USE OF POLYMYXIN B IN A CAPTURE ENZYME IMMUNOASSAY FOR DETECTION OF SALMONELLAE SPP. LIPOPOLYSACCHARIDE

A capture enzyme immunoassay for detection of salmonellae sp. lipopolysaccharide was developed. The assay made use of polymyxin B sulfate, passively attached to a polystyrene matrix, to capture lipopolysaccharide. Bound lipopolysaccharride was then detected with a monoclonal antibody, specific for salmonellae spp. followed by goat antimouse antibody conjugated with horseradish peroxidase.
The analytical sensitivity of the assay was approximately 1 ng/ml of lipopolysaccharide. The results are comparable to those obtained with a competitive enzyme immunoassay previously developed. The sensitivity of the polymyxin B assay decreased to 4–5 ng/ml when the salmonellae spp. lipopolysaccharide was mixed with 1–100 μg/ml of Escherichia coli lipopolysaccharide, while this level of heterogeneous lipopolysaccharide, did not decrease the sensitivity of the competitive enzyme immunoassay.
The polymyxin B capture assay was advantageous in that polymyxin B is a standardized reagent that is relatively inexpensive and does not require extensive preparation or containment facilities. The assay is robust; however, because of the light sensitivity of polymyxin B, its stickiness to other reagents and interference by other lipopolysaccharides, this assay requires careful attention to detail and may therefore be an unsuitable assay for field use.     

Composition and implications of diverse lipids in New Zealand Geothermal sinters

Microbial adaptations associated with extreme growth environments, including high temperatures and low pH, are of interest to astrobiologists and origin of life researchers. As part of a survey of microbial lipids present in terrestrial geothermal settings, we examined four silica sinters associated with three different hot spring areas of the Taupo Volcanic Zone (TVZ), New Zealand. Dominant bacterial lipids include free fatty acids, 1,2‐diacylglycerophospholipids, 1,2‐di‐O‐alkylglycerols, 1‐O‐alkylglycerols, wax esters, alkanols, alkan‐1,2‐diols and various hopanoids, whereas dominant archaeal lipids include both archaeol and glycerol dialkyl glycerol tetraethers. Although many of these compounds occur in other settings, in the TVZ sinters their distributions (with high abundances of β‐OH fatty acids and high‐molecular‐weight (> C₁₈) fatty acyl components) and carbon isotopic compositions (ranging from ?40 to +4, with up to 25 variability in a single sample) are unusual. In addition, we have identified a range of unusual compounds, including novel macrocyclic diethers and hopanoids. The distributions of these compounds differ among the study sites, suggesting that, where preserved in ancient sinters, they could serve as an important tool in studying past hydrothermal environments.     

Molecules,morphology and minute hooded beetles: a phylogenetic study with implications for the evolution and classification of Corylophidae (Coleoptera: Cucujoidea)

Eight genes (nuclear: 18S, 28S, H3, CAD; mitochondrial: 12S, 16S, COI, COII) and morphology were used to infer the evolutionary history of Corylophidae, some of the smallest free‐living insects. The study included 36 corylophid exemplars, representing approximately 60% of the known generic diversity of the family and 16 cucujoid outgroup taxa. Multiple partitioning strategies, molecular datasets, combined datasets and different taxon sampling regimes using maximum likelihood and mixed‐model Bayesian inference were utilized to analyse these data. Most results were highly concordant across analyses. There was strong agreement across (i) partitioning strategies, (ii) maximum likelihood and Bayesian inference analyses of the molecular data, and (iii) Bayesian inference of the molecular data alone and Bayesian inference of the combined morphological and molecular data when all terminal taxa were included. When a strict taxon sampling protocol was employed so that only single generic exemplars were included, deep relationships were affected in the resulting phylogenetic hypotheses. Under such narrow sampling strategies, deep phylogenetic relationships were also sensitive to the choice of generic exemplars. Although it is often challenging to obtain single representatives for many taxa in higher‐level phylogenetic analyses, these results indicate the importance of using denser taxon sampling approaches even at the specific level for genera included in such studies. Molecular data alone support Anamorphinae (Endomychidae) strongly as the sister group of Corylophidae. In combined data analyses, Coccinellidae is recovered as the sister group to Corylophidae. In all analyses, Corylophidae and the subfamily Corylophinae are recovered as monophyletic. The monophyly of Periptyctinae was untested, as only a single species was included. All included corylophine tribes were recovered as monophyletic with the exception of Aenigmaticini; Aenigmaticum Matthews forms the sister group to Orthoperus Stephens and Stanus?lipiński et al. is recovered as the sister group of Sericoderus Stephens. Stanus tasmanicus?lipiński et al. is transferred to a new genus, Pseudostanus Robertson, ?lipiński & McHugh gen.n. incertae sedis. We propose a new tribe, Stanini Robertson, ?lipiński & McHugh trib.n. for Stanus bowesteadi?lipiński et al. and a new concept of Aenigmaticini sensu.n. to include only the nominate genus. Anatomical transitions associated with corylophid miniaturization are highlighted. Key phenotypic modifications and elevated rates of substitution in nuclear rRNA genes are evident in a subgroup of Corylophinae that includes the most diminutive species. Other taxonomic and evolutionary implications are discussed in light of the results.     

Population structure in the catfish Trichogenes longipinnis: drift offset by asymmetrical migration in a tiny geographic range

Based on population genetic theory and empirical studies of small populations, we expect that species with very small ranges (narrow endemics) will exhibit reduced genetic diversity, increasing their susceptibility to the negative effects of genetic homogeneity. Although this pattern of reduced diversity applies to most narrow endemics, conservation biologists have yet to identify a general pattern for the degree of spatial population genetic structure expected in species with very small ranges. In part, this is because the degree of population structure within narrow endemics will be highly variable depending on the equilibrium between the homogenizing effects of dispersal and the diversifying effects of drift and local selection in small populations, thus precluding general predictions about the relative importance of small range, small population sizes, and habitat patchiness for maintaining genetic diversity in narrowly-distributed species. We document a striking example of high population structure in the tiny geographic range of a stream-dwelling catfish, Trichogenes longipinnis , endemic to the Atlantic Forest of Brazil. The maintenance of this diversity results from a combination of asymmetrical and limited dispersal, and drift in small populations. Our results highlight the need to understand population structure, and not only overall genetic diversity, of narrowly-distributed species for their conservation planning.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 259–274.     

Microsatellite DNA markers for population genetic studies in the dinoflagellate Karenia brevis

Nine nuclear‐encoded microsatellites from an enriched genomic DNA library of the HAB (harmful algal bloom) dinoflagellate Karenia brevis were isolated and characterized. The microsatellites include five perfect (three dinucleotide and two trinucleotide) and four imperfect (two dinucleotide and two trinucleotide) repeat motifs. Gene (haplotype) diversity ranged from 0.153 to 0.750 among a sample of 13 isolates; the number of alleles among the isolates ranged from two to six and pairwise tests of genotypic disequilibria were nonsignificant. The microsatellites developed in this study will provide insight into the genetic diversity of this HAB species and tools that may prove useful in predicting source populations and physiological parameters of individual K. brevis blooms.     

Shed skin as a source of DNA for genotyping seals

Obtaining a sufficient number of DNA samples from ice‐breeding marine phocids, in a noninvasive manner, has proven difficult and has limited the ability to use molecular genetics on these species. We evaluate the ability to genotype ringed seals using a novel source of DNA, skin cells shed by the seal as it moults on sea ice. We found that shed skin samples yielded a lower quantity and purity of DNA compared to tissue samples. Nevertheless, the shed skin cells were a viable source of DNA for microsatellite analysis; we found no significant difference in allelic diversity or heterozygosities between tissue samples and shed skin cells. This source of DNA should allow the rapid collection of a large number of noninvasively collected DNA samples in ice‐breeding phocids.