Directly labelling locus‐specific primers for microsatellite analysis is expensive and a common limitation to small‐budget molecular ecology projects. More cost‐effective end‐labelling of PCR ...products can be achieved through a three primer PCR approach, involving a fluorescently labelled universal primer in combination with modified locus‐specific primers with 5′ universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of available universal primers suitable for co‐amplifying large numbers of size overlapping loci and without requiring locus‐specific PCR conditions to be modified. In this study, we report a suite of four high‐performance universal primers that can be employed in a three primer PCR approach for efficient and cost‐effective fluorescent end‐labelling of PCR fragments. Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ °C) that enhance primer versatility and enable higher annealing temperatures to be employed compared with commonly used universal primers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co‐amplify multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single and multiplex PCR across a wide range of taxa.
In an exploratory, block-randomised, parallel, double-blind, single-centre, placebo-controlled superiority study (ISRCTN12562026, funded by Cultech Ltd), 220 Bulgarian participants (30 to 65 years ...old) with BMI 25-34.9 kg/m
received Lab4P probiotic (50 billion/day) or a matched placebo for 6 months. Participants maintained their normal diet and lifestyle. Primary outcomes were changes in body weight, BMI, waist circumference (WC), waist-to-height ratio (WtHR), blood pressure and plasma lipids. Secondary outcomes were changes in plasma C-reactive protein (CRP), the diversity of the faecal microbiota, quality of life (QoL) assessments and the incidence of upper respiratory tract infection (URTI). Significant between group decreases in body weight (1.3 kg, p < 0.0001), BMI (0.045 kg/m
, p < 0.0001), WC (0.94 cm, p < 0.0001) and WtHR (0.006, p < 0.0001) were in favour of the probiotic. Stratification identified greater body weight reductions in overweight subjects (1.88%, p < 0.0001) and in females (1.62%, p = 0.0005). Greatest weight losses were among probiotic hypercholesterolaemic participants (-2.5%, p < 0.0001) alongside a significant between group reduction in small dense LDL-cholesterol (0.2 mmol/L, p = 0.0241). Improvements in QoL and the incidence rate ratio of URTI (0.60, p < 0.0001) were recorded for the probiotic group. No adverse events were recorded. Six months supplementation with Lab4P probiotic resulted in significant weight reduction and improved small dense low-density lipoprotein-cholesterol (sdLDL-C) profiles, QoL and URTI incidence outcomes in overweight/obese individuals.
Insecticide resistance evolves extremely rapidly, providing an illuminating model for the study of adaptation. With climate change reshaping species distribution, pest and disease vector control ...needs rethinking to include the effects of environmental variation and insect stress physiology. Here, we assessed how both long‐term adaptation of populations to temperature and immediate temperature variation affect the genetic architecture of DDT insecticide response in Drosophila melanogaster. Mortality assays and behavioural assays based on continuous activity monitoring were used to assess the interaction between DDT and temperature on three field‐derived populations from climate extremes (Raleigh for warm temperate, Tasmania for cold oceanic and Queensland for hot tropical). The Raleigh population showed the highest mortality to DDT, whereas the Queensland population, epicentre for derived alleles of the resistance gene Cyp6g1, showed the lowest. Interaction between insecticide and temperature strongly affected mortality, particularly for the Tasmanian population. Activity profiles analysed using self‐organizing maps show that the insecticide promoted an early response, whereas elevated temperature promoted a later response. These distinctive early or later activity phases revealed similar responses to temperature and DDT dose alone but with more or less genetic variance depending on the population. This change in genetic variance among populations suggests that selection particularly depleted genetic variance for DDT response in the Queensland population. Finally, despite similar (co)variation between traits in benign conditions, the genetic responses across population differed under stressful conditions. This showed how stress‐responsive genetic variation only reveals itself in specific conditions and thereby escapes potential trade‐offs in benign environments.
RNA interference (RNAi) technology is being developed as a weapon for pest insect control. To maximize the specificity that such an approach affords we have developed a bioinformatic web tool that ...searches the ever-growing arthropod transcriptome databases so that pest-specific RNAi sequences can be identified. This will help technology developers finesse the design of RNAi sequences and suggests which non-target species should be assessed in the risk assessment process.
http://rnai.specifly.org
crobin@unimelb.edu.au.
The apicoplast and mitochondrion of the malaria parasite
Plasmodium falciparum are important intracellular organelles and targets of several anti-malarial drugs. In recent years, our group and others ...have begun to piece together the metabolic pathways of these organelles, with a view to understanding their functions and identifying further anti-malarial targets. This has involved localization of putative organellar proteins using fluorescent reporter proteins such as green fluorescent protein (GFP). A major limitation to such an approach is the difficulties associated with using existing plasmids to genetically modify
P. falciparum. In this paper, we present a novel series of
P. falciparum transfection vectors based around the Gateway™ recombinatorial cloning system. Our system makes it considerably easier to construct fluorescent reporter fusion proteins, as well as allowing the use of two selectable markers. Using this approach, we localize proteins involved in isoprenoid biosynthesis and the posttranslational processing of apicoplast-encoded proteins to the apicoplast, and a protein putatively involved in the citric acid cycle to the mitochondrion. To confirm the localization of these proteins, we have developed a new immunofluorescence assay (IFA) protocol using antibodies specific to the apicoplast and mitochondrion. In comparison with published IFA methods, we find that ours maintains considerably better structural preservation, while still allowing sufficient antibody binding as well as preserving reporter protein fluorescence. In summary, we present two important new tools that have enabled us to characterize some of the functions of the apicoplast and mitochondrion, and which will be of use to the wider malaria research community in elucidating the localization of other
P. falciparum proteins.
Over the last 40 years, many types of population genetic markers have been used to assess the population structure of the pest moth species Helicoverpa armigera. While this species is highly vagile, ...there is evidence of inter-continental population structure. Here, we examine Z-chromosome molecular markers within and between Chinese and Australian populations. Using 1352 polymorphic sites from 40 Z-linked loci, we compared two Chinese populations of moths separated by 700 km and found virtually no population structure (n = 41 and n = 54, with <1% of variation discriminating between populations). The levels of nucleotide diversity within these populations were consistent with previous estimates from introns in Z-linked genes of Australian samples (π = 0.028 vs. 0.03). Furthermore, all loci surveyed in these Chinese populations showed a skew toward rare variants, with ten loci having a significant Tajima's D statistic, suggesting that this species could have undergone a population expansion. Eight of the 40 loci had been examined in a previous study of Australian moths, of which six revealed very little inter-continental population structure. However, the two markers associated with the Cyp303a1 locus that has previously been proposed to be a target of a selective sweep, exhibited allele structuring between countries. Using a separate dataset of 19 Australian and four Chinese moths, we scanned the molecular variation distributed across the entire Z-chromosome and found distinct blocks of differentiation that include the region containing Cyp303a1. We recommend some of these loci join those associated with insecticide resistance to form a set of genes best suited to analyzing population structure in this global pest.
WD 1953-011 is an isolated, cool (7920 ± 200 K) magnetic white dwarf (MWD) with a low average field strength (∼70 kG), and a higher than average mass (∼0.74 M⊙). Spectroscopic observations taken by ...Maxted et al. showed variations of equivalent width in the Balmer lines, unusual in a low-field white dwarf. Here we present V-band photometry of WD 1953-011 taken at seven epochs over a total of 22 months. All of the data sets show a sinusoidal variation of approximately 2 per cent peak-to-peak amplitude. We propose that these variations are due to a star-spot on the MWD, analogous to a sunspot, which is affecting the temperature at the surface, and therefore its photometric magnitude. The variations have a best-fitting period over the entire 22 months of 1.4418 d, which we interpret as the rotational period of the white dwarf.
The recognition and invasion of human erythrocytes by the most lethal malaria parasite Plasmodium falciparum is dependent on multiple ligand-receptor interactions. Members of the erythrocyte ...binding-like (ebl) family, including the erythrocyte binding antigen-175 (EBA-175), are responsible for high affinity binding to glycoproteins on the surface of the erythrocyte. Here we describe a paralogue of EBA-175 and show that this protein (EBA-181/JESEBL) binds in a sialic acid-dependent manner to erythrocytes. EBA-181 is expressed at the same time as EBA-175 and co-localizes with this protein in the microneme organelles of asexual stage parasites. The receptor binding specificity of EBA-181 to erythrocytes differs from other members of the ebl family and is trypsin-resistant and chymotrypsin-sensitive. Furthermore, using glycophorin B-deficient erythrocytes we show that binding of EBA-181 is not dependent on this sialoglycoprotein. The level of expression of EBA-181 differs among parasite lines, and the importance of this ligand for invasion appears to be strain-dependent as the EBA-181 gene can be disrupted in W2mef parasites, without affecting the invasion phenotype, but cannot be targeted in 3D7 parasites.
Genetic polymorphisms underlying adaptive shifts in thermal responses are poorly known even though studies are providing a detailed understanding of these responses at the cellular and physiological ...levels. The Frost gene of Drosophila melanogaster is a prime candidate for thermal adaptation; it is up‐regulated under cold stress and knockdown of this gene influences cold resistance. Here we describe an amino‐acid INDEL polymorphism in proline repeat number in the structural component of this gene. The two main repeats, accounting for more than 90% of alleles in eastern Australia, show a strong clinal pattern; the 6P allele was at a high frequency in tropical locations, and the 10P allele was common in temperate populations. However, the frequency of these alleles was not associated with three different assays of cold resistance. Adult transcription level of Frost was also unrelated to cold resistance as measured through post chill coma mobility. The functional significance of the proline repeat polymorphism therefore remains unclear despite its clinal pattern. The data also demonstrate the feasibility of using Roche/454 sequencing for establishing clinal patterns.
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