Showing papers in "Virginia journal of science in 2001"

A New Error Analysis for Brun's Constant

Abstract: Enumeration of the twin primes, and the sum of. their reciprocals, is extended to 3 x 1015 , yielding the count 1r2 (3 x 1015 ) = 3310517800844. A more accurate estimate is obtained for Brun 's constant, B2 = 1.90216 05823 ± 0.00000 00008. Error analysis is presented to support the contention that this estimate produces a 95 % confidence interval for B2 • In addition, published values of the count 1r(x) of primes, obtained previously by indirect means, are verified by direct count to x = 3 x 1015 . MATHEMATICS SUBJECT CLASSIFICATION 2000 (MSC2000) Primary: Secondary:

Ice Storm Damage to Virginia Coastal Plain Forests During the Christmas 1998 Ice Storm

Abstract: On December 23-25, 1998, a major ice storm struck southeastern Virginia The storm-deposited glaze ice felled trees and limbs, causing a power outage and highway blockage. Between Februmy and April, 1999, we recorded occurrence, severity, and type of damage to trees over 2.5 cm dbh in nine mostly gently sloping plots in Matoaka Woods at the College of William and Macy. Frequency and severity of damage varied with species and with size of trees. Canopy damage occurred in 75% of large Fagus grandifolia trees, but in only 6% of small Sassafras a/bidum stems. As a group, small (2.5 to 15 cm dbh) trees were less likely to be damaged than large ( 15 cm dbh) trees, but about as likely to be severely damaged. Damage type also varied among the species and size. Despite severe damage to public utilities, damage within the forest was not great. Since few trees lost their entire crown, canopy gap sizes were small, and it not clear that much change in forest composition will result from this storm. However, increased density of ground litter will contribute to greater mineral release, and this plus small gaps may promote growth of already present seedlings and saplings. INTRODUCTION On December 23, 24, and 25, 1998, a major ice storm affected southeastern Virginia. Precipitation in the form of sleet and freezing rain accumulated to 1-3 cm of ice across the region, with Williamsburg reporting 3 cm of precipitation for the three-day period. In the City of Williamsburg and surrounding counties, 400,000 customers lost power for three to ten days following the storm. Many roads, including portions of Interstate 64 near Lightfoot, VA, were rendered impassable by fallen branches and trees (NCDC 1998a,b). The storm's impact on the community was certainly severe, and much of the infrastructure damage was caused by ice-felled branches and trees along roadsides and on forest margins. Based on the degree of damage readily observable from the roads, we felt that this storm presented an ideal opportunity to detennine the effects of ice accumulation on local forests. The great damage to roadside and forest margin trees, however, was due to their peculiar location. Without adjacent vegetation of comparable height to support their accumulated weight in ice, and with either asymmetric or fuller crowns due to lack of competition for light, individuals in the open would likely be more susceptible to damage than those in the forest. Nevertheless, preliminaiy investigation-of our potential study sites indicated that, although the damage within the forest was not as heavy as on its margins, it did appear significantenough t9 provide data for a meaningful study on the dominant tree species of the area. We surmised that the College Woods (also called Matoaka Woods), a forested area owned by the College of William and Macy, was an ideal place for a small-scale 4 VIRGINIA JOURNAL OF SCIENCE investigation into the susceptibility to ice of several major tree species on the Coastal Plain of Virginia. Matoaka Woods is made up of a variety of small, homogenous stands dominated by canopy species such as tulip poplar (Liriodendron tulipifera), oaks (Quercus spp.), beech (Fagus grandifolia), and loblolly pine (Pinus taeda). The mosaic pattern of the woods (fanned and forested patches were abandoned or last timbered at various times for various reasom) has allowed for a diversity of species, and also has ensured equal representation of a broad spectrum of size classes. In this study, our primary goal was to swvey the amount and type of damage to each of the more abundant tree species in Matoaka Woods. Of secondmy interest was the comparison of damage among different size individuals of the same species. METIIODS Our field swvey was conducted in the Matoaka Woods of the College of William and Mary between February 3 and April 7, 1999. No further forest-ravaging natural phenomena occurred between the end of the Christmas storm and the completion of our swvey. Sampling sites were chosen based on the constituent species and apparent age of the dominant individuals: younger and older stands dominated by oak species~ tulip poplar, loblolly pine, and beech were sought out with the hopes of comparing damage between different aged canopy trees of the same species or genus, as well as among the different species. The sampling sites were widely spread throughout the woods. We chose to follow Seischab et al. (1993) in our methodology. We marked a 20x40-meter plot at each sampling site. Each of these was broken into four 10x20 meter subplots for ease in sampling. In each subplot, trees larger than 2.5 cm dbh were identified by species and were placed in one of two size categories: between 2.5 and 15 cm dbh and over 15 cm dbh. In general, trees in the smaller size class were subcanopy, and those in the larger size class were in the canopy. Though we took measures to avoid bias toward areas likely to be heavily damaged (such as steep slopes above ravines; W arrillow and Mou 1999), beech-dominated stands could not be found in the more level portions of the woods. Thus, in order to sample beech, it was necessary to place two plots on slopes. Effects on the results due to this difference in topography will be discussed later. Each tree swveyed was placed in a damage class between O and 7 based on percent canopy loss due to ice damage. A rating of O corresponded to no perceptible damage, 1 to~ 5 %canopy loss, 2 to 6-10%canopy loss, 3 to 11-25%canopy loss, 4 to 26-50% canopy loss, 5 to 51-75% canopy loss, and 6 to 76-99% canopy loss. A rating of 7 was given where damage was so severe that mortality was likely. Though we quantified canopy damage as an estimate of percent of canopy lost, the accuracy of our estimates was necessarily subject to error, for we were not able to observe the leafed out canopies , of deciduous trees, nor had we previously documented canopy sizes for any of the trees surveyed. However, eveiy effort was made to be consistent. We recorded the nature of the damage to each tree, noting whether each damaged tree was uprooted (symbolized by oin the tables), had its main stem broken (symbolized by I\\), had its main stem bent or bowed (C), had one or more branches completely broken from the tree ( o ), had one or more branches broken but still attached to the tree (/\\). We also noted whether the damage, of whatever type, was direct (as a result of ice accumulation on the tree in question) or secondaiy (a result of ice-laden branches, ICE STORM DAMAGE TO VIRGINIA FORESTS 5 TABLE 1. Field data for individuals ~ 1' cm dbh. See text for desaiption of damage classes and types. Sample Damage class Damage type S~ies size 0 1 2 3 4 5 6 7 o-1\\ ~ \" 0 s Pinrutaeda 53 29 2 7 3 3 1 2 6 7 2 12 Liriodendron tulipifera 47 27 5 6 2 3 2 2 15 Quercus alba 35 21 6 1 3 3 1 5 4 Fagua grandifolia 28 7 5 4 4 3 4 4 16 1 Oxydendron arboreum 23 13 4 4 1 2 5 2 Liquidambar styraciflua 15 12 1 2 Quercus velutina 11 6 2 2 1 2 1 Acerrubrum 9 4 2 2 2 4 Carya glabra 8 6 2 2 Quercus falcata 6 3 1 2 Nyssa sylvatica 5 3 2 1 Quercus rubra 4 4 Jlexopaca 3 1 2 Quercus coccinea 3 2 1 Comus jlorida 2 2 Carya tomentosa 1 1 Fraxinius americana 1 1 Prunus serotina 1 canopies, or entire trees falling on individuals below). Recently fallen live branches~ 2.5 cm at the broken base (butt end) found in the plots were tallied by species and size; any above 10 cm diameter at the base were further roted. We did not attempt to quantify the deadwood since it was impossible to distinguish dead material felled by this stonn from that previously on the ground By performing our investigation in the winter and early spring immediately following the ice storm, we were able to easily determine the most recent open wounds and fallen branches, for the infection and decay dependent on warm temperatures had not begun. We also avoided the possibility of additional damage from other natural disasters (such as windstonns, including the hurricane that struck the study area the following summer). Because no new growth had begun on bent or wounded stems, we could distinguish fresh bending from older bending or breaking, since trees previously damaged had redirected their foliage or sprouted new stems during the last growth season The lack of intervening foliage in the understory made it easier to examine damage to canopy trees, but, as mentioned previously, percent canopy loss was harder to estimate accurately without foliage. RESULTS We found no significant differences in damage between older stands and younger stands with the same dominant species. Because or' this finding, descriptions of individual plots have not been included, and all data from each species have been merged to reflect interspecific differences and differences between the canopy and understory classes. The amount and type of damage incurred by the 27 species we encountered during our survey is shown in Tables 1 (individuals ~ 15 cm dbh) and 2 (individuals< 15 cm dbh). 6 VIRGINIA JOURNAL OF SCIENCE TABLE 2. Field data for individuals < 1 Scrn dbh. See text for description of damage classes and types. Sample Damage class Damage type S~ies size 0 2 3 4 5 6 7 oI\\ ~ I\\ 0 s Liriodendron tulipi.fera 146 109 12 5 1 1 4 4 9 12 9 3 5 8 Comus florida 132 106 8 2 6 5 2 3 8 1 Cl!l2 Acerrubrum 75 46 6 5 6 2 2 5 3 4 6 10 4 5 Oxydendron arborewn 54 27 4 4 4 2 6 7 3 2 11 4 4 12 Ilexopaca 49 31 3 4 5 5 7 3 6 8 Liquidambar styraciflua 44 36 3 1 2 4 1 1 3 Fagus grandifolia 40 36 3 2 1 Nyssa sylvatica 37 31 3 2 2 1 Sassafras albidum 17 16 1 Carya glabra 14 9 2 2 2 Quercus alba 6 5 1 Pinus taeda 5 1 2 2 2 2 Carya tomentosa 3 3 Castanea dentata 2 2 Cercis canadensis 5 3 1 Quercus velutina 5 4 1 Juniperus virginiana 4 2 2 Vitis rotundif olia 2 2 Diospyros virginiana 1 1

Environmental Effects on Yield and Agronomic Traits of Common Bean (Phaseolus vulgaris L.)

Abstract: Common bean (Phaseolus vulgar is L.) demand is increasing with an alarming rate around the world, especially in Latin America, Africa, and Asia. Therefore, increased bean yield per hectare i.s the best way to meet the world demand rather than expansion of area under cultivation. The objectives of this experiment were to determine the genotypic variations for green bean and dry seed yield and magnitude of genotype x environment interaction effects on yield and yield components of common bean. Thirteen genotypes were planted during the 1992, 1994, and 1995 growing seasons. Genotypes were evaluated for green pod and seed yield and yield components at R7 and R9 growth stages. Years differed significantly for all recorded parameters at both R 7 and R9 stages. Genotypes and genotype x year interaction were also differed significantly for most measured parameters at both stages. The genotype Eagle showed the highest green pod yield, while Branco and Blue Ridge ranked second and third, respectively when averaged over the three years. Number of pods plant-, hundred pod weight and pod length were positively and significantly correlated with green pod yield. Number of pods planf showed the highest correlation (r = 0.61 **) with green pod yield. All the recorded parameters were positively significantly correlated with dry seed yield. Plant height was negative!( correlated with seed size, number of seeds planf 1 and seed weight plant . Number of pods planf 1 was positively correlated (r = 0.51 **) and seed size exhibited highest correlation value (r = 0 .48**) with seed yield. Seed size and number of pods plant -I can be effectively used for indirect selection of green pod yield and dry seed yield of common bean.

An Examination of the First Sediment Cores From Mountain Lake, Giles County, Virginia, for Diatoms and Pollen

Abstract: Mountain Lake, Virginia is a small, unique, oligotrophic, subalpine ecosystem in the southern Appalachians. Its geology, origin, climate, and history have influenced its morphometry, and therefore its sedimentology and algal flora. Radiocarbon dates establish specific Mountain Lake sediment ages at 1800, 4100, and 6100 years BP. Sediment core analysis suggests at least 6 prolonged periods when Mountain Lake probably was nearly dry or very small in size. These individual low-water periods (at approximately 100, 400, 900, 1200, 1800 and 4100 years BP) are evidenced by changes in diatom and pollen content, sedimentary erosion features, and the presence of wood fragments, plant fibers, and abundant Sphagnum and fem spores. The ratio of planktonic-to-benthic diatom taxa was used to estimate approximate past water depths from sediment. One or more of these low-water intervals may correspond to a drier climate coincident with solar activity minima. Resolution of prolonged low water intervals probably has been enhanced by the continuous loss of water through the crevice or fault at the lake bottom. The sediment core record suggests also that some eutrophication has occurred during the 20th century, in parallel with anthropogenic impacts (i.e. increased sedimentation, induced eutrophication, and diatom diversity changes). These findings provide the first published diatom, pollen and sedimentology-based paleolimnology for this lake.

Comparison of Spawning and Non-Spawning Substrates in Nests of Species of Exoglossum and Nocomis (Actinopterygii: Cyprinidae)

Abstract: Percent composition of pebble size classes from spawning and non-spawning substrates of nests were used to test the hypothesis that distribution of pebble sizes is random in nests of Exoglossum laurae, Exoglossum maxi/lingua, Nocomis leptocephalus, Nocomis micropogon, Nocomis platyrhynchus, and Nocomis raneyi in Virginia. In nests of the two species of Exoglossum, spawning areas (i.e., upstream bases of nests) contain significantly greater amounts of the 6.0 mm size class, and significantly smaller amounts of the 2.5 nun size class of stones than do non-spawning areas. Spawning areas (i.e., pits) in nests of N. leptocephalus contain significantly more 6.0 mm pebbles than non-spawning areas, whereas spawning areas (i.e., troughs) in nests of N. micropogon, N. platyrhynchus, and N. raneyi are composed of significantly more 6.0 and 11.3 mm pebbles than non-spawning areas. In all Nocomis species, there was significant selection against the largest (23 .0 mm) size class of pebbles in spawning areas. Male Exoglossum and Nocomis expend significant amounts of time reorganizing substrate material in spawning areas of their nests before and during spawning. Reorganizing pebbles results in relatively unifonn sizes of substrate material at spawning areas in nests of each species. We propose tl1at selection of 6.0 and 11.3 mm size classes for spawning areas is related to spawning behaviors and enhancement of survival of eggs and larvae in nests. Sizes larger than 11.3 mm intetfere with spawning behaviors, and sizes smaller than 6.0 mm form a compacted substrate, which can crush eggs and larvae during trough or pit reshaping, and impede water flow, and consequently, aeration of eggs and larvae. INTRODUCTION Reighard (1943), Lobb and Orth (1988), Maurakis et al. (1991a,b~ 1992a; 1998), and Vives (1990) present qualitative and quantitative infonnation of overall composition of pebble nests constructed by males in species of Exoglossum (E. laurae and E. maxi/lingua), and Nocomis (N. biguttatus, N. /eptocephalus, N. micropogon, N. platyrhynchus, and N. raneyi). However, no attention is given to composition of substrates at specific sites in nests where spawning occurs. In Exoglossum and Nocomis, a completed nest is typically a dome-shaped mound of pebbles (Van Duzer, 1939; Lachner, 1952; Maurakis, 1991b), and tl1e spawning act is restricted to a specific site that accounts for only a small portion of the total nest substrate. In Exoglossum, 26 SUBSTRATES OF Exoglossum AND Nocomis the spawning act occurs at the upstream base of the nest (Van Duzer, 1939; Maurakis, 1991b). In Nocomis, spawning occurs either in a pit (N. leptocephalus, Maurakis et al. , 1991a, Sabaj et al., 2000) or in the upstream half of a trough (N. micropogon, N. platyrhynchus, N. raneyi; Maurakis et al., 199 la, Maurakis, 1998; Sabaj et al., 2000) located on the upstream slope of the nest. Direct obseIVations and reviews of video recordings of nest construction and breeding behaviors of males in N. platyrhynchus and N. raneyi in Virginia by Maurakis (1998) reveal that considerable time is spent by males in reorganizing substrate material where spawning occurs, and that apparent sizes of substrate material are smaller than those in non-spawning areas of nests. Objectives of our study are to analyze percent composition of size classes of pebbles from spawning and non-spawning substrates to test whether the distribution of pebble sizes is random in nests of E. laurae, E. maxi/lingua, N. /eptocephalus, N. micropogon, N. platyrhynchus, and N raneyi in Virginia. MATERIALS EXAMINED The state, drainage, collection number (EGM=Eugene G. Maurakis), locality, collection date, and number of nests in parenthesis for Exoglossum and Nocomis species are: Exoglossum laurae. Virginia: New, EGM-V A-439 B, Craig Co., Clemmons fann, Sinking Cr., St. Rt. 42, 28 May 1999, (9). Exoglossum maxi/lingua. Virginia: James, EGM-VA-438, Craig Co., Johns Cr., Co. Rt. 658 at Maggie, 13 May 1999, (6); EGM-VA-440, Craig Co., Jolms Cr., Co. Rt. 632, 0.3 km upstream of Maggie, 28 May 1999, (6); EGM-VA-443 , Rockbridge Co., South R. , Co. Rt. 608 Bridge, 3 km S of Vesuvius, 29 May 1999, (2). Nocomis leptocephalus. Virginia: Rappahannock, EGM-V A-427, Faquier Co., Thumb Run, Co. Rt. 688, 29 May 1998, (2); James, EGM-VA-428, Albemarle Co., Stockton Cr., Co. Rt. 691 , 50 m. from US 250, 3.2 km W ofYanay Mills, 3 June 1998, (5); EGM-VA-429, Albemarle Co., confluence of Mechum R. and Lickinghole Cr., US Rts. 240 and 250 Jct. , 3 June 1998, (2); EGM-V A-438, Craig Co., Jolms Cr., Co. Rt. 658 at Maggie, 13 May 1999, (l ); EGM-VA-445, Nelsen Co., RockfishR., Co. Rt. 612, 1 mi S. of St. Rt. 151, 29 May, 1999, (3); New, EGM-VA-437, Craig Co., Clemmons farm, Sinking Cr., St. Rt. 42, 13 May 1999, (6); EGM-VA-439 B, Craig Co., Clemmons farm, Sinking Cr., St. Rt. 42, 28 May 1999, (2). Nocomis micropogon. Virginia: Potomac, EGM-VA-426, Loudon Co., Catoctin Cr., Co. Rt. 664 at Taylorsville, 27 May 1998, (6); James, EGM-VA-444, Albemarle Co., confluence of Mechum R. and Lickinghole Cr. at US Rts. 240 and 250 Jct., 29 May 1999, (2); EGM-V A-445, Nelsen Co., Rockfish R. , Co. Rt. 612, 1.6 km S of St. Rt. 151 , 29 May 1999, (1 ). Nocomis platyrhynchus. Virginia: New, EGM-VA-416, Montgomery-Floyd Co. Line, Little R. , St. Rt. 8 Brigde and 1 km upstream on dirt road, 16 May 1998, (2); EGM-VA-417, Montgomery Co., Little R. at Co. Rt. 693 and 613 Jct., E of Snowville about 8 km W of Riner, 16 May 1998, (2); EGM-V A-439 A, Montgomery Co., Little R. at Jct. Co. Rt. 693 and 613, about 8 km W of Riner, 13 May 1999 ( 4 ); EGM-V A-441 , Montgomery-Floyd Co. Line, Little R. , St. Rt. 8, under bridge and along Little Camp Rd, 28 May 1999, (4). VIRGINIA JOURNAL OF SCIENCE 27 Nocomis raneyi. Virginia: James, EGM-VA-424, Rockbridge Co., Mawy R. at US Rt. 60 at Ben Salem wayside, 23 May 1998, (4); EGM-VA-442, Rockbridge Co., Maury R. at US Rt. 60 at Ben Salem wayside, 28 May 1999, (7). MATERIALS AND METHODS Pebble samples of spawning and non-spawning substrates in nests of each species were collected in a I-liter plastic beaker. Spawning and non-spawning substrate samples were air-dried and sifted through five custom-built wire sieves. Mesh sizes of sieves (23.0, 11.3, 6.0, 2.5, and0.8 mm) were determined by commercially available prefabricated screen sizes and provide a more detailed account of nest composition than standard sieve samplers described by Hynes (1970). Material (<0.8 mm) that sifted through the smallest size mesh was collected in a pan Weights of materials in each sieve size and pan were used to calculate the percentage of material per mesh and pan size. Percentages of size classes (based on weights) were used in electivity indices (Ivlev, 1961) to calculate the relative proportion of each pebble size class in spawning areas and non-spawning areas of nests. The equation E = (n -p) I (n + p) (where E = pebble size selection, n = the percentage of a particular pebble size in the spawning area of the nest, and p = the percentage of a particular pebble size in the non-spawning area of the nest) was used to determine if selection of pebble size for the spawning area was nonrandom. Electivity index values range from 1 to 1. Values closer to one indicate a greater selection of a particular pebble size. Percentages and electivity values were transfonned to arcs in equivalents. Differences in average percentages of each size class of stones between spawning and non-spawning areas in nests of each species were tested with a t-test (SAS, 1996). Differences in average percentages and those of electivity values among pebble size classes of spawning and non-spawning areas in nests of each species were detennined with a General Linear Model and Duncan's Multiple Range Test (SAS, 1996).

Mammals of Fort A. P. Hill, Caroline County, Virginia and Vicinity

Abstract: Fort A.P. Hill (APH) is a 30,329 ha military training installation (U.S. Army) located in the upper Coastal Plain of Caroline County, Virginia. It was formed in 1941 and named in honor of Civil War Confederate Lt. General Ambrose Powell Hill. The current landscape includes a mosaic of habitats that range from old fields to hardwood forests. Forty species of mammals are known to exist on or near the installation. These include one marsupial, five insectivores, 9 chiropterans, one lagomorph, 12 rodents, 10 carnivores, and one cervid. We have studied many of the species on APH since 1997. In this paper w,e describe the physical environment of the area and 7 important habitats used by mammals. We also summarize the ecology and natural history of each species and provide statistical summaries of original measurements from mammals caught on the installation. The results of several recent studies on APH allow us to describe habitat affiliations and relative abundance of most of the mammals native to the mid-Atlantic region. Old fields and clearcuts support a total of 20 species, including several found predominately in this habitat. Pine stands and pine plantations support the fewest number of mammal species (17) of any habitat on the installation. Mixed pine and hardwood forests, hardwood forests, and riparian forests support the largest number of species (29-36). With the possible exception of pine plantations, the habitat mosaic found on APH provides abundant resources for mammal communities. We also include an evaluation of age and health attributes of the deer population and describe the hunting program on the base. Number of deer harvested annually 1985-2000 varied from 460 to 1765. Management activities since 1996 when the deer population exceeded carrying capacity have improved herd health. Because much of Caroline County and eastern Virginia is in extensive agriculture and the remaining hardwood forests are Current address: Deparbnent of Biological Sciences, Old Dominion University, Norfolk, VA 23529. Current e-mail address: abell008@odu.edu. 164 VIRGINIA JOURNAL OF SCIENCE being clearcut, APH is becoming a valuable habitat island for the mammalian fauna of the upper Coastal Plain of Virginia and the mid-Atlantic region.

Isolation and Fusion of Cotton Protoplasts

Abstract: Protoplasts were isolated from five species of Gossypium. · Protoplast yield and viability were affected by incubation conditions, osmolarity, purification procedures, and cell source. Using an optimized procedure, highly viable protoplasts were isolated from cell suspensions, callus cultures, and leaf tissue of G. hirsutum, G. arboreum, G. k/otzschianum, G. harlmessii, and G. herbaceum. Protoplasts of G. harknessii were enucleated and successfully fused with protoplasts of G. hirsutum. IN1RODUCTION Modification of plants through tissue culture methods, such as somatic cell fusion has the potential of providing immediate benefits to agriculture (Evans and Sharp, 1986), because genetic characteristics can be transferred by somatic cell fusion without the necessity of isolation and identification of the genes responsible for the trait. Many aspects of plant improvement through somatic cell hybridization have been reviewed previously (Evans and Flick, 1983; Schieder, 1982). An important plant breeding tool is male sterility. Through sexual crosses, Meyer (1975) demonstrated that Gossypium harlmessii Brandegee cytoplasm in the mclear background of G. hirsutum L. resulted in plants with CMS. Production of these cotton plants required extensive backcrossing, and the seed set was limited Although the genetic basis for cytoplasmic male sterility (CMS) is not understood (Boeshore et al., 1985; Jigeng and Yi-mong, 1983; Levings and Pring, 1979), this trait has been transferred to a male fertile plant through protoplastfusion (Zelceret al., 1978) and subsequent hybrid regeneration. 1k fusion of G. harlmessii cytoplasts with G. hirsutum protoplasts should also produce G. hirsutum plants exhibiting CMS. As a first step in this process, a technique to rapidly obtain good yields of highly-viable protoplasts suitable for protoplast fusion was developed. Protoplast yields and viability exceeded other published accounts (Bhojwani et al., 1977; El-Shihy and Evans, 1983; Finer and Smith, 1982; Firoozabady and DeBoer, 1986; Khasanov and Butenko, 1979). Additionally, cytoplasts were prepared and fused with these protoplasts. MATERIALS AND ME1HODS Plant Material Cotton plants G. hirsutum L. var. Coker 310, Stoneville 213, and Paymaster 145, were grown in potting soil in an environmentally-controlled grvwth chamber which 1 Address all correspondence to:Dr. Michael H. Renfroe, Department of Biology MSC 7801, James Madison University, Harrisonburg. VA 22807 58 VIRGINIA JOURNAL OF SCIENCE was progranuned for 12 h of light with a temperature of 30 C and 12 h of darkness at 20 C. Plants were watered as necessaiy and fertilized with Osmocote slow-release fertilizer. Young, rapidly-expanding leaves were selected as source leaves for protoplast isolation Callus cultures of G. hirsutum L., G. harknessii Brandg., G. arboreum L., G. herbaceum L., and G. klotzschianum Anderss. were maintained on a medium consisting of: rnacrormtrients and micronutrients of Murashige and Skoog ( 1962) (MS salts); 2 mg/L NAA; 1 mg!L 2-iP; 30 g/L glucose; a vitamin mixture consisting of 1 mg!L thiarnine-HCl; 0.5 mg/L pyridoxine-HCl; 0.5 mg/L nicotinic acid; and 100 mg!L myo-inositol (Smith et al., 1977). Media were sterilized by autoclaving for 15 min at 121° C. Prior to autoclaving and addition ofagar, the pH was adjusted to 5.7-5.8 and medium was solidified with 0.8% Difeo Bacto agar. Cultures were subcultured at monthly inteivals. Suspension cultures of G. hirsutum and G. harknessii were maintained in a medium described above with the following modifications: NAA reduced to 1 mg/L; BAP, 0.1 mg/L substituted for 2-iP; agar was omitted. Suspension cultures were maintained at a 16:8 h photoperiod (601pm). Cultures were supplied with fresh medium weekly and serially subcultured eve:ry 3 weeks. · Protoplast isolation The general isolation procedure was developed using cotton cell suspension cultures of G. hirsutum. Basal isolation medium consisted of rnacronutrients of MS salt (Murashige and Skoog, 1962), 5 rnM MES (2-(N-morpholino)ethanesulfonic acid), 0. 7 M rnannitol, 5% (w/v) Cellulysin cellulose and 1 % (w/v) Macerase pectinase at a pH of 5.7. Effect of osmotic potential on protoplast isolation and viability was detennined by reduction of mannitol to 0.5 Mand 0.3 M. Effect of rnacronutrient composition on protoplast isolation and viability was detennined by testing full and half strength MS rnacronutrients and full strength macronutrients from Garnborg's ( 197 5) medium. Effects of enzyme concentration and length of incubation were tested by reduction of the enzyme concentration to 1/2 and by increasing the incubation period from 5 h to 24 h. Isolated pro top lasts were purified either by filtration through a nylon mesh with 100 nun pores, by centrifugation at 125 RCF for 6 min layered over a 20% (w/v) sucrose solution, or by a combination of filtration and centrifugation treatments. Prior to incubation in the isolation medium, cells from the suspension culture were plasmolyzed ina solution identical to the respective isolation medium without enzymes for approximately 30 min. Plasrnolyzed tissue was incubated in protoplast isolation medium for 5 hat 28° C in a water bath reciprocal shaker at 40 CPM. Protoplasts used for enucleation experiments were also isolated using this procedure. Cell counts were made using a haemocytometer. Cell viability was measured using the Evans' blue dye exclusion techniqu~ (Onyia et al., 1984). Protoplasts of the other species were obtained from callus cultures and young plant leaves using the procedure developed for cell suspension cultures. Cytoplast Preparation and Protoplast Fusion G. harknessii protoplasts were enucleated by centrifugation at 31,000 RCF for 1 h on an iso-osmotic step gradient (Lon and Potrykus, 1980). Enucleation of protoplasts to form cytoplasts was verified using epi-fluorescence microscopy (Zeiss) following ISOLATION OF COTION PROTOPLASTS 59 TABLE 1. Yield and viability of protopl$ts isolated in media of various osmotic strength after S hours incubation in protoplast isolation mediwn. Mannitol Water Potential of Yield Viability (M) Medium (MPa) (No/ml PCv8) (%) 0.3 -0.97 3.2X 10 s 96.7 0.5 -1.48 2.7 X 10 s 95.7 0.7 -2.07 1.9 X 10 s 91.9 • PCV =packed cell volwne incubation of protoplasts and cytoplasts for at least 1 h in DAPI (4,6-diamidino-2phenylindole) at 1 mg,'mL. Protoplasts of G. hirsutum were mixed with an excess of G. harknessii cytoplasts (approximately 2: 1 cytoplasts:protoplasts). A red pigmented cell line of G. hirsutum was used in some experiments to provide a visual marlcer for interspecific fusion. Protoplasts and cytoplasts were fused using the procedure by Evans (1983), modified by substitution of 0.5 M glucose for sorbitol in the enzyme wash solution. Fusion was promoted by a 50%, (w/v) PEG (mw 6000) solution (Evans, 1983). The PEG fusing solution was eluted with either a glycine buffer (50 mM glycine, 50 mM CaC12·2~0, 0.3 M glucose, pH 10.5) followed by a wash with culture media, or by a Tris buffer (5 mM Tris, 50 mM CaC~·2H20, 0.3 M glucose, pH 7 .0) followed by a wash with culture media, or eluted by washing with culture media alone (pH 5. 7-5.8). Pro top lasts and fusion products were cultured in various media based on Murashige and Skoog's (1962), Gamborg's (1975) or Kao and Michayluk's (1975) fonnulations. Liquid culture, agar or agarose-solidified media and nurse cultures were all used in an attempt to promote growth of protoplasts and fusion products. RESULTS Protoplasts of high viability were obtained from all.the Gossypium species examined and from leaf, as well as callus and suspension cultures, using this procedure. The highest yield and viability were obtained using the isolation medium with 0.3 M mannitol (Table 1). ~ the medium osmotic strength was increased, protoplast yield and viability decreased. Isolation medium containing 0.3 M mannitol was therefore selected for subsequent trials. Isolation medium macronutrient content had no effect on yield (Table 2). Similar results were obtained when macro-salts of MS were at fullor half-strength. Yield and viability were only slightly decreased by substitution of Gamborg's macronutrients. MS macronutrients at full-strength were chosen for routine use. Reduction of enzyme concentration by half had no effect on viability over a 5 h period but did decrease protoplast yield (Table 3). An increase in incubation period from 5 h to 24 h resulted in decreased total yield and decreased viability. Loss of viability was slightly greater at the lower concentration of enzymes (fable 3). A 5 h incubation period using 5% Cellulysin and 1 % Macerase was selected as the standard procedure. · Several purification procedures were compared for their effect on protoplast yield and viability (fable 4). Filtration of the protoplast suspension through a nylon mesh 60 VIRGINIA JOURNAL OF SCIENCE TABLE 2. Effect on protoplast yield of various macronutrient formulations in the protoplast isolation medium Protoplasts were isolated from suspension cultures of G. hirsutum •. Macro nutrient Yield Viability Formulation Strength (No/mlPCV) (%) Murashige & Skoog IX 4.4 X 10 5 96.2 Murashige & Skoog 0.5X 4.5 X 10 5 94.0 Gamborg IX 4.3 X 10 5 91.7 TABLE 3. Effect of enzyme concentration and incubation period on yield and viability of protoplasts. Cellulysin Mace race Incubation Yield Viability (%wlv) (%w/v) (hrs) (No/mlPCV) (%) 5.0 1.0 5 3.1X10 5 100 2.5 0.5 5 5.2 X 10 4 100 5.0 1.0 24 1.0 X 10 5 93.2 2.5 0.5 24 1.4 X 10 4 89.8 TABLE 4. Effect of protoplast yield and viability of several purification procedures by filtration, centrifugation, or their combination. Recovery% Nwnberof Initial Viability After of Pro top lasts Protoplasts/ Viability Purification Purification Method (%) mLPCVYield (%) (%) Filtration, 100 mM Mesh 100 4.0X 10 6 92.3 82.1 Floatation over Sucrose 68 1.7X 10 6 88.4 77.8 Filtration and Floatation 26 9.0X 10 5 86.9 85.7 with 100 mm pores, allowe

Effects of Collection, Transport, and Redeployment Methods on Natural Mortality of Rangia cuneata (Mactridae) Used in Biomonitoring Studies

Abstract: Sporadic, high mortality in test populations of wedge clams (Rangia cuneata) has limited the potential for using this otherwise desirable test organism in biomonitoring studies. To determine whether high mortality was due to ontogenic or experimental variables, a two-phased study was conducted. In phase I, mortality of collected and re-deployed wedge clams, subjected to varying transport conditions, was determined at 7, 14, 21 and 60 days re-deployment. The use of three transport times (1, 2, 3 hr.), two vehicle conditions ( open, closed) and three transport treatments ( open, closed, iced containers) yielded 18 test groups. Individual test group mortalities were below 10% through the 21 day re-deployment period and peaked at 13% at the 60 day re-deployment point. The low rates of mortality observed in phase I of this study indicate that reasonable collection and transport of wedge clams does not significantly increase natural mortality and suggests other parameters are more strongly correlated to test population mortality. In phase II of this study, percent survival of collected and \"acutely\" redeployed and \"acclimated\" redeployed wedge clams was determined. Acclimated re-deployment is the transfer of R. cuneata from saline to freshwater in decrements of 3-4 ppt/day in accord with recommendations in Bedford and Anderson (1972). Acute re-deployment is the placement of R. cuneata in lower salinity waters or freshwater without acclimation. Although percent survival of clams acutely deployed to the freshwater test site was significantly (p<0.05) less than the percent survival at other test sites, mortality was only 3.3 %. No significant differences (p<0.05) were recorded in the percent survival of acclimated redeployed wedge clams.

Trachelomonas spp. and Other Euglenophyceae Taxa in a Southeastern Virginia Lake

Abstract: Trachelomonas species from Lake Kilby, a resetvoir lake in southeastern Virginia are descnbed with supportive electron micrographs . The most abundant Trachelomonas species were T. hispida and T. volvocina. Other members of the Euglenophyceae occurring in this lake are identified. INTRODUCilON Within freshwater phytoplankton communities, representatives of the genus Trachelomonas Ehrenberg (phylum Euglenophyta, class Euglenophyceae) are often common and abundant. These microscopic, unicellular, and flagellated cells are encased in a lorica (test) whose characteristic smface morphology (among other factors), provides significant infonnation for their identification Representative Trachelomonas species that were identified in Lake Kilby, a relatively small resetvoir lake in southeastern Virginia, are descnbed with representative scanning electron microscope prints. A listing of other species within the Euglenophyceae category that were present in Lake Kilby are also given Lake Kilby is located in Suffolk, Virginia (76°38'W, 36°42' 40\"N) and its waters setve as a regional water supply source. Typical of other resetvoirs established within an impounded watershed segment, the lake has an elongated and generally narrow basin, mostly shallow, with a maximum depth of approximately 6-7 meters, and a surface area of 90 ha (Norman, 1985). Seasonal stratification occurs in the deeper regions, with submerged tree stumps found throughout its more shallow sections. During periods of low water level, additional water from the Potomac aquifer will be pumped into the lake (Schaf ran and Scully, 1994 ). This pumping is most common beginning in September and continuing into late fall. When this occurs the oxygen level in the lake decreases, with the total phosphorus concentrations and alkalinity increasing. In addition, as a control measure to reduce the occurrence of algal blooms, copper sulfate is often added to the lake surface between March and November. ME1HODS Water samples (500 ml) were taken from the upper 1 meter depth at 4 stations that were placed along the length of the lake. Collections were made once in June and twice in July 1996. The samples were collected with a Kemmerer sampler and presetved with Lugol's solution A modified UtennOhl method was followed where the water sample was passed through a series of settling and siphoning steps (3) to provide a 40 ml concentrate of the original water sample (Marshall and Alden, 1990). Fractions of this concentrate (based on the density of the plankton and suspended solids present) were placed in settling chambers and examined at both 315x and 500x magnification using a Zeiss (inverted) plankton microscope. Taxon concentrations were determined from this analysis. Representative water samples were then prepared for examination using a Cambridge Stereoscan model S-100 scanning electron microscope. This report 14 VIRGINIA JOURNAL OF SCIENCE emplmizes only the euglenophycean species identified in the sample analysis. Water quality variables were detennined by perso111_1Cl from the City _\"f Portsmouth Identification refereJX:es included Huber-Pestalozzi ( 1955), Conforti and ~udelman ( 1994 ), Coute and Iltis (1981), Coute and Therezien (1985), and Wolowski (1998). RESULTS 1be year 1996 may be comidered a ''wet\" year with the highest annual rainfall for this area recorded over the last decade at 65.8 inches, with a monthly mean of 5.49\". Subsequently this resulted in approximately 56 million gallons of water pumped into the lake from the Potomac aquiferfor 1996, which is considerably less than what would be added during a \"cby\" year, as occurred in 1997 when this region had 42.7 inches of rainfall and theamountofpumpingreached 181 million gallons. Forthetwo months (June-July 1996) when water samples were taken, the pH ranged from 5.7 to 6.2. The swface oxygen was from 3.1 to5.9 mg L\"1, orthophosphates 0.06 to 0.09 mg L·1, nitrates 0.07 to 0.1 mg L\"1, and nitrites remained at0.02 mg L.1 • The water's surface temperature ranged from 20.1 to 25.3 °C. The lake is considered eutrophic. During this period cell concentrations for the total Trachelomonas spp. at the four stations ranged from 2-183 x 103 cells L\"1 in June and 29-108 x 103 cell L.1 in July. These concentrations for the Euglena spp. ranged from 2-11 x 103 cells L\" 1 in June and 2-15 x 103 cellL\"1 in July, and for Phacus spp. ranged from 6-63 x 103 cells L\"1 in June and 2-88 x 103 cell L\"1 in July. The genus Trachelomonas: Following mainly Huber-Pestalozzi (1955): The genus Trachelomonas is represented by unicellular, free swimming cells, enclosed in a lorica, usually spherical, oval, or spindle shaped. The cell surface may be smooth, rough, pitted. and possess small or large si7.e punctae. The cell surface may be with, or without spines; and when present the spines may vazy in size, thickness, and location among different species. The flagellar pore may have an annular ring, or possess a collar that would vazy in its \"-!hape, length, and the presence of spines. These cells are common in shallow lakes, J)onds, and swamp waters. Trachelomonas acanthophora Stokes Lorica spindle-form, having an ellipsoidal central area with ends exlended. The anterior end contains a tubular collar, ending tenninally with the rim of the collar possessing a ring of spines (6-8) diverted outwanlly. The collar length is around 18 microns. The caudal end is more narrow, but slightly conical, tubular, with the terminus containing a ring of spines (5-6) diverted outwardly. The surface of the central body contains punctae, with thick pointed spines (3-5 microns long). Spines also on the caudal extension, with some but fewer spines on the lower part of the collar. Variability in shape and size of central cell body. Similar to T. Dastuguei Balech. See Huber-Pestalozzi (1955). Approximately 37-65 X 20-25 microns in si7.e, yellow brown in color. Figure 1. Trachelomonas acanthostoma Stokes emend. DeFlandre Represented by a sub-spherical or broadly ellipsoidal lorica, having a smooth swface, containing fine punctae. May be without a collar, or with a low collar, with the flagellar pore surrounded by 1 or 2 rings of short spines. 26-31 x 22-27 microns Trachelomonas spp. IN A VIRGINIA LAKE 15 in size. See Huber-Pestalozzi (1955) and Wolowski (1998). Color reddish brown Figure 2. Trachelomonas alisoviana Skvort7.0v Spherical to slightly ellipsoidal lorica, with the entire surface pitted, and having a flagellar pore surrounded by a distinct and raised annular rim. Flagellar pore is 1.5-2.0 microns. Size 15-20 microns. See Coute and Therezien (1985). Figure 3. Trachelomonas armata v. Stein ii Lemmennann emend. Deflandre Possesses an egg-shaped lorica, with a fairly smooth surface, containing minute pUJX:tae. The posterior end is slightly broader. The collar is represented by a low elevated rim containing a circle of spines. The spines are concentrated at both ends of the cell. Small straight spines (2 microns) at the anterior end. A cluster of long, cwved, thick spines (8-12 microns) are at the posterior end. Similar to T. armata v. longispina (Playf.) Deflandre illustrated in Prescott 1956), and T. armata v. long a Deflandre. Also see Huber-Pestalozzi (1955), Coute and Therezien (1985). Size, 35-40 x 25-30 microns. Yellow brown in color. Figure 4. Trachelomonas globularis v. Boyeri (Palmer) Conrad The lorica has a spherical shape, with short conical-shaped spines scattered over the swface, which are inter-spaced by punctae. Golden brown in color. Si7.e 9-14 microns. See Huber-Pestalozzi (1955). Figure 5. Trachelomonas hispida (Perty) Stein The lorica is ellipsoidal, with short spines and punctae over the entire surf ace of the cell. The collar is either absent, or it is only slightly developed. A vety common species. Light brown to reddish brown in color. 19-25 x 15-19 microns in size. See Huber-Pestalozzi (1955). Figure 6. Trache/omonas hispida v. coronata Lemmermann Possesses an elongated ellipsoidal shape lorica, with a swface covered with short spines. There is a slight development of a collar, with the collar rim encircled by row of short spines. 22-3 0 x 8-15 microns in si7.e. A common species, with variable ranges in size. Darlcbrownincolor. Referto Coute and lltis (1981),Huber-Pestalozzi (1955), and Wolowski (1998). Figure 7. Trachelomonas intermedia Dangeard Has a lorica that is sub-spherical to broad ellipsoidal in shape, with a collar having a low, but distinct rim, but is not raised. The cell surface is rough, dense, and contains pUIX:tae. The cell is darlc brown in color. 15-22 microns. (See Coute and Therezien, p.114, Plate 11, figs.1-3, 1985; Huber-Pestalozzi, 1955). Trachelomonas Raciborskii Woloszynska Lorica ellipsoid in shape, with no developed collar. Distinct, sparsely distributed spines, oot dense, mostly concentrated at polar ends of cell. Surface area contains pUJX:tae. Size 30-34 x 25-28 microns. Darlc brown in color. See Coute and Therezien (1985), Huber-Pestalozzi (1955). Figure 8. Trachelomonas simi/is Swirenko Ellipsoidal shaped lorica, coarse rough surface, with punctae scattered over swface, butoo spines. Possesses a thick, coarse collar, exlending (4-6 microns), bent from the vertical. Golden brown in color. 24-29 x 16-20 microns in size. See Wolowski ( 1998). Figure 9. I 16 VIRGINIA JOURNAL OF SCIENCE Trachelomonas superba Swirenko emend. Deflandre Lorica ellipsoidal, with small punctae over surface. Different length conicalshaped spines over the surface, being longer at the polar ends. Possesses a low collar encircled by ring of spines. Reddish brown in color. Size 30-36 x 23-28 microns. See Coute and Therezien (1985). Figure IO. Trachelomonas volvoclna Ehrenberg Lorica globular, spherical shaped, with a surface that is generally smooth, lacking spines. The collar is either lacking, or the collar is slightly developed With no collar, flagellar pore is surrowxled by slightly raised annular region. Very conunon. Diameter 7-28 mi