Showing papers in "Virginia journal of science in 1999"

PCR and FISH Detection Extends the Range of Pfiesteria piscicida in Estuarine Waters

Abstract: PCR and fluorescent in situ hybridization probes were used to assay for the presence of the dinoflagellate Pfiesteria piscicida in 170 estuarine water samples collected from New York to nothem Florida. 20% of samples tested positive for the presence of P. piscicida, including sites where fish kills due to Pjiesteria have occurred and sites where there was no historical evidence of such events. The results extend the lmown range of P. piscicida northward to Long Island, New York. The results also suggest that P. piscicida is a common, and normally benign, inhabitant of estuarine waters of the eastern

A Cohort Study to Determine the Epidemiology of Estuary-Associated Syndrome

Abstract: INTRODUCTION Estuary-Associated Syndrome (EAS) is the name given to a potential illness characterized primarily by changes in an individual's cognitive abilities, including acute onset of memory loss or the sudden inability to solve simple problems. Other possible signs of illness include respiratory symptoms, skin rash, or gastrointestinal distress. This illness appears to arise following exposure to toxin produced by Pjiesteria piscicida, or other toxic dinoflagellates, that resides in estuary waters. In 1988, researchers at the College of Veterinary Medicine, North Carolina State University, observed the unusual death offish in laboratory tanks following exposure to water from the Pamlico River Estuary in North Carolina (Smith et al., 1988). A toxic dinoflagellate was identified in association with the fish deaths and researchers at North Carolina State University were able to reproduce the fish toxicity in a laboratory setting (Burkholder et al., 1992). The organism was named Pjiesteria piscicida (Lewitus et al., 1995; and Steidinger et al., 1996). In 1995, this dinoflagellate was found in the Chesapeake Bay (Lewitus et al., 1995) and more recently in creeks and rivers of Maryland and Virginia (Marshall, personal communication 1999; Grattan et al., 1998). It is now generally recognized that there is a complex of Pjiesteria-like dinoflagellates, including P. piscicida and an estimated ten or 11 similar organisms. These have been referred to as Pjiesteria-complex organisms and more recently as Pjiesteria-like organisms (PLOs). PLOs have a complex life cycle and reside in different forms in the sediment or the water column of estuarine waters. They appear to require live finfish or their secreta for transformation to a toxic phase with subsequent release of a powerful exotoxin(s). Because of the many different forms for these organisms, speciation is difficult and the accepted method depends on scanning electron microscopy (SEM). Screening for PLOs in water samples is currently done using a light microscope with SEM performed if high concentrations of PLOs are seen. Different laboratories have developed molecular methods to identify these organisms in water samples and these methods are being tested (Oldach et al., 1998; Rublee et al., 1999). Work is also ongoing to develop a test to analyze released toxins.

Growth and Feeding Studies on the Algal Feeding Stage of a Pfiesteria-like Dinoflagellate

Abstract: The dinoflagellate Cryptoperidiniopsis sp. was isolated from sediment samples taken from Virginia estuaries, and established in culture for subsequent growth and feeding studies. The maximum abundance, or yield, of Cryptoperidiniopsis is exponentially related to the concentration of algal prey and is saturated at about 4.00 x 10 mL-. Salinity from 10-20 ppt and temperature between 15-25 C have no effect on the yield of this form of Cryptoperidiniopsis. Light intensity has a secondary effect in that the algal prey reproduces more quickly in higher light as they are being grazed. Growth rates of Cryptoperidiniopsis were highest with a cxyptophyte, Cryptomonas, as food, but growth was also demonstrated utilizing both diatoms and chlorophytes. Cryptoperidiniopsis sp. is similar to Pjiesteria in that it feeds myzocytotically with a peduncle, is similar in size and shape, has a complex life cycle, and is distinguished only by plates hidden under membranes. INIRODUCTION Over the past decade, the importance of heterotrophic dinoflagellates in coastal waters has received increased attention. Studies have found they are abundant (Lessard 1991; Jeong, 1999) and they capture a variety of prey by several different means (Hansen and Calabo, 1999; Schnepf and Elbrachter, 1992). Some feed on other dinoflagellates (Hansen and Nielsen, 1997; Jeong et al., 1997) as ·well as zooplankton (Jeong 1994). One of the most exireme examples is Pjiesteria piscicida, which can feed heterotrophically, but also has the capability to survive through photosynthesis by ingesting and using the chloroplasts of other algae (Lewitus et al., 1999). This dinoflagellate has been documented to possess a widespread distribution in turbid estuaries (Bmkholder et al., 1995; Burkholder and Glasgow, 1997; Steidinger et al., 1996). Prior to this study we isolated several dinoflagellates from sediments coming from Virginia estuaries (Marshall et al., 1998; Marshall et al., 1999) and these included a strain of Cryptoperidiniopsis (identification confirmed by Drs. Karen Steidinger through SEM analysis and Parke Rublee with a genetic probe) which was common in our samples (Figure 1). Dr. Steidinger indicated this strain is morphologically distinct from the one known species, C. brodyii, first found in Florida, which differed morphologically by a slight variation in its apical plates. The objectives of this study were to: 1 Department of Biological Sciences, Old Dominion University, Norfolk, Virginia 23529 2 Department of Ocean, Earth, and Atmospheric Sciences, Old Dominion University, Norfolk, Virginia 23529 338 VIRGINIA JOURNAL OF SCIENCE FIGURE 1. Cryptoperidiniopsis sp. A. (Upper picture) Ventral view of the motile vegetative stage; B. (Lower picture) Lobose amoeboid stage. DINOFLAGELLATE GROWTH AND FEEDING 339 1. To determine the relationship of Cryptoperidiniopsis in our cultures to several environmental factors. These included light intensity, prey concentration, salinity, and temperature; 2. To determine the growth rate of these cells; and 3. To identify any prey preferences this species may have. METHODS A series of sediment samples (250 mL) were taken with a petite ponar grab in 1998 by personnel from the Virginia Department of Health's Shellfish Sanitation Division and the Virginia Department of Environmental Quality as part of the Pfiesteria Monitoring Program in Virginia. Sub-samples from these sediment samples were incubated with:f/2-Si medium in a 50 mL Falcon tissue culture flask. To each sediment incubation, 5 mL of the food source Cryptomonas (CCMP 767 Provesali-Guillard) were added. Dinoflagellates were subsequently isolated from these incubations and identified. Cultures of Cryptoperidiniopsis (strain DEQ-002) were tested for their growth response to four factors: light, concentration of prey, salinity, and temperature. Triplicate 250 mL Falcon tissue culture flasks were used for both the experimental (dinoflagellates and prey) and control (only prey). All exi,eriments were conducted in incubators, in order to achieve constant temperature and fixed light conditions. To each flask, 5 mL of dinoflagellates and 10 mL of Cryp_tomonas were added. The Cryptomonas inoculum has a concentration of 1.891 x 10 mLwhile the dinoflagellates were at 1.338 x 10 mLFlasks were filled to 100 mL withf/2-Si medium at 15 ppt (except for those adjusted.for the salinity experiment). The medium was created by diluting water from the mouth Chesapeake Bay with double de-ionized water and passing it throu~ a 0.2 um glass filter. The initial concentrations in the flasks were 1.89 x 10 mLfor the prey and 6.69 x 10 mLfor the dinoflagellates. All studies included triplicate culture sets. Concentration of ~rey was investigated on three levels. These were 2X (1.89 x 10 mL-1), IX (0.95 x 10 mL-1), and 8X (7.55 x 10 mL-). The growth of Cryptoperidiniopsis was observed under three different set temperatures: 15C, 20C, and 25C. Light concentration was also varied on three levels. This was achieved by leaving one set of triplicates over time in a direct line with the incubator lights while another set was wrapped once with mesh screening. A third set of triplicates was wrapped twice in mesh screening to prevent more light from reaching the cells. RESULTS The Cryptoperidiniopsis in our study is very similar to Pfiesteria piscicida. It feeds myzocytotically with a peduncle, possesses similar size and shape, has a complex life cycle which includes both cyst and amoeboid stages, and has its distinguishing plates hidden under layers of membranes (Seaborn and Marshall, 1998). This species was found in 27 of 51 tested sediment samples from the Virginia portion of Chesapeake Bay. Scanning electron microscopy reveals the Virginia strain is slightly, yet consistently, different from the one know species C. brodyii. The initial concentration of Cryptomonas prey had a significant effect on the maximum Cryptoperidiniopsis abundance (Figure 2). This response was similar where light and temperature were controlled. The intensity of light had a significant effect 340 VIRGINIA JOURNAL OF SCIENCE on the maximum dinoflagellate yield (Figure 3). The growth rate of Cryptoperidiniopsis (1.43) is high when compared to otherdinoflagellates suchasPjiesteria piscicida (Figure 4 ). Peak dinoflagellate abundances and growth rates did not differ for temperatures between 15-25C. Maximum dinoflagellate abundances and growth rates did not significantly differ for salinities from 10-20 ppt Cryptoperidiniopsis grew at a much high rate when feeding on the cryptophyte, Cryptomonas, as opposed to other algal prey (Figure 5). CONCLUSIONS The incubated sediment samples yielded a strain of Cryptoperidiniopsis which is common from Virginia tidal waters. Preliminary data of food preferences suggest this dinoflagellate has a higher growth rate when feeding on cryptophytes as opposed to other algal prey. This result is similar to the cryptophyte feeding preferences shown by Pfiesteria piscicida (Glasgow et al., 1998). Both dinoflagellates feed in a similar fashion through the use of a peduncle and have complex life cycles including the formation of amoeboid stages. The concentration of cryptophytes is an important factor which determines the abundance and duration of Cryptoperidiniopsis in the ·water column. The dinoflagellate exhibits robust growth in salinities from 10-20 ppt and in temperatures between 15-25C. LIIBRA TURE CITED BUikholder, J.M., Glasgow Jr., H.B., and Hobbs, C.W. 1995. Fish kills linked to a toxic ambush-predator dinoflagellate: distribution and environmental conditions. Mar. Ecol. Frog. Ser., 124: 43-61. Burkholder, J.M. and Glasgow Jr., H.B. 1997. The ichthyotoxic dinoflagellate, Pjiesteria piscicida: Behavior, impacts, and environmental controls. Limnol. Ocenogr., 42: 1052-1075. Glasgow, H.B., Lewitus, A.J., and Burkholder, J.M. 1998. Feeding behavior of the ichthyotoxic estuarine dinoflagellate, Pfiesteria piscicida, on amino acids, algal prey, and fish vs. mammalian erythrocytes., UNESCO 4 pp. Hansen, P.J. and Calabo, A.J. 1999. Phagotrophic mechanisms and prey selection in free-living dinoflagellates, J. Euk. Microb., 46: 382-389. Hansen, P.J. and Nielsen, T.G. 1997. Mixotrophy feeding of Fragilidium subglobosum (Dinophyceae) on three species of Ceratium: effects of prey concentration, prey species and light intensity. Mar. Ecol. Frog. Ser., 147:187-196. Jeong, 1994. Predation by the heterotrophic dinoflagellate Protoperidnium cf divergens on copepod eggs and early naupliar stages. Mar. Ecol. Frog. Ser., 114: 203-208. Jeong, H.J. 1999. The ecological roles of heterotrophic dinoflagellates in marine planktonic community. J. Euk. Microb., 46: 390-396. Jeong, H.J., Lee, C.W., Yih, W.H., and Ki~ J.S. 1997. Fragilidium cf mexicanum, a thecate mixotrophic dinoflagellate which is prey for and a predator on co-occurring thecate heterotrophic Protoperidinium cf divergens. Mar. Ecol. Frog. Ser., 151: 299-305. Lessard, E.J. 1991. The trophic role of heterotrophic dinoflagellates in diverse marine environments. Mar. Microb. Fd. Web., 5:49-58. DINOFLAGELLATE GROWTH AND FEEDING 341 Predicting Dino Max from Initial Prey (Incubator) 35 --------------------------~ :i25-!------------~.,,e.=------------=-----1 e 0 20 +--------71\"\"~---+----------------l 0 0 .... .!':. 15 .J__ _ __A,.__J'.:..._ ____________________ ----J ! C: i5 10 .L_------------------1R = 0.84 Adj. R2 = 0.81 5 +-------------------1p-value = 0.0005 ,__ _ __, 0-!----------,-----------..---------1 0 100 200 300 400 500 600

Conservation Status of the Southern Appalachian Herpetofauna

Abstract: Seventy one species of amphibians (55 salamanders, 16 anurans) and 46 species of reptiles (15 turtles, 8 lizards, 23 snakes) inhabit a five state area (Kentucky, Nort:11 Carolina, Tennessee, Virginia, West Virginia) in the southern Appalachian region bordered by the Potomac River, the Blue Ridge Mountains, and the western margin of the Appalachian Plateau. Of these, 4 7.9% of the amphibian fauna and 52.2% of the reptilian fauna are listed as being of conservation concern by federal, state, and Natural Heritage programs in all or a portion of their ranges in this region. The Shenandoal1 salamander (Plethodon shenandoah) is listed as Endangered and the Cheat Mountain salamander (Plethodon nettingi) is listed as Threatened under the U.S. Endangered Species Act. Nine others are classified as federal species at risk. State endangered species number 1-3 (per state), threatened 1-4, and special concern or declining 6-19. Three to 6 species per state are additionally listed as natural heritage Sl and 2-13 as S2. We review the existing and potential threats to species and populations (e.g., timbering, urbanization, collection for the wildlife trade, acid precipitation, introduced species) and provide an assessment of the conservation status of the southern Appalachian herpetofauna based on land ownership. Presented in the Appalachian Biogeography Symposium, June 25-29, 1995, 14 VIRGINIA JOURNAL OF SCIENCE

Monitoring Results for Pfiesteria piscidida and Pfiesteria-like Organisms from Virginia Waters in 1998

Abstract: Results of an extensive 1998 monitoring program for the presence of Pflesteria-lik.e organisms (PLO) in Virginia estuaries indicate these dinoflagellates are widely distributed in both the water column, and as cysts in the sediment, however Pflesteria piscicida was not detected at this time. The highest concentrations of PLO were in estuaries along the Virginia shore line of the Potomac River, and in western Chesapeake Bay estuaries from the Little Wicomico River to the Rappahannock River. The most common PLO included Cryptoperidiniopsis sp. and Gymnodinium galatheanum. The lowest PLO concentrations were at ocean side locations. PLO were also present throughout the water column at stations in the lower Chesapeake Bay, being most abundant in waters above the pycnocline. INTRODUCTION Pflesteria piscicida is a predatoiy dinoflagellate which is capable of toxin production and has been associated with both massive fish kills and human illness (Burkholder et al., 1995; Glasgow et al., 1995). This species has been identified in several estuaries along the U.S. east coast, with its most extensive developmentto date in North Carolina estuaries (Burkholder et al., 1995). There are several dinoflagellate species that resemble Pflesteria piscicida in size, morphology, and some even have similar life cycle stages. These species have been placed in a categoiy called the Pflesteria-like organisms (PLO). They consist of a variety of gymnodinioid type cells that may include besides Pflesteria, species within the genera Gymnodinium, Cryptoperidiniopsis, Gyrodinium,Amphidinium, and others (Burkholder, 1997; Steidinger et al., 1997). An earlier designation for this group was Pflesteria complex organisms (PCO), with the term toxic Pflesteria complex (TPC) referring to those Pflesteria species known to produce ichthyotoxins. Bwkholder et al. ( 1999) have also identified both toxin producing and non-toxin producing populations of this species. Since the recognitionofmimte motphologicalfeatures of aP. pisicida cell is necessaiy for its identification, light microscopy alone is not adequate to distinguish this species from other PLO (and 1PC) cells (Steidinger et al., 1996). However, light microscopy is commonly used as the initial step to identify those cells that can be placed in the PLO categoiy. The enumeration of these cells from a water sample will give what is termed the \"presumptive cell counts\" for P. piscicida. These counts do not by themselves indicate the presence of Pflesteria, only that this species may be included in this assemblage. When these counts exceed a predetermined concentration level of concern, subsequent steps are then followed to determine if P. piscicida is the dominant constituent of these counts and if a toxic population of this species is present. Bwkholder et al. (1995) indicate cell concentrations of the toxic 288 VIRGINIA JOURNAL OF SCIENCE P. piscicida 250 cells/mL are generally lethal to fish. If presumptive PLO cell counts of this magnitude occur, further examination using scanning electron microscopy (SEM) is then recommended to provide identification of the dominant species. If P. piscicida is present, then a fish bioassay will determine if this is a toxin producing population (Burlcholder et al., 1999). The earliest notation of Pfiesteria piscicida in Virginia is given by Burlcholder et al. (1995) who identified these cells in the lower reach of the York River. Rublee et al.(1999) using a genetic probe in 1998 have identified P. piscicida in Mosquito Creek, which is located on the Virginia ocean side of the Delmalva Peninsula. However, no fish bioassays were run in either of these cases to detennine if they were toxin producers. In response to the rising concern regarding the presence of P. piscicida reported in Maryland estuaries during 1997, the Virginia Task Force on Pfiesteria established a monitoring plan for Virginia waters. This included the examination of water and sediment samples for P. piscicida during occurrences of fish kills, or when there was a high incidence of fish bearing lesions. This PLO analysis was conducted at the Old Dominion University Phytoplankton Analysis Laboratory. Neither of these events were common in 1997, and there were only several occasions when the examination of water samples identified PLO cell concentrations greater than 250 cells/mL (Marshall et al., 1998; Marshall and Seabom,1998). These locations were in the Pokomoke River(Virginia region), Rappahannock River, and Great Wicomico River. None of these events, proved to be associated with P. piscicida (based on representative SEM analyses by JoAnn Burkholder and Karen Steidinger). The fish bioassays by Dr. Burkholder also gave negative results for P. piscicida. Among these sites, the area that received special attention in 1997 was the Pokomoke River in Maryland, which was the site of a fish kill that was associated with toxic Pfiesteria. Although this river originates in Maryland, its lower reach forms the border between Virginia and Maryland, with its southern shoreline in Virginia Subsequent water analysis and fish bioassays of this lower region did not reveal Pfiesteria cells in 1997 (Marshall and Seaborn, 1998). One of the major questions that remained at the close of 1997 centered on what extent is P. piscicida and other PLO species present in Virginia estuaries. In order to gain information regarding the distribution and abundance of Pfiesteria-like organisms in Virginia estuaries, a broad based monitoring program was established in 1998 under the sponsorship of the Virginia Department of Environmental Quality and the Virginia Department of Health. This monitoring program emphasized two plans for PLO sample analysis. The first indicated that water and sediment samples would be examined for PLO during significant fish kill events, or when there was a high incidence of fish having lesions. For instance, if more than 20% of a certain fish population had lesions, and there were at least 50 fish in the count, this would warrant sample analysis for PLO. The second approach involved monitoring representative estuaries in Virginia for PLO. In both plans, any high concentrations of PLO would initiate subsequent SEM analysis aid toxic fish bioassays. There were originally two major objectives of this study. The first was to identify the presence and distribution of Pfiesteria and other PLO in Virginia estuaries. The other was to determine if there are relationships in the abundance and distnbution patterns of PLO to water quality conditions at these sites. Many of these PLO have co-existed with P. piscicida during fish kill events (Burkholder et al. 1997; Steidinger, MONITORING FOR Pfiesteria 289 1997). This infers similar environmental conditions and locations that support PLO development may also apply to the more elusive Pfiesteria spp. In support of this second goal, personnel from the Virginia Department of Environmental Quality (VDEQ) analyzed water samples taken during each collection for a broad survey of water quality parameters. This data in relation to the PLO abundance are presented by Weber and Marshall (1999). In general, they found no high correlations between these two sets of parameters. This may be a result of the low cell concentrations and variety of many of these PLO over the 6 month period. Rather than having a single species to relate to these water quality parameters, the PLO were composed of a group of species that may have had different environmental cues and requirements for their development. Also, due to the multiple life stages associated with the PLO, relationships between these different stages and the environmental variables that would influence their development, may not be clearly defired with only one year of data. Other related reports regarding PLO results in the VirginiaPfiesteria monitoring program, are those by Marshall et al. (1998a; 1998b; 1999), Seaborn and Marshall (1999), and Seaborn et

Pfiesteria piscicida and Dinoflagellates Similar to Pfiesteria

Abstract: This Article is brought to you for free and open access by the Biological Sciences at ODU Digital Commons. It has been accepted for inclusion in Biological Sciences Faculty Publications by an authorized administrator of ODU Digital Commons. For more information, please contact digitalcommons@odu.edu. Repository Citation Marshall, Harold G., \"Pfiesteria piscicida and Dinoflagellates Similar to Pfiesteria\" (1999). Biological Sciences Faculty Publications. 111. https://digitalcommons.odu.edu/biology_fac_pubs/111

Field Sampling and Necropsy Examination of Fish

Abstract: This paper presents an overview of observational and fish sampling tech­ niques for investigating fish lesions, morbidity and mortality. These sam­ pling techniques and investigations are much like detective work and require attention to detail, common sense, technical proficiency and experience. To solve the mystery of a fish kill, the investigator must use available evidence and clues to piece together a series of events that often have long since passed. The cause of these field events may be chemical, biological or physical; more often, it is some combination of these. An initial categorization approach may be used to reduce the great number of possible causes of a fish kill to something more reasonable. Through proper observations, the most probable cause may be placed in one of four broad categories (although additional secondary relationships should also be recognized). These broad categories include oxygen related, toxics or water quality related, disease or population related and trauma related events, and may be based on defined criteria Caution should be taken on making etiologic generaliz.ations since many types of lesions or mortality events may appear similar. This paper provides support for making consistent observations; taking photographs, tissue and water samples; classifying external lesions and choosing appropriate ne­ cropsy methods. A bibliography is provided to reference information perti­ nent to fish kill investigations and fish disease, anatomy and taxonomy. IN1RODUCTION In order to determine the cause offish lesions, morbidity and mortality it is essential to make detailed field observations and examinations and specimen collections. The field investigator, whose job it is to determine the cause of such environmental events, frequently lacks needed historical information about the event in question. Although the science of fish disease and mortality investigation is not new, the diagnostic methodologies are constantly being refined and updated, particularly since they are applied under many different field circumstances. Fish lesions or mortalities may be 1 University of Maryland School of Medicine, Aquatic Pathobiology Center, Department of Pathology, 10 South Pine Street, Baltimore, MD 21201 2 Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, 8077 Greenmead Drive, College Park, MD 20740 3 U.S. Food and Drug Administration, Center for Veterinary Medicine, Office of Research, 8401 Muirkirk Road, Laurel, MD 20708 4 Louisiana Department of Environmental Quality, Barataria-Terrebonne National Estuary Program, 320 Audobon Drive, Thibodaux, LA 70310 5 Maryland Department of the Environment, Technical and Regulatory Services Administration, 416 Chinquapin Round Road, Annapolis, MD 21401 6 Maryland Department of Natural Resources, Cooperative Oxford Laboratory, 904 South Morris Street, Oxford, MD 21654

Virginia's Pfiesteria Monitoring Program: Water Quality

Abstract: ABSTRACT During the summer and early fall of 1998, 34 estuarine stations in Virginia were sampled for Pfiesteria like organisms (PLOs) and associated water quality conditions. Stations were sampled either bimonthly (20 stations) or monthly (14 stations) from June to October 1998. At each station, a set of live and Lugol' s preserved samples were collected for presumptive counts of PLOs and water quality conditions were determined. Water quality paran1eters measured included standard field parameters, nutrients (total, dissolved, and particulate), chlorophyll a, and conventional water quality indicators. AlthoughPLOs were relatively low in 1998 as compared to 1997, presumptive PLO counts revealed higher PLO levels at stations located in the Northern Neck area (Potomac Embayments, Rappallannock River, and other smaller watersheds). The water quality parameters of pH, dissolved ox·ygen, and temperature were correlated with higher PLO counts, while several nutrients, salinity, and turbidity ,vere negatively correlated with PLOs. In the August 1997, relatively minor fish kills in the Pocomoke River and the Pocomoke Sound on the Virginia and Maryland border were attnbuted to the toxic dinoflagellate, Pfiesteria piscicida. Pfiesteria piscicida was first identified as a fish-killing dinoflagellate in fish tank at North Carolina State University (Noga et al., 1993). Pjiesteria piscicda has since been implicated in large widespread fish kills in North Carolina's estuaries (Burkholder et al., 1992, 1995).

Cartilage Regeneration on a Large Articular Surface Facilitated by Stress Shielding

Abstract: An animal model 3 for the study of articular cartilage regeneration in-vivo facilitated by stress-shielding is introduced. TI1e object of the model is to test the hypothesis that some fonn of cartilaginous tissue will grow upon a large joint surface in vivo with the joint in nonnal motion The model utilizes the known capability of immature cells to differentiate. The source of cells is bleeding subchondral bone. In addition, the model provides a mechanically shielded environment in which cell differentiation and maturation can occur. TI1e study showed that a substantial amount of tissue will grow in the animal model only when the new tissue is relieved of the nonnaljoint stresses. The characteristics of the new tissue were observed after 12 weeks of growth. Gross observation showed that the new tissue grew to completely surround the shielding devices and covered the entire articular surface. The new tissue grew to the height of the shielded area (2 to 3mrn.). Histologic evidence indicated the new growth was largely fibrous in nature but with some areas of newly differentiated chondrocytes. Bio mechanical analyses quantified the tissue as being a soft, penneable neocartilage: biochemical evaluations dem­ onstrated increased hydration \\Vith small amom1ts of proteoglycans. These characteristics are inferior to nonnal cartilage. Never the less, the tissue quality is as good or better than that obtained in other models and it grew to cover a significantly larger articulating surface tl1an all other ex--perimental models. Material obtained in this experiment provides a baseline of data for future experiments designed to manipulate the new tissue using tissue engi­ neering methods and to learn how the new tissue will tolerate exposure to reintroduced nonnal stress. Key tenns: cartilage, in vivo, repair, interdisciplinary

Comparative Taphonomy and Paleoecology of a Glaciomarine Fauna, Carboniferous (Westphalian- Namurian) La Capilla Fm., Argentina

Abstract: The Carboniferous La Capilla Fm. of the Calingasta-Uspallata basin of western Argentina contains a low diversity fauna inhabiting a continental shelf under glacial ice fronts advancing from the east. Distal glaciomarine sediments on these ice-influenced shelves of Gondwana are most commonly interpreted as being deposited under quiet, low-energy conditions. Ta­ phonomic and paleoecologic analysis of a sample of the fauna reveals the following: low species richness, yet comparable equitability to coeval, tropi­ cal faunas; low articulation ratios and high pedicle valve dominance for brachiopods; diverse corrasion modes, about half relatively high categories; one hundred percent fracturing of brachiopod shells, with carinate fracture types dominant, and no evidence of epibiont coverage ofbrach.iopod shells, despite presence of encrusting organisms in the fauna. Collectively, the data indicate long residence time on the seafloor, with strong episodes of rework­ ing contrary to the lo,Y-energy hypothesis. Modem analogues of continental shelves reworked by currents to depths of 250 meters exist in Antarctica. The existence of similarly preserved faunas in the coeval, marginal basins of southern Gondwana needs to be confinned.

Water Quality Relationships to Concentrations of Pfiesteria-like organisms in Virginia Estuaries for 1998.

Abstract: A series of statistical analyses were performed to identify the relationship between abundance of dinoflagellates grouped as Pfiesteria-like organisms and a set of 25 water quality variables from May through October of 1998 at 41 estuarine locations. Although regions were identified in relation to seasonal density of cells present, there were no strong relationships to specific water quality variables. Factors that may have influenced these results included: a) several species were included in the group analyzed and this composite did not respond as a unit to changing environmental conditions; b) cell concentrations were low and there were a large number of zero counts; and; c) there were no marked changes involving increasing abundance during the study that could be related to environmental factors. INTRODUCTION Phytoplankton populations in Virginia estuaries include an assemblage of many diatoms, chlorophytes, cyanobacteria, dinoflagellates, and other less dominant algal components (Marshall, 1994; Marshall and Borchardt, 1998). Included among the dinoflagellates are those species that are recognized as Pfiesteria-lik.e organisms (PLO). They have motile cells (e.g. zoospores) that are similar in size and morphology to the toxin producing dinoflagellate Pfiesteria piscicida (Burkholder and Glasgow, 1997; Steidinger et' al., 1997). This category may include members of the genera Pfiesteria, Gymnodinium, Gyrodinium, and others. The most favorable environmental conditions that have been associated with Pfiesteria piscicida have been nutrient rich waters, salinities around 15 ppt, temperatures >26 °C, and in estuaries with low flushing rates (Burlcholderetal., 1995; Magnien et al., 1999). Other direct and indirect relationships to nitrogen and phosphorus concentrations have been discussed, including the influence high nutrient levels will have on the development of algae preyed upon by Pfiesteria (Burkholder et al., 1992; Fensin and Burldlolder, 1996; Burkholder and Glasgow, 1997). To date Pfiesteria piscicida has been reported in Virginia from the York River (Burldlolder et al., 1995) and from Mosquito Creek, located on the Virginia ocean side of the Delmaiva peninsula (Parke Rublee, personal communication). The 1998 presence of Pfiesteria-lik.e organisms (PLO) in Virginia estl,laries is presented by Marshall et al. (1999). Since PLO organisms have been found in the water column when Pfeisteria piscicida is present, their general relationships to water quality parameters gain additional significance in identifying conditions that may favor the development of P. piscicida. In 1998, an extensive six month swvey regarding the abundance and distribution of PLO in Virginia estuaries was conducted. At the same time, water quality parameters were also determined The objectives of this study were to apply a 366 VIRGINIA JOURNAL OF SCIENCE series of regression analysis · procedures to identify relationships that existed between PLO abundance and specific water quality parameters. ME'IlIODS This study is based on too use of regression analysis statistics to identify relationships of water quality parameters to concent.ratiom of Pfiesteria-like organisms. Water samples for too PLO analysis were collected by personnel from the Virginia Department of Environmental Quality (VDEQ) from June through October 1998 at widely distributed stations in Virginia estuarine riveIS, creeks {fable 1). These stations included 20 which were sampled twice a month as part of the Virginia Department of Health COHORT monitoring program, with another 14 stations sampled monthly as a component of tre VDEQ monitoring plan. During each sampling date, water samples were collected for water quality measurements that included 25 chemical and physical parameters to be analyzed by VDEQ (Table 2). Two sets of water samples were collected at each site for the PLO analysis, one set was preserved with Lugol 's solution, the otoor set did not have a preservative added. In this study, only the preserved sample PLO data are included. It will be noted later that a comparison of too station data for too two sets indicated slightly higher cell concentratiom were in samples preserved with Lugo I's solution. The lower cell concentrations in the non-preserved samples are likely due to the transfonnation of many of the motile zoospores present to either cyst or amoeba stages of tooir life cycle. This change may be easily accomplished during the transport period from the water site to the laboratoiy by any agitation to the water sample. The preseIVed water samples provided a more accurate cell count estimate of the motile zoo spores at the time of collection. From each water sample, an aliquot was placed in a plankton counting cell, and only those recognizable PLO cells were counted using light microscopy at 400x magnification. Concentrations were given as numbers of cells/mL (MaIShall et al., 1999). The VDEQ provided results of the water quality analysis. To facilitate analysis and to generate broader conclusions of the data, the sites were divided into categories based upon location (Figure 1). Table I lists each of too stations by river code, DEQ site number, location, station type, and co-ordinates. The primacy divisions were by river basin and include the James (JW), Piankatank (PKW), Potomac (PW), · Rappahannock (R W), and Yolk Rivers (YW). The Chesapeake Bay was divided into the eastern (CBE) and western (CBW) Bay. The stations listed as Chesapeake Bay east, or Chesapeake Bay west were stations within minor tributaries and bays. There were also two larger bay categories listed as Ingram Bay (IBW) and Mobjack Bay (MBW). There was also one site located along the Atlantic coastline of the Eastern Shore of Virginia (A 1). Data Analysis A ranked correlation matrix was made to determine relationships between cell abundance and water quality variables. The individual variables were chosen based upon their significance, their correlation with other variables, and tre number of observations for the variable. Regressiom were run on the variables with significant correlations against PLO concentrations. If a variable was correlated with another variable, it was considered unlikely to provide infonnation in the regression model and would likely result in multi-collinearity. The presence of missing values decreased the total number of observations used in too regression model. To resolve this condition, an aibitraiy limit of 2500 water quality observations for the variable of interest was made for inclusion of a variable into the model As variables are added to a regression model, too ability of the model to predict the dependent variable improves am WATER QUALITY RELATIONSHIPS OF PLO 367 TABLE l. Water Quality and Cohort Station Locations, with River code and coordin~ River Station Station Code Abreviation DEQ station id Location Type Latitude Longitude AT AT2 7FLLOOOS0 Folly Creek WQ 37.68444 -7S.60S8 CBE CBEl 7NSS00060 Nusawadox Creek COHORT 37.47417 -7S.9S17 CBE CBE2 70CH00160 Occahanock Creek COHORT 37.SSlll -7S.9106 CBE CBE3 70CN00192 Onancock Creek COHORT 37.72833 -7S.8047 CBE CBE4 7POCOOOOO Pocomoke River COHORT 37.96389 -7S.6478 CBE CBE7 7KNS00040 Kings Creek WQ 37.27944 -76.00')7 CBE CBE8 7PUN00212 Pungoteague Creek WQ 37.66472 -7S.8289 CBW CBWl 1ALIS00420 Little Wicomico COHORT 37.897S -76.3011 CBW CBW2 1ALIS00200 Little Wicomico COHORT 37.88861 -76.2686 CBW CBW3 1ALIS00200 Little Wicomico COHORT 37.88861 -76.2686 CBW CBW6 71NDOOOS0 Indian Creek WQ 37.68389 -76.3306 CBW CBW7 71ND00261 Indian Creek WQ 37.70333 -76.3S39 CBW CBW8 7BBY00288 Lynnhaven River WQ 36.897S -76.0378 IBW IBW14 7BLS00073 Balls Cr I Gr Wicomico R Trib WQ 37.84SS6 -76.3822 IBW IBWlS 7COC00161 Cockrell Cr I Gr Wicomico R Trib WQ 37.83722 -76.2794 IBW IBW19 7GWR00889 Great Wicomico River WQ 37.87028 -76.4197 IBW IBW20 7GWR0048S Great Wicomico River WQ 37.84833 -76.3672 1W 1W 1 2WWKOOOOO Warwick River COHORT 37.072S -76.S414 1W 1W2 2WBE00444 W estem Branch Eliz.abeth River WQ 36.82917 -76.39S8 1W 1W3 2JMS032S9 James River WQ 37.20667 -76.6S17 1W 1W4 2PGN00119 Pagan River WQ 36.99639 -76.S842 MBW MBWl 7NOR00638 North River COHORT 37.43944 -76.4431 MBW MBW2 7NOR00269 North River COHORT 37.41S -76.4106 MBW MBW3 7NOR00676 North River COHORT 37.44444 -76.44S8 MBW MBW4 7WAR00282 Ware River COHORT 37.38S83 -76.4492 MBW MBWS 7WAROOS77 Ware River COHORT 37.40333 -76.4897 PKW PKW2 7PNK01S49 Piankatanlc River WQ 37.S4806 -76.S089 PKW PKW3 7PNKOOS36 Piankatanlc River WQ 37.S2972 -76.3728 PW PW l 1ALOW00473 Lower Machodoc Creek COHORT 38.09861 -76.6S39 PW PW2 1ALOW0013S Lower. Machodoc Creek COHORT 38.13944 -76.6492 PW PW3 1ANOM00472 Nomini Creek COHORT 38.10222 -76.7172 PW PW4 1ANOM00162 Nomini Creek COHORT 38.14028 -76.7244 PW PW13 1AMON00191 Monroe Bay WQ 38.24278 -76.9678 RW RWl 3CRR00338 Corrotoman River COHORT 37.69333 -76.4733 RW RW2 3CRR00138 Corrotoman River COHORT 37.66S83 -76.4797 RW RW3 3LANOOOOO Lancaster Creek COHORT 37.79264 -76.64S6 RW RW4 3RPP04302 Rappahannock River COHORT 37.92194 -76.83S3 RW RWlS 3URB00100 Urbanna Creek, Rt 227 WQ 37.62931 -76.S698 RW RW16 3URB001S0 Urbanna Creek WQ 37.62278 -76.S819 RW RW7 3CTR00106 Carter Creek WQ 37.66472 -76.43S6 YW YWl 8SRHOOOOO Sarah Creek COHORT 37.2S361 -76.4828 l AT=Atlantic sites, MBW=MobJack Bay sites, 1W = James River sites, YW=Yorlc River sites, CBE=CHesapeake Bay East sites, IBW=Ingram Bay sites, PKW=Piankatanlc sites, RW=Rappahannock Rivers~. CBW=Chesapealce Bay West Sites, PW=Potomac River sites. 2 Latitude and Longitude are in decimal degrees. 368 VIRGINIA JOURNAL OF SCIENCE TABLE 2. List of environmental parameters analyzed.

Possible Predation Scars on Rectithyris subdepressa (Stoliczka, 1872), Late Cretaceous (Maastrichtian) Kallankurichi Fm., India

Abstract: A single specimen of the terebratulid brachiopod, Rectithyris subdepressa (Stoliczka, 1872) from the Late Cretaceous (Maastrichtian) Kallankurichi Fonnation of southern India was found with durophagous predation traces. This occurrence is significant as it is possibly the first documentation of elasmobranch shark predation on brachiopods from the Mesozoic. INTRODUCTION Predator prey relationships are difficult to document in the fossil record (Boucot, 1990), though numerous cases of predation scars on fossil brachiopod shells are known (Alexander, 1981;1986). Among numerous potential predators on invertebrates, shell piercing and crushing sharks are most notable for leaving well defined puncture holes (Hansen and Mapes, 1990). In a preliminary review of predation upon brachiopods throughout the Phanerozoic, Alexander (1985) found high frequencies of predation (up to 75% of shells per species) for Paleozoic brachiopods, but could document no single instance of predation on Mesozoic brachiopods. He hypothesized that the post-Paleo­ zoic decline in predation might reflect several phenomena, including a shift in duro­ phagy from bracb.iopods to molluscs, as the bivalves increasingly replaced the brachiopods in most shelf environments (Gould and Calloway, 1980; Walsh, 1996). Articulate brachiopods today have repellant, noxious-tasting flesh that insures that they are commonly not chosen as prey in modem marine environments (Thayer, 1981; 1985). Onset of this adaptation may be linked to the brachiopod/bivave replacement, or may have a Paleozoic history (cf. Thayer and Allmon, 1991). RESULTS The Late Cretaceous (Maastrichtian) Kallankurichi Formation crops out in the Tancem Mines area of the state of Tamil Nadu, India (Figure 1). A diverse fauna of bivalve oysters, echinoids, brachiopods, gastropods and ammonites has been described (Stoliczka, 1872; Radulovic and Ramamoorthy, 1992) from the arenaceous limestones (Sundram and Rao, 1986). These sediments may be interpreted as representing shal­ low-water, marine deposition in high-energy environments. Trace fossils attributable to predation are termed Praedichnia, and in the case of shark predation upon brachiopods, are usually expressed as holes in the skeleton not located at the cardinal margins (Ruggiero, 1990). A single specimen of the large (up to 74 111m length) terebratulid brachiopod, RectithJris subdepressa (Stoliczka, 1872) was collected from the Mines that has three holes on the pedicle valve (Figure 2) that are possibly Praedichnia traces. 46 VIRGINIA JOURNAL OF SCIENCE Tamil Nadu • Tancern Mines

Synthesis and Crystal Structure of Tetrachloro (1,10-phenanthroline) platinum (IV)

Abstract: We report the crystal structure determination of tetrachloro( l ,10-phenan­ throline)platinum(IV). X-ray data indicate there is little steric repulsion between the cx.-hydrogens on the phenanthroline ligand and the chloride ligands in the equatorial plane. BACKGROUND Coordination complexes of the type M(N-N)x, [M = rhodium (Barton, J. K, 1989), (Fleisher, Watenn� Turro & Barton, 1986), (Pyle, Long & Barton, 1989), nickel (Mack & Derv� 1990), copper (Barton 1986), (Pope, & Sigman 1984) ruthenium or rhodiwn (Barton, J. K, 1989), (Barton 1986), N-N = 1,10-phenanthroline, its substi­ tuted derivatives, or bipyridine and its substituted derivatives], have been used as molecular probes of secondary DNA structure and as indicators of DNA sequence by acting as artificial nucleases. The metal-phenanthroline interaction holds the phenan­ throline ligand in a rigid confonnation thus allowing for site specific intercalation of the ligand into the DNA. The site specificity and the strength of the interactions are functions of both the substitution on the ligands and the characteristics of the coordi­ nated metal center. An X-ray structural detennination of the complex M(N-N)x allovvs one to obtain information regarding the three dimensional structure of the site in the DNA tc which it binds. Although there have been many studies of DNA interactions with these types of coordination complexes, no studies to date have involved similar octal1edral Pt(IV) probes. It is our intention to report the synthesis and DNA-studie� of [Pt(phen)/+ [phen = 1, 10-phenanthroline]. Thus far, the synthesis of [Pt(phen)3] 4 · has not been reported. The syntheses of [PtCliDIMP)] and [PtC12(DIMP)2][Cl)i (DIMP = 4,7-Dimethyll , 10-phenanthroline) and [Pt( en)(phen)2][Fe(CN)6] have been reported and characterized solely on microanalysis data (Power, 1975), (Kolobova, 1983). DNA-interactions with these complexes have not yet been reported. Author to whom correspondence regarding synthetic studies should be addressed. Present address: Department of Chemistry, Virginia ;..Jilitary Institute, Lexington VA 24450. 2 Present address: Department of Chemistry, Sweet Briar College, Sweet Briar VA,, 24595. 3 Present address: Department of Biology, Washington and Lee University, Lexington VA, 24450. 4 Present address: Department of Chemistry, Sweet Briar College, Sweet Briar VA ,, 24595. 5 Author to whom correspondence regarding crystallographic data should be addressed. Present address: Department of Chemistry, Purdue University, West Lafayette IN, 47907. 52 VIRGINIA JOURNAL OF SCIENCE

Three-Dimensional Reconstructions of Tadpole Chondrocrania from Histological Sections

Abstract: Reconstructing three dimensional structures (3DR) from histological sections has always been difficult but is becoming more accessible with the assistance of digital imaging. We sought to assemble a low cost system using readily available hardware and software to generate 3DR for a study of tadpole chondrocrania. We found that a combination of RGB can1era, stereomicro­ scope, and Apple Macintosh PowerPC computers running NIH Image, Object Image, Rotater, and SURFdriver software provided acceptable reconstruc­ tions. These are limited in quality primarily by the distortions arising from histological protocols rather than hardware or software.