Showing papers in "Virginia journal of science in 2009"

Phytoplankton Blooms: Their Occurrence and Composition Within Virginia's Tidal Tributaries

Abstract: Sporadic algal bloom development within a IO year monitoring program in Virginia tidal tributaries of Chesapeake Bay is reviewed. These blooms were common events, characteristically producing a color signature to the surface water, typically short lived, occurring mainly from spring into autumn throughout different salinity regions of these rivers. and were produced primarily by dinoflagcilatcs. The abundance threshold levels that would identify bloom status from a non-bloom presence were species specific, varied with the taxon's cell size, and ranged from ca. 10 to \\() cells ml • Among the most consistent sporadic bloom producers were the dinoflagcllatcs Akashiwo sanguinea, Cochlodinium polvkrikoides, Heterocapsa rotundata, Hetcrocapsa triquetra, Karlodinium l'eneficum, Prorocentrum minimum, Scrippsiella trochoidea, the cyanobacterium lvficrocystis aeruginosa, and two categories containing several species of often unidentified Gymnodinium spp. and Gyrodinium spp. Additional bloom producers within these tributaries arc also discussed.

Graminicolous Fungi of Virginia: Fungi in Collections 2004-2007

Abstract: Fungus-grass associations recognized in Virginia from 2004 to 2007 are recorded. Many associations are new to the United States (U), eastern United States (EU), and Virginia (V); other associations extend the known distribution of those previously discovered. These reports contribute further to knowledge of the mycoflora of Virginia.

Seasonal Variation in Diet of a Marginal Population of the Hispid Cotton Rat, Sigmodon hispidus

Abstract: Cotton rats live in oldfields, habitats with a variety of mostly herbaceous plants. Based on other studies, the hispid cotton rat, Sigmodon hispidus, eats many kinds of herbaceous plants but grasses predominate. In contrast, our population of cotton rats ate many monocots but mostly they were not grasses. Our study sought to determine the diet of the cotton rat in eastern Virginia, near the northern limit of distribution on the Atlantic Coast. Fecal samples, collected each month during an on-going capture-mark-release demographic study of the rodent community, were analyzed using a standard method. A greater variety of foods (including insects) was eaten in the summer and autumn than in winter and spring. In winter, when much herbaceous vegetation is standing dead, cotton rats supplemented their diets with pine bark. Cotton rats ate significantly greater proportions of monocots in winter and spring, an apparent response to the need for more calories to compensate for greater heat loss. In summer and autumn, cotton rats enhanced their diets with significantly greater proportions of the more nutritious but harder to digest dicots. Reproductive females ate significantly more dicots and less monocots than males and non-reproductive females, whose diets were similar.

Management and Social Indicators of Soil Carbon Storage in a Residential Ecosystem, Midlothian, VA

Abstract: Soil carbon storage - defined here as carbon mass per unit ground area - is an important ecosystem service, sequestering carbon that might otherwise exist 2 in atmospheric CO . Significant attention has focused on the effects that humans have on carbon cycling, but little is known about how human behaviors and attitudes relate to lawn carbon storage. The objectives of this study were to conduct household surveys in concert with soil carbon sampling in a 10-yr-old exurban neighborhood near Richmond, Virginia to quantify differences in soil carbon storage between residential lawns and mixed pinehardwood forest fragments, and to determine how lawn management and environmental attitudes relate to soil carbon storage. Lawns stored significantly less carbon than forest fragments in the top 10 cm of soils. A significant negative relationship was observed between watering and fertilizer frequency and soil carbon storage, but the goodness-of-fit was sensitive to intra-lawn variability in soil carbon mass. Survey respondents that claimed to be environmentalists stored significantly more carbon and spent one hour less per week managing their lawns, suggesting that environmental attitudes may affect how households manage their lawns and, in turn, the quantity of soil carbon stored in residential soils.

A Standardized RNA Isolation Protocol for Yam (Dioscorea alata L) cDNA Library Construction

Abstract: For the purpose of constructing yam cDNA libraries, attempts to isolate high quality RNA using several previously reported protocols were unsuccessful. Therefore a protocol was standardized for yam total RNA isolation by using guanidium buffer at the Department of Biology, Virginia Sate University. The RNA isolated using this standardized protocol was high in quality and led to successful good quality cDNA library construction and identification of functional ESTs in yam. INTRODUCTION Yam, (Dioscorea alata L), is the main food source for over 100 million people in humid and sub-humid tropics. Its production is affected by several biotic and abiotic factors (Abang et al., 2003). Anthracnose, caused by Colletotrichum gloeosporioides, is the most severe foliar disease of water yam (Dioscorea alata L) and is a major hurdle in yam production. It is reported that anthracnose causes yield reduction up to 90% (http://annualreport.iita.org). There are no cost effective control measures and the long-term solution to the problem will be through the development of resistant genotypes (Mignouna et al., 2002. Very limited yam sequence information is available from public genome databases. A review of previous efforts to develop cDNAs towards EST development in yams revealed that housekeeping genes were prevalent in the libraries constructed using total RNA from male flowers (Mignouna et al., 2002a, b, c). It is realized that obtaining high quality, intact RNA is the first and the most critical step in conducting cDNA library construction and for further analysis of gene of interest. After many attempts of total RNA isolations from yam leaf samples using standard plant RNA isolation protocols (Verwoerd et al,1989), only 6-10 ug of total RNA was extracted from the leaves and no colonies were observed when this RNA was used for cDNA library construction. The RNA appeared as a smear on 1.1% agarose gel (Fig. 1). The most likely reason for not getting good quality RNA is the mucilagenous tissue in yam plant parts like leaf, stem and tuber. This tissue causes problem because of polyphenols, polysaccharides and other secondary metabolites that are rich in yam plant parts and are not easily removed by conventional extraction methods. The aim of this study was to establish a protocol for RNA isolation from Dioscorea alata to get high quality and high quantity RNA that is suitable for generation of molecular markers, such as EST-SSRs and SNPs. Therefore, the following article discusses successful and reproducible method of RNA isolation Virginia Journal of Science Volume 60, Number 4 Winter 2009 172 VIRGINIA JOURNAL OF SCIENCE procedure employed for yam cDNA library constrcution and ways of increasing RNA yields MATERIALS AND METHODS Tissue collection: In order to standardize the protocol for RNA isolation, the yam (source: local grocery store) were grown in the green house in pots. Fresh 1g leaf tissues are collected in 50ml BD Falcon tubes, frozen quickly in liquid nitrogen. FIGURE 1. A smear of rRNA samples of Dm-Resistant yam genotype and BmSusceptible yam genotype isolated using standard protocols on 1.1% Formaldehyde agarose gel RNA ISOLATION PROTOCOL 173 RNA isolation. Only the successful procedure of RNA isolation with the modifications to standard plant RNA isolation protocol is reported here. Solutions and solvents used: · Extraction buffer (100 ml stock): 76.424g of 8M Guanidium Hydrochloride + 425 mg of 20mM MES + 740mg of 20mMEDTA+ 35ml of DEPC water. Adjust the pHwith 10M NaOH, autoclave and store at 4°C. Add 1.38μl of âmercaptoethanol (50mM) just before use. · Phenol:Cholorform:Isoamulalcohol (24:23:1) Procedure: 1. 1g tissue ground in liquid nitrogen was homogenized in 2ml extraction buffer + 2ml Ph:Chl:IAA. {The sample was homogenized using power operated mini grinder (the steel grinder part was pre-cooled in liquid nitrogen) that perfectly fits in to the falcon tube. It was necessary to maintain frozen conditions throughout the extraction to enhance the quality of the target RNA. }. 2. The sample was centrifuged for 10 min at 10,000rpm (at 0-2 °C ). 3. To the Supernatant, Ph:Chl:IAA (equal volumes in 1:1 ratio) was added and the RNA was precipitated overnight in -20. 4. The next day the sample was centrifuged for 20 minutes at 10,000rpm (at 0-2 °C) and the pellet and was dissolved in Deionized water (Volume based on required concentration). 5. RNA was stored at -80°C.The quality of RNA was confirmed by using BIO-RAD Smartspec plus Spectrophotmeter and also by Formaldhyde agarose gel T M electrophoresis (Sambrook et al, 1989). cDNA LIBRARY CONSTRUCTION The freeze dried leaves of D. alata L genotypes, Tda 95/00328, resistant to the FGS strain of C. gloeosporioides but susceptible to the SGG strain and TDa 92-2, susceptible to the FGS and SGG strains of C.gloeosporioides were obtained from IITA, Ibadon, Nigeria. Leaves were ground in liquid nitrogen and total RNA was isolated using the standardized protocol. Total RNA thus isolated was used for the construction of cDNA library using The Creator smart cDNA library construction kit (BD Biosciences Clonetech). First strand cDNA was synthesized using SMART IV oligonucleotide followed by long distance PCR amplification to generate high yields of full-length ds cDNAs (~400 to >4000 bp) followed by Sfi I digestion and column fractionation. The cDNA fractions that match the desired size distribution (1-4kb) were selected. The Sfi I – digested cDNA was ligated to the Sfi I digested dephosphorylated pDNR-LIB Vector (Clonetech) and transformed into DH10B T1 Phase resistant bacterial cells. The chloramphenicol resistant colonies were picked and archived in 96 well plates. For preliminary round of sequencing, about 100 colonies from each library (resistant and susceptible) were randomly selected and subjected to single pass sequencing (Agencourt Biosciences). 174 VIRGINIA JOURNAL OF SCIENCE RESULTS AND DISCUSSION The quantity of total RNA is between 250 to 500μg from 1g of yam leaf tissue. The 18S and 28S ribosomal RNA bands are clearly visible in the intact leaf RNA samples Dm and Bm of yam (Fig. 2) and the quality reading on spectrophotometer were presented in the Table 1. Following quality check of the sequences, the pure quality sequences were checked for homology to sequences in GenBank using BLAST similarity search tool. Data obtained from the BLAST analysis of 100 clones from each resistant (Dm) and susceptible (Bm) accessions were compiled and interpreted with respect to the hits identified in other plant species (Table 2 and 3). This preliminary data describes the initial efforts to develop tools to annotate EST's for anthracnose disease resistance genes by constructing good quality cDNA libraries for different accessions of D.alata. From each cDNA library 6000 colonies were arrayed into 96 well plates. A total of 100 clones randomly selected each from two FIGURE 2. Intact yam rRNA samples using current protocol. RNA ISOLATION PROTOCOL 175 distinct libraries namely Dm and Bm. Of the 100 cDNA clones from each yam genotype, 10 yielded no sequence and an additional 9 produced sequences of less than 100 bp and these were not used for sequence analysis. The average length of the remaining sequences was 762 bp. Based on top Blast hits in plants, in yam type Bm, out of 100 sequences, 48 were distinct gave >400bp and were showing functional similarities. In Yam type Dm, out of 100 sequenced clones 48 were distinct, gave >400bp and 22 were duplicates of yam type 1 were observed. The genes putatively identified are shown in Table 2 and 3. The blast hits identified in different crops showed 88-100% identity and, in general, the homology of the insert sequence to the blast hit is about 400-500bp out of 700-800 bp length aligned. The genes (ESTs) identified based on sequence similarity are involved in various putative functions such as gene or protein expression, protein binding, ripening, cell wall and stress response, defense, photosynthesis, photoperiodic flowering response, cell division and proliferation, nodulation, and secondary metabolism etc. and some of them could not be classified into any of these categories. The numbers of hits showing stress/defense related function were comparatively more in resistant genotype when compared to susceptible genotype (Satya et al, 2007). Of the distinct sequences there are sequences similar to unknown protein and unknown mRNA (1-2%) not presented here. The information on hits to clone sequences (10%) in different crop species and the top blast hits to mitochondrial genes and genes encoding for ribosomal protein genes (20%) were not listed in the table. By sequencing a large number of cDNAs, we can selectively avoid the clones that represent ribosomal and mitochondrial genes, and choose clones that represent genes that we wish to examine. This is a significant improvement compared to previous efforts where sequences coding for ribosomal proteins were predominant in the libraries. This achievement is attributed to quality RNA isolation. CONCLUSION Two cDNA libraries for yam, one each for resistant and susceptible genotypes, were constructed for the purpose of identifying clones that are differentially expressed in these two genotypes. Many new genes have been identified that can be useful for future studies. The sequences may also be a source of single-nucleotide polymorphisms or simple sequence repeats for molecular marker development. Preliminary analysis of 200 clones revealed homologies to known genes in several related and distant plant species. Though the numbers of hits were comparatively more in resistant genotype compared to susceptible genotype, not much distinct differences were observed between the functional hits to sequences of these two genotypes. TABLE 1. Spectrophotometer readings of quality RNA samples from yam genotypes. Sample ID ng/ìL A260 A280 A260 A280 A260 A230 Constant Cursor Pos Cursor Abs 340 raw Bm 257.6 6.438 2.9

First Records of Hypleurochilus geminatus and Centropristis philadelphica from Chesapeake Bay

Abstract: During the fall of 2007, Centropristis philadelphica (rock seabass) and Hypleurochilus geminatus (crested blenny) were collected from Chesapeake Bay. These captures are significant as they represent the first substantiated record of C. philadelphica from Chesapeake Bay and only the second and third validated records of H. geminatus. Additionally, the first record of H. geminatus from Chesapeake Bay was only recently recognized since the specimen had been previously misidentified as Parablennius marmoreus (seaweed blenny). The collection of seven individuals of H. geminatus in 2007, from two locations, indicates that the species may be resident within the Chesapeake Bay estuary. INTRODUCTION The Chesapeake Bay, an ecotone between the Atlantic Ocean and the rivers of Maryland and Virginia, experiences extreme seasonal temperature changes and contains a range of habitats. Species richness is typical of such ecological systems and is evident by the estuary’s diverse and dynamic fish fauna, which includes permanent residents, spawning migrants, and seasonal visitors (Murdy et al. 1997). The fish fauna of Chesapeake Bay has been surveyed extensively since the early 1900’s (Hildebrand and Schroeder 1928; Massman 1962; Massman and Mansueti 1963; Musick 1972; Murdy et al. 1997) yet warmwater species uncommon to the estuary continue to be encountered (Halvorson 2007). Two such species, Centropristis philadelphica (rock seabass) and Hypleurochilus geminatus (crested blenny), were collected in Chesapeake Bay during the fall of 2007 by the Virginia Institute of Marine Science (VIMS) Juvenile Fish and Blue Crab Trawl Survey. MATERIALS AND METHODS Five-minute bottom tows were conducted in lower Chesapeake Bay with a 9.14 m otter trawl (38.11 mm stretched mesh body, 6.35 mm cod-end liner, and a tickler chain) off the 8.5 m R/V Fish Hawk. Fish were identified and measured to the nearest mm (total length for H. geminatus and total length centerline for C. philadelphica). Voucher specimens were deposited in the Ichthyological Collection, Virginia Institute of Marine Science, Gloucester Point, Virginia (H. geminatus-VIMS 11776, C. philadelphicaVIMS 11979). Hydrological measurements (water temperature, salinity) were taken with a YSI 600Q (YSI Incorporated, Yellow Springs, Ohio). Virginia Journal of Science Volume 60, Number 3 Fall 2009 (804) 684-7751, aimeehal@vims.edu 1 142 VIRGINIA JOURNAL OF SCIENCE RESULTS On September 6 , 2007, five individuals of H. geminatus (39-78 mm) were th captured in Chesapeake Bay at 37o17.13’N, 76o 03.11’W, near Cape Charles, Virginia (Figure 1; Table 1). Water depth at this station was 7 m and the bottom water temperature and salinity were 26.59oC and 23.64‰, respectively. Two additional specimens (34-37 mm) were collected on November 14 , 2007, at 36o58.43’N, th 76o16.59’W, near the entrance to Hampton Roads, in 5.5 m of water (Figure 1; Table 1). The bottom water temperature was 13.63oC and bottom salinity was 22.79‰. A single specimen of C. philadelphica (210 mm) was collected November 5 , th 2007 at 36o58.76’N, 76o07.16’W, approximately 1 km upstream of the first tunnel of the Chesapeake Bay Bridge-Tunnel (Figure 1; Table 1). Water depth was 13.4 m and the bottom water temperature and salinity were 17.22oC and 24.54‰, respectively. DISCUSSION The crested blenny (Hypleurochilus geminatus) is a subtropical species often found in association with oyster reefs, shell bottoms (Dahlberg 1972; Crabtree and Middaugh 1982; Lehnert and Allen 2002), and marine growths attached to pilings and rocks (Hildebrand and Cable 1938). They feed on free swimming organisms as well as sessile FIGURE 1. Collection locations of Centropristis philadelphica (\") in 2007 and Hypleurochilus geminatus (Ä) in 1993 and 2007 in Chesapeake Bay. CHESAPEAKE BAY FISH 143 growths (Hildebrand and Cable 1938), with their diets primarily consisting of crustaceans and algae, followed by hydroids and polychaetes (Lindquist and Chandler 1978; Lindquist and Dillaman 1986). Hildebrand and Cable (1938) determined that North Carolina specimens of H. geminatus spawn from May to September and the larvae are mainly surface dwelling until 10-15 mm in length, at which time they change their habitat preference. The largest fish collected in their study was a 72 mm male, with the largest female measuring 58 mm (Hildebrand and Cable 1938). Although the range of H. geminatus encompasses the waters of New Jersey to the eastern central coast of Florida (Williams 2002), the only collections north of North Carolina have occurred sporadically off New Jersey (Fowler 1914; Allen et al. 1978; Able 1992; Able and Fahay 1998). Hypleurochilus geminatus was not reported in earlier studies of Virginia waters, including Chesapeake Bay and its tributaries (Hildebrand and Schroeder 1928; Massman 1962; Massman and Mansueti 1963; Musick 1972; Murdy et al. 1997) and the seaside coasts and inlets (Schwartz 1961; Richards and Castagna 1970; Cowan and Birdsong 1985; Norcross and Hata 1990; Layman 2000). Ditty et al. (2005) erroneously reported that Hildebrand and Cable (1938) obtained larvae of H. geminatus from Chesapeake Bay. Ongoing baywide surveys, including the Chesapeake Bay Multispecies Monitoring and Assessment Program (ChesMMAP) (James Gartland, Virginia Institute of Marine Science, Gloucester Point, Virginia, personal communication) and the Chesapeake Bay FisheryIndependent Multispecies Survey (CHESFIMS) (Miller and Loewensteiner 2008), have yet to encounter this species, nor do specimens from Chesapeake Bay exist in the VIMS Ichthyological Collection or the U. S. National Museum (USNM) fish collection (L. Palmer, Smithsonian Institution, pers. comm.). The captures in 2007 are not the first records of H. geminatus collected from Chesapeake Bay. Murdy et al. (1997) reported a single specimen of Parablennius marmoreus (seaweed blenny) captured in June 1993 (VIMS specimen 09086). Upon Table 1. Table of species showing the number of specimens, year collected, and collection location (latitude and longitude). Species Year Collected Number of specimens Latitude Longitude Centropristis philadelphica 2007 1 36o58.76N 76o07.16W Hypleurochilus geminatus (reported by Murdy et al. 1997 as Parablennius marmoreus) 1993 1 37o16.63N 76o03.43W Hypleurochilus geminatus 2007 5 37o17.13N 76o03.11W Hypleurochilus geminatus 2007 2 36o58.43N 76o16.59W 144 VIRGINIA JOURNAL OF SCIENCE further evaluation in 2007, it was determined that this specimen had been misidentified and is indeed H. geminatus. Interestingly, this specimen was collected at 37o16.63’N, 76o03.43’W (Figure 1; Table 1), within 1 km from the location where five individuals were collected in September 2007. The collection of a single misidentified H. geminatus in 1993 is the first documented occurrence of this species in Chesapeake Bay and the subsequent capture of seven individuals during 2007 indicates that not only has this species extended its range to include the estuary, but that an established population might exist off Cape Charles, VA . The smallest member of the genus Centropristis, C. philadelphica is a fast growing, short-lived species (Link 1980) that attains a maximum length of 300 mm (Heemstra et al. 2002). This protogynous hermaphrodite inhabits a range of depths over various substrates, including hard bottoms, rocky reefs, and the preferred softer mud bottoms (Miller 1959; Link 1980). Spawning occurs offshore between February and July (peak April-May) off North Carolina (Link 1980) and from late March to May in the Gulf of Mexico (Miller 1959). Ross et al. (1989) described C. philadelphica as a “euryphagic benthic carnivore” and their study of Gulf of Mexico specimens found a diet dominated by shrimps, crabs, mysids, and fishes, agreeing with Links’ (1980) findings that crustaceans, fishes, and mollusks were the most frequent prey. The range of C. philadelphica includes Cape Henry, Virginia, to Palm Beach, Florida, as well as the Gulf of Mexico (Miller 1959; Heemstra et al. 2002). Centropristis philadelphica was not reported in earlier studies of Chesapeake Bay and its tributaries (Hildebrand and Schroeder 1928; Massman 1962; Massman and Mansueti 1963; Musick 1972; Murdy et al. 1997) nor the Virginia seaside coasts and inlets (Schwartz 1961; Richards and Castagna 1970; Cowan and Birdsong 1985; Norcross and Hata 1990; Layman 2000). Ongoing baywide surveys including the ChesMMAP (James Gartland, Virginia Institute of Marine Science, Gloucester Point, Virginia, personal communication) and the CHESFIMS (Miller and Loewensteiner 2008) have yet to encounter this species, nor are there specimens from Chesapeake Bay in the VIMS Ichthyological Collection or the U. S. National Museum (USNM) fish collection (L. Palmer, Smithsonian Institution, pers. comm.). The individual collected in November 2007 represents the first substantiated record for C. philadelphica from Chesapeake Bay. The Northeast Fisheries Science Center (NEFSC) trawl survey’s most northerly validated record of C. philadelphica is a 100 mm standard length specimen from 37o28’N, 74o25’W, approximately 100 km east of Parramore Island, Virginia, in the Atlantic Ocean (William Kramer, NOAA Fisheries Service, Woods Hole, Massachusetts, personal communication). Both of these occurrences are slightly north of the published northern range boundary of Cape Henry, Virginia. Nearly twenty years ago, Kennedy (1990) predicted that climate change would cause “poleward estuaries to resemble neighboring estuaries that are located in the direction of the equator.” As such, he stated that Chesapeake Bay could become as warm as southeast Atlantic coast estuaries and that warmwater or subtropical species would move north from these neighboring estuaries and occupy Chesapeake Bay (Kennedy 1990). Interestingly, the VIMS Juvenile Fish and Blue Crab Trawl Survey, which has sampled Chesapeake Bay and its tributaries since 1955, has recently documented an increase in the diver