Seed Transmission of Broad Bean Stain Virus in the Wild Legume Vicia Palestina Boiss

Abstract Seed-transmissibility of brood bean stain virus (BBSV) was investigated in a number of wild legume species. Genninating axes of seeds coliected from BBSV -infected plants were tested by the enzyme-linked immunosorbent assay (ELISA). The virus was found to be seedtransmitted in Vida pal«stina.

“Who decides?” Security and privacy in the wild

Even though web security protocols are designed to make computer communication secure, it is widely known that there is potential for security breakdowns at the human-machine interface. This paper examines findings from a qualitative study investigating the identification of security decisions used on the web. The study was designed to uncover how security is perceived in an individual user's context. Study participants were tertiary qualified individuals, with a focus on HCI designers, security professionals and the general population. The study identifies that security frameworks for the web are inadequate from an interaction perspective, with even tertiary qualified users having a poor or partial understanding of security, of which they themselves are acutely aware. The result is that individuals feel they must protect themselves on the web. The findings contribute a significant mapping of the ways in which individuals reason and act to protect themselves on the web. We use these findings to highlight the need to design for trust at three levels, and the need to ensure that HCI design does not impact on the users' main identified protection mechanism: separation.

The N170 observed ‘in the wild’: robust event-related potentials to faces in cluttered dynamic visual scenes

As a social species in a constantly changing environment, humans rely heavily on the informational richness and communicative capacity of the face. Thus, understanding how the brain processes information about faces in real-time is of paramount importance. The N170 is a high temporal resolution electrophysiological index of the brain's early response to visual stimuli that is reliably elicited in carefully controlled laboratory-based studies. Although the N170 has often been reported to be of greatest amplitude to faces, there has been debate regarding whether this effect might be an artifact of certain aspects of the controlled experimental stimulation schedules and materials. To investigate whether the N170 can be identified in more realistic conditions with highly variable and cluttered visual images and accompanying auditory stimuli we recorded EEG 'in the wild', while participants watched pop videos. Scene-cuts to faces generated a clear N170 response, and this was larger than the N170 to transitions where the videos cut to non-face stimuli. Within participants, wild-type face N170 amplitudes were moderately correlated to those observed in a typical laboratory experiment. Thus, we demonstrate that the face N170 is a robust and ecologically valid phenomenon and not an artifact arising as an unintended consequence of some property of the more typical laboratory paradigm.

Mapping quantitative trait loci in a wild population using linkage and linkage disequilibrium analyses

Historical information can be used, in addition to pedigree, traits and genotypes, to map quantitative trait locus (QTL) in general populations via maximum likelihood estimation of variance components. This analysis is known as linkage disequilibrium (LD) and linkage mapping, because it exploits both linkage in families and LD at the population level. The search for QTL in the wild population of Soay sheep on St. Kilda is a proof of principle. We analysed the data from a previous study and confirmed some of the QTLs reported. The most striking result was the confirmation of a QTL affecting birth weight that had been reported using association tests but not when using linkage-based analyses. Copyright © Cambridge University Press 2010.

Formation of specialized propagules resistant to desiccation and cryopreservation in the threatened moss Ditrichum plumbicola (ditrichales, bryopsida)

Background and Aims Successful cryopreservation of bryophytes is linked to intrinsic desiccation tolerance and survival can be enhanced by pre-treatment with abscisic acid (ABA) and sucrose. The pioneer moss Ditrichum plumbicola is naturally subjected to desiccation in the field but showed unexpectedly low survival of cryopreservation, as well as a poor response to pre-treatment. The effects of the cryopreservation protocol on protonemata of D. plumbicola were investigated in order to explore possible relationships between the production in vitro of cryopreservation-tolerant asexual propagules and the reproductive biology of D. plumbicola in nature. Methods Protonemata were prepared for cryopreservation using a four-step protocol involving encapsulation in sodium alginate, pre-treatment for 2 weeks with ABA and sucrose, desiccation for 6 h and rapid freezing in liquid nitrogen. After each stage, protonemata were prepared for light and electron microscopy and growth on standard medium was monitored. Further samples were prepared for light and electron microscopy at intervals over a 24-h period following removal from liquid nitrogen and re-hydration. Key Results Pre-treatment with ABA and sucrose caused dramatic changes to the protonemata. Growth was arrested and propagules induced with pronounced morphological and cytological changes. Most cells died, but those that survived were characterized by thick, deeply pigmented walls, numerous small vacuoles and lipid droplets in their cytoplasm. Desiccation and cryopreservation elicited no dramatic cytological changes. Cells returned to their pre-dehydration and cryopreservation state within 2 h of re-hydration and/or removal from liquid nitrogen. Regeneration was normal once the ABA/sucrose stimulus was removed. Conclusions The ABA/sucrose pre-treatment induced the formation of highly desiccation- and cryopreservation-tolerant propagules from the protonemata of D. plumbicola. This parallels behaviour in the wild, where highly desiccation-tolerant rhizoids function as perennating organs allowing the moss to endure extreme environmental conditions. An involvement of endogenous ABA in the desiccation tolerance of D. plumbicola is suggested.

Protein complementation assay as a display system for screening protein libraries in the intracellular environment

A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the β-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) *Escherichia coli* proteins were used as model interaction partners for developing the system. These proteins drove effective β-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other β-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent β-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wild-type Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.

PCR bias Toward the wild-type k-ras and p53 sequences: Implications for PCR detection of mutations and cancer diagnosis

PCR-based cancer diagnosis requires detection of rare mutations in k- ras, p53 or other genes. The assumption has been that mutant and wild-type sequences amplify with near equal efficiency, so that they are eventually present in proportions representative of the starting material. Work on factor IX suggests that this assumption is invalid for one case of near- sequence identity. To test the generality of this phenomenon and its relevance to cancer diagnosis, primers distant from point mutations in p53 and k-ras were used to amplify wild-type and mutant sequences from these genes. A substantial bias against PCR amplification of mutants was observed for two regions of the p53 gene and one region of k-ras. For k-ras and p53, bias was observed when the wild-type and mutant sequences were amplified separately or when mixed in equal proportions before PCR. Bias was present with proofreading and non-proofreading polymerase. Mutant and wild-type segments of the factor V, cystic fibrosis transmembrane conductance regulator and prothrombin genes were amplified and did not exhibit PCR bias. Therefore, the assumption of equal PCR efficiency for point mutant and wild-type sequences is invalid in several systems. Quantitative or diagnostic PCR will require validation for each locus, and enrichment strategies may be needed to optimize detection of mutants.

Population genetic structure and patterns of dispersal in the Giant Long-Armed Prawn, Macrobrachium lar (Fabricius, 1798) (Decapoda : Palaemonidae)

The Giant Long-Armed Prawn, Macrobrachium lar is a freshwater species native to the Indo-Pacific. M. lar has a long-lived, passive, pelagic marine larval stage where larvae need to colonise freshwater within three months to complete their development. Dispersal is likely to be influenced by the extensive distances larvae must transit between small oceanic islands to find suitable freshwater habitat, and by prevailing east to west wind and ocean currents in the southern Pacific Ocean. Thus, both intrinsic and extrinsic factors are likely to influence wild population structure in this species. The present study sought to define the contemporary broad and fine-scale population genetic structure of Macrobrachium lar in the south-western Pacific Ocean. Three polymorphic microsatellite loci were used to assess patterns of genetic variation within and among 19 wild adult sample sites. Statistical procedures that partition variation implied that at both spatial scales, essentially all variation was present within sample sites and differentiation among sites was low. Any differentiation observed also was not correlated with geographical distance. Statistical approaches that measure genetic distance, at the broad-scale, showed that all south-western Pacific Islands were essentially homogeneous, with the exception of a well supported divergent Cook Islands group. These findings are likely the result of some combination of factors that may include the potential for allelic homoplasy, through to the effects of sampling regime. Based on the findings, there is most likely a divergent M. lar Cook Islands clade in the south-western Pacific Ocean, resulting from prevailing ocean currents. Confirmation of this pattern will require a more detailed analysis of nDNA variation using a larger number of loci and, where possible, use of larger population sizes.

Impacts of traditional husbandry practices on exploitable levels of genetic diversity in cultured 'Tra' catfish (Pangasianodon hypophthalmus) in the Mekong Delta, Vietnam

Sutchi catfish (Pangasianodon hypophthalmus) – known more universally by the Vietnamese name ‘Tra’ is an economically important freshwater fish in the Mekong Delta in Vietnam that constitutes an important food resource. Artificial propagation technology for Tra catfish has only recently been developed along the main branches of the Mekong River where more than 60% of the local human population participate in fishing or aquaculture. Extensive support for catfish culture in general, and that of Tra (P. hypophthalmus) in particular, has been provided by the Vietnamese government to increase both the scale of production and to develop international export markets. In 2006, total Vietnamese catfish exports reached approximately 286,602 metric tons (MT) and were valued at 736.87 $M with a number of large new export destinations being developed. Total value of production from catfish culture has been predicted to increase to approximately USD 1 billion by 2020. While freshwater catfish culture in Vietnam has a promising future, concerns have been raised about long-term quality of fry and the effectiveness of current brood stock management practices, issues that have been largely neglected to date. In this study, four DNA markers (microsatellite loci: CB4, CB7, CB12 and CB13) that were developed specifically for Tra (P. hypophthalmus) in an earlier study were applied to examine the genetic quality of artificially propagated Tra fry in the Mekong Delta in Vietnam. The goals of the study were to assess: (i) how well available levels of genetic variation in Tra brood stock used for artificial propagation in the Mekong Delta of Vietnam (breeders from three private hatcheries and Research Institute of Aquaculture No2 (RIA2) founders) has been conserved; and (ii) whether or not genetic diversity had declined significantly over time in a stock improvement program for Tra catfish at RIA2. A secondary issue addressed was how genetic markers could best be used to assist industry development. DNA was extracted from fins of catfish collected from the two main branches of the Mekong River inf Vietnam, three private hatcheries and samples from the Tra improvement program at RIA2. Study outcomes: i) Genetic diversity estimates for Tra brood stock samples were similar to, and slightly higher than, wild reference samples. In addition, the relative contribution by breeders to fry in commercial private hatcheries strongly suggest that the true Ne is likely to be significantly less than the breeder numbers used; ii) in a stock improvement program for Tra catfish at RIA2, no significant differences were detected in gene frequencies among generations (FST=0.021, P=0.036>0.002 after Bonferroni correction); and only small differences were observed in alleles frequencies among sample populations. To date, genetic markers have not been applied in the Tra catfish industry, but in the current project they were used to evaluate the levels of genetic variation in the Tra catfish selective breeding program at RIA2 and to undertake genetic correlations between genetic marker and trait variation. While no associations were detected using only four loci, they analysis provided training in the practical applications of the use of molecular markers in aquaculture in general, and in Tra culture, in particular.

AtBAG7, an Arabidopsis Bcl-2-associated athanogene, resides in the endoplasmic reticulum and is involved in the unfolded protein response

The Bcl-2-associated athanogene (BAG) family is an evolutionarily conserved, multifunctional group of cochaperones that perform diverse cellular functions ranging from proliferation to growth arrest and cell death in yeast, in mammals, and, as recently observed, in plants. The Arabidopsis genome contains seven homologs of the BAG family, including four with domain organization similar to animal BAGs. In the present study we show that an Arabidopsis BAG, AtBAG7, is a uniquely localized endoplasmic reticulum (ER) BAG that is necessary for the proper maintenance of the unfolded protein response (UPR). AtBAG7was shown to interact directly in vivo with themolecular chaperone, AtBiP2, by bimolecular fluorescence complementation assays, and the interaction was confirmed by yeast two-hybrid assay. Treatment with an inducer of UPR, tunicamycin, resulted in accelerated cell death of AtBAG7-null mutants. Furthermore, AtBAG7 knockouts were sensitive to known ER stress stimuli, heat and cold. In these knockouts heat sensitivity was reverted successfully to the wild-type phenotype with the addition of the chemical chaperone, tauroursodexycholic acid (TUDCA). Real-time PCR of ER stress proteins indicated that the expression of the heat-shock protein, AtBiP3, is selectively up-regulated in AtBAG7-null mutants upon heat and cold stress. Our results reveal an unexpected diversity of the plant's BAG gene family and suggest that AtBAG7 is an essential component of the UPR during heat and cold tolerance, thus confirming the cytoprotective role of plant BAGs.

Book review of Donald, S.H., & Gammack, J.G. 2007. Tourism and the Branded City : Film and Identity in the Pacific Rim. Aldershot : Ashgate.

In the decade since the destination branding literature emerged (see for example Pritchard & Morgan 1998, Dosen & Vransevic 1998), only a few books have been published. These are Morgan et al.’s (2002, 2004) edited volumes of international case studies and conceptual papers, and Baker’s (2007) practitioner perspective on branding small cities in the USA. This work by Stephanie Donald and John Gammack is the first research-based text related to destination branding, and is a welcome and timely addition to the field. In the foreword to the first issue of Place Branding and Public Policy, editor Simon Anholt (2004, p. 4) suggested “almost nobody agrees on what, exactly, branding means”, when he described place branding practice as akin to the Wild West. Indeed, this lack of theory was one of the motivators for the authors of this text. Tourism and the Branded City is part of Ashgate’s New Directions in Tourism Analysis series, edited by Dimitri Ioannides. The aim of the series is to address the gap in published theory underpinning the study of tourism, with a particular interest in non-business disciplines such as Sociology, Social Anthropology, Human and Social Geography, and Cultural Studies...

Studies of assisted reproduction in the spotted grass frog Limnodynastes tasmaniensis: ovulation, early development and microinjection (ICSI)

Assisted Reproductive Technologies (ART) offer a wide range of techniques that have the potential to augment efforts to conserve and manage endangered amphibians and improve wild and captive population numbers. Gametes and tissues of species nearing endangered or extinct status can be cryopreserved and stored in gene banks, to provide material that can be utilised in the future as ART methods are refined. The Spotted Grass Frog, Limnodynastes tasmaniensis, is an abundant amphibian species in South-Eastern Australia of the family Myobatrachidae, that is suitable for the development of ART systems that can be applied to the threatened and endangered myobatrachid and other amphibian species native to Australia. The aim of this study was to advance the understanding of ovulation, fertilisation and embryo nic development of Lim. tasmaniensis and in vitro manipulations of reproduction and development for use in the development of advanced ART procedures such as intracytoplasmic spermatozoon injection (ICSI), androgenesis and nuclear transfer. Ovulation in amphibians can be induced by protocols utilising natural or synthetic hormones. All protocols tested on Lim. tasmaniensis in this study required two injections and the most effective protocols continued to require a first injection of pituitary extracts to induce ovulation. The second injection was, however, successfully replaced by synthetic chorionic gonadotrophin at a threshold dosage of 100 iu and halved the number of cane toads required to source the pituitaries. A combination of LHRH and Pimozide offered a less effective protocol, that did not require the use of pituitary extracts, and avoided the risk of pathogen transfer associated with unsterilised pituitary extracts. Unfertilised eggs of Lim. tasmaniensis were exposed to media of various osmolalities to determine media effects on eggs and their surrounding jelly layers that might impact on egg viability and fertilisability. Osmolality had no effect upon the egg diameter, however, rapid swelling of the jelly layers occurred within 15 minutes of exposure to various media treatments and plateaued from 30-90 minutes without further expansion. Swelling of the jelly layers was increased in hypotonic media (2.5% SAR, H2O) and minimised in the isotonic media (100% SAR). The optimal conditions for the culture of Lim. tasmaniensis eggs were identified as a holding media of 100% SAR, followed by a medium change to 2.5% SAR at insemination. This sequence of media minimised the rate of swelling of the jelly layers prior to contact with the spermatozoa, and maximised the activation of spermatozoa and eggs throughout fertilisation and embryonic development. Embryos of Lim. tasmaniensis were cultured at four temperatures (13 C, 17 C, 23 C and 29 C), to determine the effect of temperature on cleavage and embryonic development rates. Embryonic development progressed through a sequence of stages that were not altered by changes in temperature. However cleavage rates were affected by changes in temperature as compared with normal embryonic growth at 23 C. Embryonic development was suspended at the lowest temperature (13 C) while embryonic viability was maintained. A moderate decrease in temperature (17 C) slowed cleavage, while the highest temperature (29 C) increased the cleavage rate, but decreased the embryo survival. Rates of embryonic development can be manipulated by changes in temperature and this method can be used to source blastomeres of a specific size/stage at a predetermined age or halt cleavage at specific stages for embryos or embryo derived cells to be included in ART procedures. This study produced the first report of the application of Intracytoplasmic Spermatozoon Injection (ICSI) in an Australian amphibian. Eggs that were activated by microinjection with a single spermatozoon (n=50) formed more deep, but abnormal, cleavage furrows post-injection (18/50, 36%), than surface changes (12/50, 24%). This result is in contrast to eggs injected without a spermatozoon (n=42), where the majority of eggs displayed limited surface changes (36/42, 86%), and few deep, abnormal furrows (3/42, 7%). Three advanced embryos (3/50, 6%) were produced by ICSI that developed to various stages within the culture system. Technical difficulties were encountered that prevented the generation of any metamorphs from ICSI tadpoles. Nevertheless, when these blocks to ICSI are overcome, the ICSI procedure will be both directly useful as an ART procedure in its own right, and the associated refinement of micromanipulation procedures will assist in the development of other ART procedures in Lim. tasmaniensis. A greater understanding of basic reproductive and developmental biology in Lim. tasmaniensis would greatly facilitate refinement of fertilisation by ICSI. Assisted Reproductive Technologies, in conjunction with gene banks may in the future regenerate extinct amphibian species, and assist in the recovery of declining amphibian populations nationally and worldwide.

Comparison of antigen detection and quantitative PCR in the detection of chlamydial infection in koalas (Phascolarctos cinereus)

The gold standard method for detecting chlamydial infection in domestic and wild animals is PCR, but the technique is not suited to testing animals in the field when a rapid diagnosis is frequently required. The objective of this study was to compare the results of a commercially available enzyme immunoassay test for Chlamydia against a quantitative Chlamydia pecorum-specific PCR performed on swabs collected from the conjunctival sac, nasal cavity and urogenital sinuses of naturally infected koalas (Phascolarctos cinereus). The level of agreement for positive results between the two assays was low (43.2%). The immunoassay detection cut-off was determined as approximately 400 C. pecorum copies, indicating that the test was sufficiently sensitive to be used for the rapid diagnosis of active chlamydial infections.

A strong quantitative trait locus for wing length on chromosome 2 in a wild population of great reed warblers

Wing length is a key character for essential behaviours related to bird flight such as migration and foraging. In the present study, we initiate the search for the genes underlying wing length in birds by studying a long-distance migrant, the great reed warbler (Acrocephalus arundinaceus). In this species wing length is an evolutionary interesting trait with pronounced latitudinal gradient and sex-specific selection regimes in local populations. We performed a quantitative trait locus (QTL) scan for wing length in great reed warblers using phenotypic, genotypic, pedigree and linkage map data from our long-term study population in Sweden. We applied the linkage analysis mapping method implemented in GRIDQTL (a new web-based software) and detected a genome-wide significant QTL for wing length on chromosome 2, to our knowledge, the first detected QTL in wild birds. The QTL extended over 25 cM and accounted for a substantial part (37%) of the phenotypic variance of the trait. A genome scan for tarsus length (a bodysize-related trait) did not show any signal, implying that the wing-length QTL on chromosome 2 was not associated with body size. Our results provide a first important step into understanding the genetic architecture of avian wing length, and give opportunities to study the evolutionary dynamics of wing length at the locus level. This journal is© 2010 The Royal Society.