Mating success and oviposition of a butterfly are not affected by non-lethal tissue sampling

Non-lethal methods of tissue sampling are increasingly used for genetic studies of insect species and the effects of this approach have long been assumed to be minimal. Tissue removal has the potential to influence insect reproductive behaviours such as mate recognition, courtship or oviposition but the effects of non-lethal sampling on reproductive success have not been widely and adequately tested. Here, we test potential effects of both wing-clipping and leg removal on reproductive behaviours of the cabbage white butterfly (Pieris rapae). We conducted a total of 93 male and 59 female mating trials, and found no significant differences in mating success between treated (i.e., tissue removed) and control individuals in either sex. We also monitored the number and location of eggs laid by 58 females. We found no significant differences in egg-laying behaviour among leg removed and control individuals. Power analysis indicated that we had sufficient statistical power to detect moderate effects of treatment on both mating and oviposition. Our study provides the most comprehensive examination to date of the effects of non-lethal sampling on reproductive behaviours in a butterfly/insect species, and supports the contention that tissue sampling is non-detrimental. To fully comprehend the general impacts of tissue sampling on butterfly reproductive behaviour however, additional similar studies need to be conducted on a variety of species with differing mating behaviours. Only through meta-analysis, may it be possible to detect more subtle effects of tissue removal which cannot be revealed within a single study due to sample size limitations.     

Apodemia mormo in Canada: population genetic data support prior conservation ranking

Like many species, the Mormon Metalmark butterfly (Apodemia mormo) has been given conservation ranking in Canada based on limited data. This species is widespread across western North America, but has only two populations in Canada: an “endangered” population in the Similkameen valley of British Columbia, and a “threatened” population in Grasslands National Park (GNP) in Saskatchewan. Here we present genetic data from 1498 base pairs of the cytochrome oxidase I gene sequence and six novel microsatellite loci in order to assess (1) whether the two populations are related, (2) the degree to which they are genetically diverse and demographically stable, and (3) what their relationships are to the nearest unranked populations of A. mormo across the Canada-United States border. Our principal conclusion is that the two populations are not closely related genetically. We also found that the British Columbia population is genetically depauperate and, with the exception of the nearest neighboring populations across the border, not recently genetically connected to other populations in the Pacific Northwest. In comparison, the Saskatchewan population is genetically diverse, and gene flow occurs with several other eastern populations. Population structure was not detected within either the British Columbia or the Saskatchewan populations. This research supports the prior conservation rankings of both populations and provides new insight that will help to inform future management decisions for the Canadian populations of this charismatic butterfly.     

Loss of the Cytoskeletal Protein Pdlim7 Predisposes Mice to Heart Defects and Hemostatic Dysfunction

The actin-associated protein Pdlim7 is essential for heart and fin development in zebrafish; however, the expression and function of this PDZ-LIM family member in the mammal has remained unclear. Here, we show that Pdlim7 predominantly localizes to actin-rich structures in mice including the heart, vascular smooth muscle, and platelets. To test the requirement for Pdlim7 in mammalian development and function, we analyzed a mouse strain with global genetic inactivation of Pdlim7. We demonstrate that Pdlim7 loss-of-function leads to significant postnatal mortality. Inactivation of Pdlim7 does not disrupt cardiac development, but causes mild cardiac dysfunction in adult mice. Adult Pdlim7 ^-/- mice displayed increased mitral and tricuspid valve annulus to body weight ratios. These structural aberrations in Pdlim7 ^-/- mice were supported by three-dimensional reconstructions of adult cardiac valves, which revealed increased surface area to volume ratios for the mitral and tricuspid valve leaflets. Unexpectedly, we found that loss of Pdlim7 triggers systemic venous and arterial thrombosis, leading to significant mortality shortly after birth in Pdlim7 ^+/- (11/60) and Pdlim7 ^-/- (19/35) mice. In line with a prothrombotic phenotype, adult Pdlim7 ^-/- mice exhibit dramatically decreased tail bleed times compared to controls. These findings reveal a novel and unexpected function for Pdlim7 in maintaining proper hemostasis in neonatal and adult mice.     

Chromosomal location of wheat genes of the carotenoid biosynthetic pathway and evidence for a catalase gene on chromosome 7A functionally associated with flour b* colour variation

Knowledge of molecular and genetic mechanisms controlling wheat grain quality characteristics is significant for improving flour for end-product functionality. Flour b* colour is an important quality trait for breeding wheat varieties to produce grain for specific market requirements. The degree of flour yellowness is due to the accumulation of carotenoids in grain, particularly lutein. Flour b* is under polygenic control and quantitative trait loci (QTL) have frequently been reported on chromosome 7AL. Analysis of carotenoid genes showed that phytoene synthase (PSY) co-located to the QTL on 7AL but other genes at this locus are also thought to contribute flour b* colour variation. This study used the wheat genome survey sequence and identified the chromosomal location of all wheat carotenoid genes, but none other than PSY were located on 7AL and, therefore, other genes may control flour b* colour variation including oxidative genes that degrade carotenoids. An investigation of EST bin mapped to 7AL identified a gene encoding a catalase enzyme (Cat3-A1) that was phylogenetically related to other plant class III enzymes, co-located to the QTL for flour b* colour variation on 7AL in three mapping populations and expressed during seed development. Therefore, Cat3-A1 was functionally associated with flour b* colour variation. Catalase acts upon hydrogen peroxide as a substrate and it was postulated that Cat3-A1 alleles control varying degrees of bleaching action on lutein in developing wheat grain. Markers for Cat3-A1 developed in this study can be used in conjunction with other candidate gene markers including phytoene synthase and lycopene-ε-cylase to develop a molecular signature for selecting lines with specific flour b* colour values in wheat breeding.     

Is Consumer Response to Plain/Standardised Tobacco Packaging Consistent with Framework Convention on Tobacco Control Guidelines? A Systematic Review of Quantitative Studies

Background and Objectives

Standardised or ‘plain’ tobacco packaging was introduced in Australia in December 2012 and is currently being considered in other countries. The primary objective of this systematic review was to locate, assess and synthesise published and grey literature relating to the potential impacts of standardised tobacco packaging as proposed by the guidelines for the international Framework Convention on Tobacco Control: reduced appeal, increased salience and effectiveness of health warnings, and more accurate perceptions of product strength and harm.

Methods

Electronic databases were searched and researchers in the field were contacted to identify studies. Eligible studies were published or unpublished primary research of any design, issued since 1980 and concerning tobacco packaging. Twenty-five quantitative studies reported relevant outcomes and met the inclusion criteria. A narrative synthesis was conducted.

Results

Studies that explored the impact of package design on appeal consistently found that standardised packaging reduced the appeal of cigarettes and smoking, and was associated with perceived lower quality, poorer taste and less desirable smoker identities. Although findings were mixed, standardised packs tended to increase the salience and effectiveness of health warnings in terms of recall, attention, believability and seriousness, with effects being mediated by the warning size, type and position on pack. Pack colour was found to influence perceptions of product harm and strength, with darker coloured standardised packs generally perceived as containing stronger tasting and more harmful cigarettes than fully branded packs; lighter coloured standardised packs suggested weaker and less harmful cigarettes. Findings were largely consistent, irrespective of location and sample.

Conclusions

The evidence strongly suggests that standardised packaging will reduce the appeal of packaging and of smoking in general; that it will go some way to reduce consumer misperceptions regarding product harm based upon package design; and will help make the legally required on-pack health warnings more salient.     

Optimization and Pharmacological Validation of a Leukocyte Migration Assay in Zebrafish Larvae for the Rapid In Vivo Bioactivity Analysis of Anti-Inflammatory Secondary Metabolites

Over the past decade, zebrafish (Danio rerio) have emerged as an attractive model for in vivo drug discovery. In this study, we explore the suitability of zebrafish larvae to rapidly evaluate the anti-inflammatory activity of natural products (NPs) and medicinal plants used in traditional medicine for the treatment of inflammatory disorders. First, we optimized a zebrafish assay for leukocyte migration. Inflammation was induced in four days post-fertilization (dpf) zebrafish larvae by tail transection and co-incubation with bacterial lipopolysaccharides (LPS), resulting in a robust recruitment of leukocytes to the zone of injury. Migrating zebrafish leukocytes were detected in situ by myeloperoxidase (MPO) staining, and anti-inflammatory activity was semi-quantitatively scored using a standardized scale of relative leukocyte migration (RLM). Pharmacological validation of this optimized assay was performed with a panel of anti-inflammatory drugs, demonstrating a concentration-responsive inhibition of leukocyte migration for both steroidal and non-steroidal anti-inflammatory drugs (SAIDs and NSAIDs). Subsequently, we evaluated the bioactivity of structurally diverse NPs with well-documented anti-inflammatory properties. Finally, we further used this zebrafish-based assay to quantify the anti-inflammatory activity in the aqueous and methanolic extracts of several medicinal plants. Our results indicate the suitability of this LPS-enhanced leukocyte migration assay in zebrafish larvae as a front-line screening platform in NP discovery, including for the bioassay-guided isolation of anti-inflammatory secondary metabolites from complex NP extracts.     

A benign approach to the preparation of freshwater bryozoan statoblasts for scanning electron microscopy (SEM) imaging

Abstract

Several different species of freshwater Bryozoa, belonging to the genera Plumatella, Rumarcanella and Fredericella, were detected within the Northern Mallee Pipeline (NMP) system in Victoria, Australia, that required definitive identification. These organisms produce asexual buds called statoblasts, with valves composed of sclerotised chitin that bear minute micro-ornamentations of considerable taxonomical significance. Imaging and analysis of these distinctive micro-ornamentations using scanning electron microscopy (SEM) is often employed for species identification. Meticulous preparation of statoblast samples is therefore required that necessitates the removal of adhering debris, dehydration and drying—whilst mitigating specimen damage and distortion. This technical note describes an approach whereby each of these three steps have been individually designed to be as benign as possible, using mild detergent/sonication to remove debris, a gradual and gentle dehydration procedure using ethanol, and critical point drying. For the overall process, these methods are chosen to optimise control and to minimise the use of harsh and hazardous chemicals.     

DISTRIBUTION,POPULATION SIZE AND CONSERVATION OF HARTLAUB'S GULL LARUS HARTLAUBIL

Williams, A. J., Steele, W. K., Cooper, J. & Crawford, R. J. M. 1990. Distribution, population size and conservation of Hartlaub's Gull Lorus hurtlaubii. Ostrich 61: 66–76.

Hartlaub's Gull Larus hartlaubii is endemic to southern Africa, where it breeds between Swakopmund, Namibia and Dyer Island, southwestern Cape Province, South Africa. The species has been re breeding at 48 localities within this range. Between 1984 and 1989 an estimated 12000 pain brered at 31 localities. Twenty-eet percent of the population breeds at Robben Island off the Cape Peninsula, sQuth Africa. Hartlaub's Gull frequently has low breeding success and is considered endangered in Narmbia, where 12% of the poulation occurs. However, the population is increaslng around the urbanmd Cape Peninsula where HartLub's Gull has the potential to become a pest species.     

Activation of the Endoplasmic Reticulum Calcium Sensor STIM1 and Store-Operated Calcium Entry by Rotavirus Requires NSP4 Viroporin Activity

Rotavirus nonstructural protein 4 (NSP4) induces dramatic changes in cellular calcium homeostasis. These include increased endoplasmic reticulum (ER) permeability, resulting in decreased ER calcium stores and activation of plasma membrane (PM) calcium influx channels, ultimately causing a 2- to 4-fold elevation in cytoplasmic calcium. Elevated cytoplasmic calcium is absolutely required for virus replication, but the underlying mechanisms responsible for calcium influx remain poorly understood. NSP4 is an ER-localized viroporin, whose activity depletes ER calcium, which ultimately leads to calcium influx. We hypothesized that NSP4-mediated depletion of ER calcium activates store-operated calcium entry (SOCE) through activation of the ER calcium sensor stromal interaction molecule 1 (STIM1). We established and used a stable yellow fluorescent protein-expressing STIM1 cell line (YFP-STIM1) as a biosensor to assess STIM1 activation (puncta formation) by rotavirus infection and NSP4 expression. We found that STIM1 is constitutively active in rotavirus-infected cells and that STIM1 puncta colocalize with the PM-localized Orai1 SOCE calcium channel. Expression of wild-type NSP4 activated STIM1, resulting in PM calcium influx, but an NSP4 viroporin mutant failed to induce STIM1 activation and did not activate the PM calcium entry pathway. Finally, knockdown of STIM1 significantly reduced rotavirus yield, indicating STIM1 plays a critical role in virus replication. These data demonstrate that while rotavirus may ultimately activate multiple calcium channels in the PM, calcium influx is predicated on NSP4 viroporin-mediated activation of STIM1 in the ER. This is the first report of viroporin-mediated activation of SOCE, reinforcing NSP4 as a robust model to understand dysregulation of calcium homeostasis during virus infections.     

Divergence in coloration and ecological speciation in the Anolis marmoratus species complex

Adaptive divergence in coloration is expected to produce reproductive isolation in species that use colourful signals in mate choice and species recognition. Indeed, many adaptive radiations are characterized by differentiation in colourful signals, suggesting that divergent selection acting on coloration may be an important component of speciation. Populations in the Anolis marmoratus species complex from the Caribbean island of Guadeloupe display striking divergence in the colour and pattern of adult males that occurs over small geographic distances, suggesting strong divergent selection. Here we test the hypothesis that divergence in coloration results in reduced gene flow among populations. We quantify variation in adult male coloration across a habitat gradient between mesic and xeric habitats, use a multilocus coalescent approach to infer historical demographic parameters of divergence, and examine gene flow and population structure using microsatellite variation. We find that colour variation evolved without geographic isolation and in the face of gene flow, consistent with strong divergent selection and that both ecological and sexual selection are implicated. However, we find no significant differentiation at microsatellite loci across populations, suggesting little reproductive isolation and high levels of contemporary gene exchange. Strong divergent selection on loci affecting coloration probably maintains clinal phenotypic variation despite high gene flow at neutral loci, supporting the notion of a porous genome in which adaptive portions of the genome remain fixed whereas neutral portions are homogenized by gene flow and recombination. We discuss the impact of these findings for studies of colour evolution and ecological speciation.