genetic elements was uncovered during a search of the pupfish genome for transposable elements to be used as molecular markers for population analyses. The description of this element family, named "Swimmer 1" (SW1), provides new insights into the evolution of long interspersed nuclear elements (LINEs) in vertebrates. Therefore, a full characterization of the SW1 element family was undertaken in the Japanese medaka (Oryzias latipes) as well as in the pupfish genome. The Japanese medaka is a model organism widely used for genetic and developmental biology studies.
- Reproductive Isolation and Genetic Divergence in a Young "Species Flock" of Pupfishes (Cyprinodon sp.) from San Salvador Island, BahamasBunt, Thomas Michael (Virginia Tech, 2001-05-08)The study of the process of speciation is instrumental to understanding the species diversity observed today. Diverging populations are intriguing, because speciation has not reached an endpoint, yet the process that may eventually lead to distinct species can be studied. Systems that contain many putative species and/or parallel divergences, such as many species flocks and species pairs, are extraordinary examples of divergence and therefore are critical to the understanding of the speciation process. A "miniature" species flock of pupfish (Cyprinodon variegatus) discovered in lakes on San Salvador Island, Bahamas has evolved in less than 6 000 years, and is, therefore, important to the study of the pace of evolutionary processes. The San Salvador Island pupfish flock is composed of a normal form, which resembles coastal C. variegatus, and bulldog and bozo morphs, which diverge ecologically and morphologically from the normal morph. In Chapter 1, I sequenced the mtDNA control region and used haplotype frequency analyses to assess the level of differentiation between sympatric normals and bulldogs sampled from Osprey Lake and Little Lake on San Salvador Island. The bozo morph was too rare to include in the study. I also included samples of normals that occur in lakes without bulldog and bozo morphs to assess any differences between lakes on the island. All haplotype frequency comparisons for sympatric normals and bulldogs were highly significant, which suggests these morphs are distinct populations in sympatry and, therefore, have characteristics of biological species. Further, an estimation of Time for Speciation supports geological data that suggest this fauna is very young (6 000 years). The San Salvador Island pupfish species flock is, therefore, the youngest known species flock and presents an important model system for the study of how morphological and ecological divergence can promote speciation in Cyprinodon. In Chapter 2, I first compared the San Salvador Island pupfishes to other Bahamian C. variegatus populations to assess the level of inter- and intra-island pupfish population differentiation in the Bahamas. The mtDNA control region was sequenced for bulldogs and normals from San Salvador Island and normals sampled from New Providence and Exuma Islands. San Salvador Island bulldogs were found to be distinct from all normal populations sampled, and comparisons of shared haplotypes suggest they originated on San Salvador Island rather than any of the other islands sampled. This was intriguing, because a "bulldog-like" morph has recently been observed in a lake on New Providence Island, which suggests parallel divergences may be occurring throughout the Bahamas. I also sequenced the mtDNA cytochrome b gene to assess the phylogeography of C. variegatus. Populations were sampled from the Bahamas and the east coast of North America, and the results suggest the Bahamas were only recently colonized by the Southern coastal lineage of C. variegatus. A distinct Northern lineage of C. variegatus, which may warrant species designation, was also supported by the cytochrome b data. Overall, the results supported a San Salvador Island origin for the Little Lake and Osprey Lake bulldog morphs, and also suggest the Bahamian C. variegatus populations are very young.
- Responses of Superoxide Dismutases to Oxidative Stress in Arabidopsis thalianaErturk, Hatice Neval (Virginia Tech, 1998-12-10)Superoxide dismutases (SODs) catalyze the dismutation of superoxide radicals to oxygen and hydrogen peroxide. Mn SOD is localized in mitochondria, Cu-Zn SOD is in the cytosol and chloroplast, and Fe SOD is in chloroplasts. The effects of a chloroplast-localized oxidative stress, caused by methyl viologen or 3-(3, 4-dichlorphenyl)-1-1′ dimethylurea (DCMU) on SOD populations were investigated. A cloned Arabidopsis thaliana Fe SOD gene was expressed in Escherichia coli and was purified from transformed cells. This protein was used to raise antibodies against A. thaliana Fe SOD which in turn were used to quantify the effects of oxidative stress on Fe SOD protein. Effects of oxidative stress on enzyme activity were measured in native gels. Fe SOD responded to oxidative stress with an increase in activity, but not in antibody reactive protein. Two novel forms of Fe SOD activity, with faster migration rates in activity gels, were detected. Mn SOD, a mitochondrial enzyme, responded to the stress with an increase in activity. In contrast, the activity or amount of Cu-Zn SOD protein did not respond to this oxidative stress. In light of these results, we propose that SODs respond to oxidative stress at the enzyme and gene levels. Mitochondrial Mn SOD responded to a chloroplast-localized stress with an increase in activity, suggesting either that the site of action for methyl viologen is not exclusively in the chloroplast or that there are other signals among the compartments of the cell. Fe SOD, but not Cu-Zn SOD responded to stress, suggesting that Fe SOD may be the stress responsive enzyme in this organelle. Evolutionary relationships among different isoforms were investigated based on the known primary, secondary, and tertiary structures of these isoforms. The three dimensional structure of A. thaliana Fe SOD was modeled by using structures of crystallized E. coli and Pseudomonas ovalis Fe SODs as templates. Comparison of prokaryotic Fe SOD with eukaryotic isoforms showed that Fe and Mn SODs are structurally homologous, whereas Cu Zn SOD is not.