Characterization of microbial communities from coastal waters using microarrays

Molecular methods, including DNA probes, were used to identify and enumerate pathogenic Vibrio species in the Chesapeake Bay; our data indicated that Vibrio vulnificus exhibits seasonal fluctuations in number. Our work included a characterization of total microbial communities from the Bay; development of microarrays that identify and quantify the diversity of those communities; and observation of temporal changes in those communities. To identify members of the microbial community, we amplified the 16S rDNA gene from community DNA isolated from a biofilm sample collected from the Chesapeake Bay in February, 2000. The resultant 75 sequences were 95% or more similar to 7 species including two recently described Shewanella species, baltica and frigidimarina, that have not been previously isolated from the Chesapeake. When the genera of bacteria from biofilm after culturing are compared to those detected by subcloning amplified 16S fragments from community DNA, the cultured sample exhibited a strong bias. In oysters collected in February, the most common bacteria were previously unknown. Based on our 16S findings, we are developing microarrays to detect these and other microbial species in these estuarine communities. The microarrays will detect each species using four distinct loci, with the multiple loci serving as an internal control. The accuracy of the microarray will be measured using sentinel species such as Aeromonas species, Escherichia coli, and Vibrio vulnificus. Using microarrays, it should be possible to determine the annual fluctuations of bacterial species (culturable and non-culturable, pathogenic and non-pathogenic). The data may be applied to understanding patterns of environmental change; assessing the health of the Bay; and evaluating the risk of human illness associated with exposure to and ingestion of water and shellfish.     

Effects of commercial diazinon and imidacloprid on microbial urease activity in soil and sod

Diazinon [O,O-diethyl O-2-isopropyl-6-methyl(pyrimidine-4-yl) phosphorothioate] and imidacloprid [1-(1-[6-chloro-3-pyridinyl]methyl)-N-nitro-2-imidazolidinimine] are applied to lawns for insect control simultaneously with nitrogenous fertilizers such as urea, but their potential effect on urease activity and nitrogen availability in turfgrass management has not been evaluated. Urease activity in enzyme assays, washed cell assays, and soil slurries was examined as a function of insecticide concentration. Intact cores from field sites were used to assess the effect of insecticide application on urease activity in creeping bentgrass (Agrostis palustris Huds.) and bluegrass (Poa pratensis L.) sod. Bacterial urease from Bacillus pasteurii and plant urease from jack bean [Canavalia ensiformis (L.) DC.] were unaffected by the insecticides. Both insecticides inhibited the growth of Proteus vulgaris, a urease-producing bacterium, but only diazinon significantly reduced urease activity in washed cells; neither insecticide inhibited urease activity in sonicated cells. Neither diazinon nor imidacloprid inhibited urease activity in Woolper soil (fine, mixed, mesic Typic Argiudoll) slurries, but diazinon slightly inhibited urease activity in Maury soil (fine, mixed, semiactive, mesic Typic Paleudalf) slurries. Imidacloprid had no effect on urease activity in creeping bentgrass or bluegrass sod at up to 10 times the commercial application rate. Diazinon briefly, but significantly, reduced urease activity in bluegrass sod. Co-application of imidacloprid and urea appears to be benign with respect to urease activity in soil and sod. Diazinon, in contrast, appears to have a significant, short-term, inhibitory effect on the microbial urease-producing community, but that effect depends on soil type.     

Evaluation of techniques used in the preparation of diesel extract samples for mutagenicity studies

Diesel exhaust particles were used to compare methods and techniques used in the preparation of particulate matter for microbial mutagenesis testing. Investigated in this study were extraction, concentration, and solvent exchange methodologies as they affected recovery of mutagenic material from diesel samples using a Salmonella typhimurium plate incorporation assay. Solvent removal methods applicable for use in determining the mass concentration of extracts were also evaluated. Results indicated that particle samples Soxhlet extracted with dichloromethane yielded higher levels of mutagenic activity than did comparative samples utilizing sonication. No difference was seen between rotary evaporation or Kuderna-Danish macro concentration of extracts to volumes > 50 mL. In comparison of micro concentration techniques to volumes < 10 mL, vortex evaporation was found to be more efficient than a modified micro Kuderna-Danish method in recovery of mass and mutagenicity. Solvent exchanged samples were found to yield higher recoveries of mutagenic activity than samples taken to dryness and then reconstituted in the bioassay solvent. A dry mass weighing procedure utilizing desiccation was found to be more acceptable than either the use of an infrared heat lamp or nitrogen blowdown for solvent removal.     

Extraction of DNA from amniotic fluid cells for the early prenatal diagnosis of genetic disease

Ten-ml samples of amniotic fluid were taken from pregnancies being terminated at 8–14 weeks' gestation. DNA was extracted from the amniotic cells by sequential centrifugation and analysed using the polymerase chain reaction (PCR). Fifteen samples were analysed for evidence of maternal contamination using Mfd5 oligo-nucleotide primers for repeat polymorphisms. Ten amniotic fluid samples were tested for the Delta-F₅₀₈ deletion characteristic of cystic fibrosis to demonstrate a diagnostic application for the technique. In each case, DNA extracted from fetal tissue from the same pregnancy was included in the controls. In 14 of the 15 cases tested with the Mfd5 primers, both the amniotic fluid DNA and the fetal DNA showed no evidence of contaminating DNA. In one case, neither the amniotic fluid cells nor the fetal cells yielded results. In nine of the ten cases tested with the Delta-F₅₀₈ primers, the amniotic fluid cell DNA provided accurate information about the genetic status of the fetus; in the tenth, the fetal DNA failed to amplify. The results indicate that adequate DNA can be extracted from amniotic fluid from 8 weeks' gestation onward and these samples are suitable for prenatal diagnosis using PCR.     

Erythroid-specific antibodies enhance detection of fetal nucleated erythrocytes in maternal blood

Fetal nucleated erythrocytes (NRBC) in maternal blood are a non-invasive source of fetal DNA for prenatal genetic screening. We compared the effectiveness of three monoclonal antibodies for the separation of fetal cells from maternal blood by flow sorting. Mononuclear blood cells from 49 healthy pregnant women were incubated with antibody to CD 71, CD 36, and/or glycophorin A (GPA), employed singly or in combination with each other. These monoclonal antibodies recognize surface antigens on haematopoietic precursor cells. Successful isolation of fetal cells was defined as detection of Y chromosomal sequences in maternal blood from women carrying male fetuses, with absence of Y sequences when female fetuses were carried. Thus, gender prediction accuracy was used as a measure of fetal cell separation. Using anti-CD 71 to isolate fetal cells, gender prediction was 57 per cent correct; with anti-CD 36, it was 88 per cent correct. Anti-GPA, an erythrocyte-specific antigen, used alone or in combination with anti-CD 71 or 36, improved gender prediction to 100 per cent. We conclude that antibody to GPA improves the retrieval of fetal NRBC from maternal blood, permitting genetic analysis by the polymerase chain reaction.     

Transcervical CVS sample size: Correlation with placental location,cytogenetic findings,and pregnancy outcome

Data from 2907 transcervical CVS cases performed on singleton pregnancies were reviewed retrospectively and villus sample size was correlated with cytogenetic results, placental location, maternal age at the expected date of confinement (EDC), gestational age at the time of sampling, birth weight, gestational age at the time of delivery, and pregnancy outcome. No correlation was noted between villus sample size and maternal age, gestational age at sampling, gestational age at delivery, birth weight, or pregnancy outcome. An inverse correlation between villus sample size and percentage of abnormal cytogenetic findings was statistically significant (X² = 8·53, p <0·01). The percentage of small samples was greater when the placenta was anterior, lateral, or fundal than when the placenta was posterior.     

Graded recruitment in a ponerine ant

Summary (1) The giant tropical ant, Paraponera clavata, exhibits graded recruitment responses, depending on the type, quantity, and quality of a food source. More ants are initially recruited to a large prey or scavenge item than to a large quantity of sugar water. (2) Individual ants encountering prey items gauge the size and/or unwieldiness of the item, regardless of the weight, when determining whether to recruit. (3) The trail pheromone of this species is often used as an orientation device by individual ants, independent of recruitment of nestmates. (4) It is proposed that the foraging behavior of P. clavata represents one of the evolutionary transitions from the independent foraging activities of the primitive ants to the highly coordinated cooperative foraging activities of many higher ants.     

Feeding rates and diel vertical migration of copepods near South Georgia: comparison of shelf and oceanic sites

Seventeen Longhurst Hardy Plankton Recorder profiles were taken over a diel cycle in January 1990 to study the feeding of four major copepods over the South Georgia shelf. Ontogenetic changes in vertical migration were followed and feeding cycles determined by gut fluorometry for Calanoides acutus Stage CV, Calanus sinillimus CV and CVI, C. propinquus CV and Rhincalanus gigas CV and CVI. In common with a neighbouring oceanic site visited two weeks later and reported elsewhere, all four species had a diel cycle of feeding and migration. The vertical distributions of C. simillimus (all stages), R. gigas (nauplii) and Euphausia frigida (postlarvae) were similar at both sites, the night being spent within the chlorophyll maximum at 15 to 30 m. However, the biomass dominants, C. acutus and R. gigas, dwelt below the chlorophyll maximum, about 30 m deeper than their oceanic counterparts. Unlike the oceanic site, feeding at the shelf site was not restricted to darkness, but increased 6 to 10 h before nightfall and finished at dawn; the intervening period coincided with sinking and digestion. Daylight feeding may have been induced by the shorter night, lower light levels or greater food requirements at the shelf site, despite planktonic predators being over three times more abundant. Daily ration estimates for R. gigas at both sites were only 2% body carbon per day. These low values contrast with its smaller competirors, whose rations were in the range 5.6 to 27%.     

Speech recognition as a means of enumeration in the analysis of biological samples

Voice recognition by computer using the Key tronic KB5152V keyboard with an IBM PC was found to reduce the time taken for identification and notation of zooplankton samples by approximately 50%, with an accuracy of 98%. This was achieved after only 1 to 2 wk training and the accuracy was further improved with general usage of the technique. The time saving was due to removing the requirement of recording the results on paper. Further time-savings were obtained by eliminating the process of manually entering the data into a computer. A further advantage of this technique is that with appropriate software all data processing and presentation (statistics, graphs, tables, etc.) can be carried out at the bench immediately on completion of a set of analyses. The technique is, of course, not restricted to zooplankton sorting but can be applied to a wide range of manual and enumeration processes at the bench.     

Distribution and variability in abundance ofSchistomysis spiritus (Crustacea: Mysidacea) in the Bristol Channel in relation to environmental variables,with comments on other mysids

Mysids are important components of the zooplankton biomass of estuaries and coastal regions. Twelve species were identified from the Bristol Channel and Severn Estuary (British Isles). The most abundant species wasSchistomysis spiritus (Norman), the majority of which occurred in the Channel within a salinity range of 27 to 34 S. The seasonal distribution and numerical abundance of this species are described over the period November 1973 to February 1975. The peak of abundance ofS. spiritus occurred in the Inner Channel in September (mean of 14 individuals m^-3, ca. 250 individuals m^-2 for this sub-region) when it represented 76% of the omnivore biomass (g C m^-3); for the 364 d from 4 November 1973, the species contributed 43% of the total integrated omnivore standing stock. This peak of biomass was the product of the development of the juveniles from the reproductive period in the spring. Correlation analyses were carried out betweenS. spiritus biomass and 10 physical and biotic variables for 2 mo, November 1973 and September 1974. Temperature and salinity, which are simple indices of seasonal change, exhibited significant correlations with the mysid's abundance in both months. These correlations do not necessarily imply causal relationships or mechanisms between the distribution and abundance of the species and these variables. Clearly, in such a complex environment as an estuarine ecosystem a single variable is unlikely to control the abundance of a species, it is more the result of the combined influence of a number of variables acting in concert.