Examining the phylogeny of the Australasian Lymnaeidae (Heterobranchia: Pulmonata: Gastropoda) using mitochondrial, nuclear and morphological markers

We examined the species groups relationships of the freshwater snail genus Austropeplea using mitochondrial, nuclear and morphological markers in addition to traditional methods of shell shape analysis. Based primarily on the results of a combined molecular and morphological analysis, samples of the nominal species A. tomentosa form distinct lineages. The New Zealand populations of A. tomentosa are a very distinct lineage from any of the Australian populations attributed to A. tomentosa. Furthermore, within the Australian group, three lineages, south Australia, Tasmania and eastern Australia, appear to have undergone recent and/or rapid speciation events. Samples assigned to A. lessoni were resolved as two distinct lineages, representing the eastern and northern Australian populations. Kutikina hispida was resolved within the Australian A. tomentosa clade. Molecular results for A. viridis suggests that it is also composed of at least two distinct lineages that could be treated as species. Incongruence observed between the single mitochondrial, nuclear and morphological topologies highlight the importance of using a number of different datasets in the delimitation of species-group taxa.     

Long‐term stability of entomopathogenic nematode spatial patterns in soil as measured by sentinel insects and real‐time PCR assays

Quantitative real‐time PCR (qPCR) techniques are being increasingly used to provide accurate and reliable methods to identify and quantify cryptic organisms in soil ecology. Entomopathogenic nematode (EPN) diversity in Florida is known to be extensive and our phylogenetic studies of the D2D3 and ITS regions showed the occurrence of an additional species‐complex in the Steinernema glaseri‐ group in widely separated locations of the peninsula. To address ecological studies, we developed and used qPCR assays to detect and quantify six species of EPN that are naturally distributed in Florida citrus orchards (Steinernema diaprepesi, Steinernema riobrave, Heterorhabditis indica, Heterorhabditis zealandica, Heterorhabditis floridensis and an undescribed species in the S. glaseri group) and an exotic species, S. glaseri. Species‐specific primers and TaqMan® probes were designed from the ITS rDNA region. No nonspecific amplification was observed in conventional or qPCR when the primers and probes were tested using several populations of each of the Florida species and other exotic EPN species. Standard curves were established using DNA from pure cultures. We optimised a protocol for extracting nematodes and DNA from soil samples that can detect one EPN added to nematode communities recovered by conventional extraction protocols. A survey of an 8‐ha orchard in April 2009 compared the EPN spatial patterns derived from qPCR to that obtained by baiting soil samples with Galleria mellonella larvae. The patterns were also compared to those derived from the same site in 2000–01 by repeatedly (12 sampling events) baiting soil in situ with caged larvae of the root weevil Diaprepes abbreviatus. The qPCR assay was more efficient than the Galleria baiting method for detecting the EPN species composition in population mixtures. Moreover, the spatial patterns of EPN in this orchard were remarkably stable over the course of nearly a decade. The pattern of H. zealandica detected at the site 8 years earlier was related to those derived by qPCR (P = 0.002) and from sample baiting (P = 0.02). The spatial pattern of H. indica derived from qPCR, but not that from sample baiting, was also related to the earlier pattern (P = 0.01). The qPCR assay developed here is a fast, affordable and accurate method to detect and quantify these EPN species in soil and offers great potential for studying the ecology of EPN.     

The TGV transgenic vectors for single-copy gene expression from the Escherichia coli chromosome

Plasmid-based cloning and expression of genes in Escherichia coli can have several problems: plasmid destabilization; toxicity of gene products; inability to achieve complete repression of gene expression; non-physiological overexpression of the cloned gene; titration of regulatory proteins; and the requirement for antibiotic selection. We describe a simple system for cloning and expression of genes in single copy in the E. coli chromosome, using a non-antibiotic selection for transgene insertion. The transgene is inserted into a vector containing homology to the chromosomal region flanking the attachment site for phage lambda. This vector is then linearized and introduced into a recombination-proficient E. coli strain carrying a temperature-sensitive lambda prophage. Selection for replacement of the prophage with the transgene is performed at high temperature. Once in the chromosome, transgenes can be moved into other lysogenic E. coli strains using standard phage-mediated transduction techniques, selecting against a resident prophage. Additional vector constructs provide an arabinose-inducible promoter (P(BAD)), P(BAD) plus a translation-initiation sequence, and optional chloramphenicol-, tetracycline-, or kanamycin-resistance cassettes. These Transgenic E. coli Vectors (TGV) allow drug-free, single-copy expression of genes from the E. coli chromosome, and are useful for genetic studies of gene function.     

Difficulties in location and acceptance of phloem sap combined with reduced concentration of phloem amino acids explain lowered performance of the aphid Rhopalosiphum padi on nitrogen deficient barley (Hordeum vulgare) seedlings

Effects of nitrogen deficiency in hydroponically grown barley seedlings (Hordeum vulgare L.) on the development and reproduction of the aphid Rhopalosiphum padi (L.) (Hemiptera: Aphididae) were investigated.Plant growth was significantly reduced in seedlings grown without nitrogen. Aphid intrinsic rate of increase (r _m) was also significantly lower on these plants compared with that on plants grown with 8 mol m^–3 nitrogen. Phloem sap was collected from seedling stems by aphid stylectomy and amino acids quantified by HPLC. There was a significant reduction in the concentration of non-essential amino acids as a group, but not of essential amino acids. Electrical penetration graphs (EPG) indicated that aphids reached the phloem more quickly and fed for longer on plants grown with nitrogen. This is the first reported study in which this combination of techniques has been used to understand the interactions of an aphid and plant under different environmental conditions.     

Sentinel systems on the razor's edge: effects of warming on Arctic geothermal stream ecosystems

The Earth is experiencing historically unprecedented rates of warming, with surface temperatures projected to increase by 3–5 °C globally, and up to 7.5 °C in high latitudes, within the next century. Knowledge of how this will affect biological systems is still largely restricted to the lower levels of organization (e.g. species range shifts), rather than at the community, food web or ecosystem level, where responses cannot be predicted from studying single species in isolation. Further, many correlational studies are confounded with time and/or space, whereas experiments have been mostly confined to laboratory microcosms that cannot capture the true complexity of natural ecosystems. We used a ‘natural experiment’ in an attempt to circumvent these shortcomings, by characterizing community structure and trophic interactions in 15 geothermal Icelandic streams ranging in temperature from 5 °C to 45 °C. Even modest temperature increases had dramatic effects across multiple levels of organization, from changes in the mean body size of the top predators, to unimodal responses of species populations, turnover in community composition, and lengthening of food chains. Our results reveal that the rates of warming predicted for the next century have serious implications for the structure and functioning of these fragile ‘sentinel’ ecosystems across multiple levels of organization.     

Synthesis,Biological Evaluation,and Molecular Docking of Novel Thiazoles and [1,3,4]Thiadiazoles Incorporating Sulfonamide Group as DHFR Inhibitors

2‐(1‐{4‐[(4‐Methylphenyl)sulfonamido]phenyl}ethylidene)thiosemicarbazide ( 3 ) was exploited as a starting material for the synthesis of two novel series of 5‐arylazo‐2‐hydrazonothiazoles 6a  –  6j and 2‐hydrazono[1,3,4]thiadiazoles 10a  –  10d , incorporating sulfonamide group, through its reactions with appropriate hydrazonoyl halides. The structures of the newly synthesized products were confirmed by spectral and elemental analyses. Also, the antimicrobial, anticancer, and DHFR inhibition potency for two series of thiazoles and [1,3,4]thiadiazoles were evaluated and explained by molecular docking studies and SAR analysis.     

Haplotype and phenotype analysis of six recurrent BRCA1 mutations in 61 families: results of an international study.

Several BRCA1 mutations have now been found to occur in geographically diverse breast and ovarian cancer families. To investigate mutation origin and mutation-specific phenotypes due to BRCA1, we constructed a haplotype of nine polymorphic markers within or immediately flanking the BRCA1 locus in a set of 61 breast/ovarian cancer families selected for having one of six recurrent BRCA1 mutations. Tests of both mutations and family-specific differences in age at diagnosis were not significant. A comparison of the six mutations in the relative proportions of cases of breast and ovarian cancer was suggestive of an effect (P = .069), with 57% of women presumed affected because of the 1294 del 40 BRCA1 mutation having ovarian cancer, compared with 14% of affected women with the splice-site mutation in intron 5 of BRCA1. For the BRCA1 mutations studied here, the individual mutations are estimated to have arisen 9-170 generations ago. In general, a high degree of haplotype conservation across the region was observed, with haplotype differences most often due to mutations in the short-tandem-repeat markers, although some likely instances of recombination also were observed. For several of the instances, there was evidence for multiple, independent, BRCA1 mutational events.     

An Evaluation of Genetic Heterogeneity in 145 Breast-Ovarian Cancer Families

The breast-ovary cancer–family syndrome is a dominant predisposition to cancer of the breast and ovaries which has been mapped to chromosome region 17ql2-q21. The majority, but not all, of breast-ovary cancer families show linkage to this susceptibility locus, designated BRCA1. We report here the results of a linkage analysis of 145 families with both breast and ovarian cancer. These families contain either a total of three or more cases of early-onset (before age 60 years) breast cancer or ovarian cancer. All families contained at least one case of ovarian cancer. Overall, an estimated 76% of the 145 families are linked to the BRCA1 locus. None of 13 families with cases of male breast cancer appear to be linked, but it is estimated that 92% (95% confidence interval 76%–100%) of families with no male breast cancer and with two or more ovarian cancers are linked to BRCA1. These data suggest that the breast-ovarian cancer–family syndrome is genetically heterogeneous. However, the large majority of families with early-onset breast cancer and with two or more cases of ovarian cancer are likely to be due to BRCA1 mutations.