Quantitative distribution of presumptive archaeal and bacterial nitrifiers in Monterey Bay and the North Pacific Subtropical Gyre

The recent isolation of the ammonia-oxidizing crenarchaeon Nitrosopumilus maritimus has expanded the known phylogenetic distribution of nitrifying phenotypes beyond the domain Bacteria. To further characterize nitrification in the marine environment and explore the potential crenarchaeal contribution to this process, we quantified putative nitrifying genes and phylotypes in picoplankton genomic libraries and environmental DNA samples from coastal and open ocean habitats. Betaproteobacteria ammonia monooxygenase subunit A (amoA) gene copy numbers were low or undetectable, in stark contrast to crenarchaeal amoA-like genes that were broadly distributed and reached up to 6 x 10(4) copies ml(-1). Unexpectedly, in the North Pacific Subtropical Gyre, a deeply branching crenarchaeal group related to a hot spring clade (pSL12) was at times abundant below the euphotic zone. Quantitative data suggested that the pSL12 relatives also contain archaeal amoA-like genes. In both coastal and open ocean habitats, close relatives of known nitrite-oxidizing Nitrospina species were well represented in genomic DNA libraries and quantitative PCR profiles. Planktonic Nitrospina depth distributions correlated with those of Crenarchaea. Overall, the data suggest that amoA-containing Crenarchaea are more phylogenetically diverse than previously reported. Additionally, distributional patterns of planktonic Crenarchaea and Nitrospina species suggest potential metabolic interactions between these groups in the ocean's water column.     

Active sexual reproduction but no sign of genetic diversity in range-edge populations of Vanilla roscheri Rchb. f. (Orchidaceae) in South Africa

In South Africa, the wild leafless Vanilla roscheri Rchb. f. (Orchidaceae) is distributed only on the banks of the Lake Sibaya in KwaZulu-Natal Coastal Forest in the iSimangaliso Wetland Park. It is the southernmost vanilloid orchid on the African continent and a species of high conservation priority with little understanding of its reproductive biology and levels of genetic diversity. Mating systems and pollination ecology of the species were assessed by in situ experiments, in addition to genotyping plants at 16 microsatellite markers. Allogamous but self-compatible, V. roscheri depends on pollinators to achieve sexual reproduction. Its natural fruit set (26.3 %) is the highest reported for a non-spontaneously self-pollinating Vanilla species. It was associated with numerous flower visitors including two female allodapine bees (Allodapula variegata and Allodape rufogastra, Xylocopinae), captured with pollinia stored on the hind legs, and one female anthophorine bee. On the other hand, we report an absence of genetic diversity and homozygosity of the South-African populations of V. roscheri for all the genetic markers. Given the preferential outbreeding, the high natural fruit set and the pollinator/visitor richness of V. roscheri in Sibaya, a high level of genetic diversity was expected, but this was not the case. We detected a counterintuitive situation between the results of reproductive biology and genetics, making sense once the population situation on the margins of the species distribution is included. The null diversity expressed through the homozygous monomorphic markers is a consequence of the range-edge localization of the populations, which may have been subjected to severe bottlenecks (due to long distance colonization or fragmentation) along with inbreeding, in the past. The analysis highlights the complementarity of the approaches for which the implications in terms of conservation of the species in South Africa are discussed.     

In situ localized amplification and contact replication of many individual DNA molecules

We describe a method to clone and amplify DNA by performing the polymerase chain reaction (PCR) in a thin polyacrylamide film poured on a glass microscope slide. The polyacrylamide matrix retards the diffusion of the linear DNA molecules so that the amplification products remain localized near their respective templates. At the end of the reaction, a number of PCR colonies, or 'polonies', have formed, each one grown from a single template molecule. As many as 5 million clones can be amplified in parallel on a single slide. If an Acrydite modification is included at the 5' end of one of the primers, the amplified DNA will be covalently attached to the polyacrylamide matrix, allowing further enzymatic manipulations to be performed on all clones simultaneously. We describe techniques to make replicas of these polony slides, and high throughput sequencing protocols for this technology. Other applications are also discussed.     

Bovine thelaziasis in Iowa

The prevalence of developing Thelazia nematodes in face flies (Musca autumnalis) was studied for 7 yr at a beef farm in central Iowa. Juvenile nematodes were not found among flies in reproductive diapause in autumn, nor among nulliparous, overwintered flies in spring, but only among actively reproducing insects. Thus Thelazia probably do not overwinter in face flies. A mean prevalence of 2.0% infected was recorded among flies in 7 fly breeding seasons. No heterogeneity in Thelazia prevalence was detected within fly breeding seasons. The frequency distribution of Thelazia among face flies by year of occurrence was homogeneous, with a mean of 2.75 larval nematodes per infected fly. Thelazia gulosa and T. skrjabini were recovered in necropsy from the eyes of bovines from central Iowa. Prevalence among fat cattle 18-27 mo old was 15%; among cows 3-15 yr old, prevalence was only 3%. Thelazia skrjabini was found in 29 eyes and T. gulosa in 4 eyes. Infections were randomly distributed among the eyes of subject cattle. The mean worm burden was 2.3 T. skrjabini and 12 T. gulosa per infected eye. Thelaziasis is clearly enzootic in Iowa.     

The inactivation of alpha-1-proteinase inhibitor by gas-phase cigarette smoke: protection by antioxidants and reducing species

The abilities of a number of compounds of biological interest to protect alpha-1-proteinase inhibitor (alPI) against the loss of elastase inhibitory capacity (EIC) resulting from exposure to gas-phase cigarette smoke have been tested. We have identified several species that protect AlPI. Amino acids prevent the loss of EIC in a manner that correlates with their pK alpha-values; only the unprotonated amine provides protection. Catalase partially prevents the loss of EIC, suggesting that hydrogen peroxide produced from the reduction of oxygen in cigarette smoke extracts is responsible for at least some of the smoke-induced inactivation. The best protection against smoke-induced loss of EIC was provided by two biologically important antioxidant species: glutathione and ascorbic acid. Both species provide almost complete protection to alPI under the experimental conditions used. The nature of species that protect AlPI against the inactivation caused by exposure to gas-phase smoke provides clues upon which speculations about the mechanism of this inactivation may be based. The identification of protective species could lead to the development of compounds that smokers could take (for example, vitamin C) that would protect their lung tissue against the oxidative damage caused by cigarette smoke.     

The Roosting Times of the House Martin and Sand Martin

A quantitative, year-round habitat assessment has demonstrated the importance of gorse to insectivorous passerines on heathland.